Safety Evaluation of Far-UV-C Irradiation to Epithelial Basal Cells in the Corneal Limbus

Basal cells in the corneal limbus play an important role in the turnover cycle because they are the source of all cells that constitute the corneal epithelium. We examined the penetration depth of ultraviolet (UV) light in the corneal limbus and assessed the safety of Far-UV-C on stem cells in the basal area of the corneal limbus. Rats were irradiated with UV at peaks of 207, 222, 235, 254 and 311 nm while under anesthesia. The UV penetration depth in the rat corneal limbal epithelium was wavelength dependent: 311 nm UV-B and 254 nm UV-C reached the basal cells of the epithelium, and 235 nm radiation reached the middle area; however, 207 and 222 nm UV-C reached only the superficial layer of the epithelium. Porcine cornea, which is similar to the human eye in size and structure, were irradiated with 222 and 254 nm UV-C. As in rats, 222 nm UV-C reached only the superficial layer of the porcine corneal limbal epithelium. These results indicate that Far-UV-C, such as radiation of wavelengths of 207 and 222 nm, could not reach corneal epithelial stem cells, i.e. the cells remained intact. It is unlikely that the turnover of the corneal epithelium is obstructed or disrupted by exposure to Far-UV-C.     

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

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 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.     

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.     

INHIBITION OF TRANSIENT GENE EXPRESSION IN CHINESE, HAMSTER OVARY CELLS BY TRIPLET-SENSITIZED UV-B IRRADIATION OF TRANSFECTED DNA

The biological effectiveness of thymine-thymine cyclobutane dimers specifically induced by photosensitized ultraviolet-B irradiation was analyzed by host-cell reactivation of triplet-sensitized, UV-B irradiated plasmid pRSV beta gal DNA transfected into normal and repair-deficient Chinese hamster ovary cells. For comparison, pRSV beta gal DNA was also UV-C irradiated and transfected into the same cell lines. Ultraviolet endonuclease-sensitive site induction was determined after UV-C irradiation or acetophenone-sensitized UV-B irradiation of plasmid pRSV beta gal DNA. These data were used to calculate the number of cyclobutane pyrimidine dimers required to inactivate expression of the lacZ reporter gene in each irradiation condition. Transfection with UV-C-irradiated plasmid DNA resulted in a significantly greater reduction of reporter gene expression than did transfection with acetophenone-sensitized UV-B-irradiated pRSV beta gal DNA at equivalent induction of enzyme-sensitive sites. Since only a fraction of the inhibition could be accounted for by noncyclobutane dimer photoproducts, these results suggest that cytosine-containing pyrimidine cyclobutane dimers may be more effective than thymine-thymine dimers in inhibiting transient gene expression as measured in such host-cell reactivation experiments in mammalian cells.     

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.     

THE VASCULAR RESPONSE OF HUMAN SKIN TO ULTRAVIOLET RADIATION

The vascular response of human skin to 300 nm (UV-B) and 254 nm (UV-C) ultraviolet radiation was assessed using the reflectance measurement of erythema and the technique of laser Doppler velocimetry. For both wavelengths, the increase in measured Doppler blood flux varied with the increase in erythema in a quadratic manner predicted by a simple model based on the principles of fluid mechanics. This suggests that the mean red blood cell velocity increases significantly in areas of UV-B and UV-C erythema. No qualitative difference in response to these two wavelengths was demonstrated, suggesting that the same blood vessels are involved in the causation of both UV-B and UV-C erythema.     

DO PYRIMIDINE DIMER YIELDS CORRELATE WITH ERYTHEMA INDUCTION IN HUMAN SKIN IRRADIATED in situ WITH ULTRAVIOLET LIGHT (275–365 nm)?

Ultraviolet radiation produces erythema in human skin, and damages the DNA of living cells in skin. Previous work showed that broad-band UV-B (290-320 nm) radiation produced higher levels of cyclobutyl pyrimidine dimers in DNA of individuals with high UV-B sensitivity (low minimal erythema dose) than in subjects of low UV-B sensitivity [Freeman et al. (1986) J. Invest. Dermatol., 86, 34-36]. We examined the relationship between erythema induction and dimer yields in DNA of human skin irradiated in situ with narrow band radiation spanning the wavelength range 275-365 nm. We find that, in general, higher dimer yields are produced per incident photon in volunteers with higher susceptibility to erythema induced by radiation of the same wavelength.     

RELATIVE INDUCTION OF CYCLOBUTANE DIMERS and CYTOSINE PHOTOHYDRATES IN DNA IRRADIATED in vitro and in vivo WITH ULTRAVIOLET-C and ULTRAVIOLET-B LIGHT

SV40 DNA was irradiated in vitro and in vivo with UV-C (240-280 nm) and UV-B (280-320 nm) light, and damaged sites sensitive to digestion with Escherichia coli endonuclease III (endo III) and bacteriophage T4 endonuclease V (endo V) were quantified. The frequency of endo III-sensitive sites (primarily cytosine photohydrates) induced was 1-2% of the frequency of endo V-sensitive sites (cyclobutane dimers) in both purified SV40 DNA and intracellular episomal SV40 DNA. Endo III- and endo V-sensitive sites in DNA were induced in the same relative proportion at both UV-C and UV-B wavelengths. We found no evidence to support earlier inferences that intracellular conditions enhance the formation of cytosine photohydrates or other monobasic forms of DNA damage.     

Removing UV-A and UV-C radiation from UV-B fluorescent lamp emissions. Differences in the inhibition of photosynthesis in the marine alga Dunaliella tertiolecta using chromate versus cellulose acetate-polyester filters

Ultraviolet-B (UV-B; 280-320 nm)-emitting lamps unavoidably emit ultraviolet-A (UV-A; 320-400 nm) and ultraviolet-C (UV-C; <280 nm) radiation. Short-wavelength-blocking filters are generally used to limit the wave bands of UV under investigation. The widespread use of such filters means that all exposures to UV-B radiation will have a significant UV-A component. Therefore, the physiological effects unique to UV-B exposure are difficult to clearly isolate. This study presents a method to remove the UV-A and UV-C "contamination" using a liquid potassium chromate (K(2)CrO(4)) filter, thus allowing more direct assessment of the effects of UV-B exposure. Cultures of the green marine alga Dunaliella tertiolecta were grown in the absence of UV radiation. Sunlamps supplied the UV radiation for a 24 h exposure (solar radiation was not used in this study). The UV radiation was filtered either by the standard method (i.e. cellulose acetate (CA) with polyester = Mylar controls) or by a liquid filter of potassium chromate. Photosynthetic responses were compared. Major decreases in the ratio of variable to maximal fluorescence in dark-adapted cells and photosynthetic capacity were observed in CA-filtered cultures, whereas no change was observed in cells exposed to the same UV-B flux with the UV-A removed by K(2)CrO(4). The use of a CA filter with a Mylar control does not link results unequivocally to UV-B radiation. Such results should be interpreted with caution.     

Use of Uracil Thin Layer for Measuring Biologically Effective UV Dose

Abstract— Dimerization of uracil monomers in a polycrystalline state by UV radiation changes the absorption characteristics of a thin layer of the material. The change in optical density, measured by spectrophotometry in the250–400 nm range, as a function of the exposure time is evaluated in terms of the biologically effective UV dose. A statistical evaluation of a great number of uracil dosimeters irradiated with a TL01 lamp from Philips establishes the possibility of evaluating the biologically effective UV dose using a uracil dosimeter. Nonlinear regression procedures were introduced to correct the absorption spectra for contributions due to light scattering and to determine the optical density values required to calculate the UV dose expressed in H^Uunits. Comparison of cumulative daily doses and long-term monitoring measured by the uracil thin-layer dosimeter and a phage T7 dosimeter are given, which allow the determination of conversion factors between various biological dosimeters under different irradiation conditions.     

PHOTOCHEMISTRY OF URACIL. INTERSYSTEM CROSSING AND DIMERIZATION IN AQUEOUS SOLUTION

Abstract— Ultraviolet irradiation of ¹⁴C-uracil in aqueous solution results in the formation of hydrate and dimer photoproducts. The rate of dimerization increases with increasing uracil concentration, and decreases with increasing concentration of oxygen in solution. The kinetics are in agreement with a model previously proposed to account for the reactions, in which dimerization occurs by a reaction involving the triplet state of uracil, but hydration occurs from an excited singlet state. Oxygen reduces dimer formation by quenching the triplet. The quantum yield for intersystem crossing (ISC) to the triplet depends on the irradiation wavelength, increasing from 0.0014 at 280 nm to 0.016 at 230 nm. The ratio of rate constants for reaction of the triplet with oxygen and for dimerization is 1.1; the ratio of rate constants for triplet decay and for dimerization is 5.9 × 10^-5 M. The increase in ISC with photon energy suggests that ISC is favoured from excited vibrational levels. The quantum yield for hydration is about 0.002 at pH 4.5 for all wavelengths, but increases as the pH is decreased.     

UV‐photodimerization in uracil‐substituted dendrimers for high density data storage

Two series of uracil‐functionalized dendritic macromolecules based on poly (amidoamine) PAMAM and 2,2‐bis(hydroxymethylpropionic acid) bis‐MPA backbones were prepared and their photoinduced (2π+2π) cycloaddition reactions upon exposure to UV light at 257 nm examined. Dendrimers up to 4th generation were synthesized and investigated as potential materials for high capacity optical data storage with their dimerization efficiency compared to uracil as a reference compound. This allows the impact of increasing the generation number of the dendrimers, both the number of chromophores, as well as the different steric environments, on the performance of each series of dendrimers to be investigated. The (uracil)₁₂‐[G‐2]‐bis‐MPA and (uracil)₈‐[G‐1]‐PAMAM were observed to have high dimerization efficiency in solution with different behavior being observed for the PAMAM and bis‐MPA dendrimers. The dendrimers with the best dimerization efficiency in solution were then examined in the solid state as thin films cast on quartz plates, and their film qualities along with their photodimerization performance studied. High quality films with a transmission response of up to 70% in 55 s. when irradiated at 257 nm with an intensity of 70 mW/cm² could be obtained suggesting future use as recording media for optical data storage. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 4401–4412, 2007     

PHOTOCYCLOADDITION OF ACETONE TO URACIL AND CYTOSINE

Abstract— Ultraviolet irradiation (Λ > 280 nm) of uracil in aqueous acetone (1:1) produces cyclobutane uracil dimer and uracil-acetone addition product. The addition product is identified as an oxetane. The same product is also obtained from cytosine irradiated under the same conditions. The cytosine-acetone oxetane apparently undergoes deamination readily. Irradiation (Λ= 265 nm) of the oxetane in aqueous solution produces acetone and uracil with a quantum efficiency of 0–16.     

Biological Impact of Shorter Wavelength Ultraviolet Radiation-C†

Life on earth has constantly coped with the impact of solar radiation, especially solar ultraviolet radiation (solar UV). Various biological mechanisms protect us from solar UV. New devices emitting shorter wavelengths UV-C, i.e. <254 nm emitted by conventional UV germicidal lamps, have emerged. These shorter wavelength UV-C emitting devices are useful for various purposes, including microorganism inactivation. However, as solar UV-C does not reach the earth surface, biological impacts of UV-C has been studied using 254 nm germicidal lamps, and those using shorter wavelength UV-C is rarely known. To balance the utility and risk of UV-C, the biological effect of these new UV-C emitting devices must be investigated. In addition, our knowledge of biological impacts of the wavelength-dependent entire UV (100–400 nm) must be enhanced. In this review, we briefly summarize the biological impacts of shorter wavelength UV-C. Mechanisms of UV-C-induced cellular damage and factors affecting the microorganism inactivation efficiency of UV-C have been discussed. In addition, we theoretically estimate the probable photocarcinogenic action spectrum of shorter wavelength UV-C. We propose that increasing the knowledge on UV-C will facilitate the adoption of shorter wavelength UV-C emitting new devices in an optimal and appropriate manner.     

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.     

GENETIC EFFECTS OF UV-B: MUTAGENICITY OF 308 nm LIGHT IN CHINESE HAMSTER V79 CELLS

Abstract —Chinese hamster V79 cells were irradiated with 254 nm (UV-C) and 308 nm (UV-B) light, emitted by a germicidal lamp and an excimer laser, respectively. Induction of mutations at two distinct genetic loci was measured by selecting colonies resistant to 6-thioguanine or to ouabain. Unlike 6-thioguanine resistance which can be presumed to be due to many different types of genetic damage, mutation to ouabain resistance seems to result from base-pair substitution events only. Much higher doses of 308 than of 254 nm radiation are required to induce equivalent numbers of mutants. However, induction of cell inactivation and 6-thioguanine resistant mutations with the two UV sources appears to be correlated, suggesting that a common mechanism, perhaps involving the induction of pyrimidine-containing dimers, is involved. The frequency of ouabain resistant mutants per lethal event is on the other hand much higher after irradiation with the 308 nm light. This latter finding further defines a part of the UV-B spectral region which seems to induce a unique kind of DNA damage which specifically results in base-pair substitution events. Action spectra studies therefore appear necessary in the definition of the mutagenic effects of UV-B radiations in mammalian cells.     

Polymer-based triphenyl tetrazolium chloride films for ultraviolet radiation monitoring

Ultraviolet (UV) radiation monitoring films were prepared from solutions of polymers (polyvinyl, alcohol, PVA, or polyvinyl butyral, PVB), containing triphenyl tetrazolium chloride dye (TTC). These films have a pronounced response to the main UV radiation spectral regions [UV-A (400–320 nm), UV-B (320–280 nm), and UV-C (280–180 nm)] showing different sensitivities. PVA/TTC film has its maximum sensitivity in the UV-A region, while PVB/TTC film has its maximum sensitivity in the UV-C region. Both films have almost the same sensitivity in the UV-B region. The radiation-induced colour change is analysed spectrophotometrically at the maximum of the visible absorption band peaking at 492 nm wavelength. The measurement uncertainty of estimating ultraviolet radiation energy incident per unit area on the films is found to be about 3.5% (1 σ). The study of the effect of radiance exposure, incident wavelength, and storage conditions have been carried out to characterise the use of these films for actinometric monitoring artificial ultraviolet radiation sources which are used for medical and industrial applications.     

DNA as a solar dosimeter in the ocean.

Stratospheric ozone depletion may result in increased solar UV-B radiation to the ocean's upper layers and may cause deleterious effects on marine organisms. The primary UV-B damage induced in biological systems is to DNA. While physical measurements of solar UV-B penetration into the sea have been made, the effective depth and magnitude of actual DNA damage have not been determined. In the experiments reported here, UV-B-induced photoproducts (cyclobutane pyrimidine dimers) have been quantified in DNA molecules exposed to solar UV at the surface and at various depths in clear, tropical marine waters off Lee Stocking Island (23 degrees 45' N, 76 degrees 0.7' W), Exuma Cays, Bahamas. (14C)thymidine-labeled DNA or unlabeled bacteriophage phi X174 DNA was placed in specially designed quartz tubes at various depths for up to five days. Following exposure, DNA samples were removed to the laboratory where UV-B-induced pyrimidine dimers were quantified using a radiochromatographic assay, and bacteriophage DNA inactivation by solar UV-B was assayed by plaque formation in spheroplasts of Escherichia coli. Pyrimidine dimer induction was linear with time but the accumulation of dimers in DNA with time varied greatly with depth. Attenuation of dimer formation with depth of water was exponential. DNA at 3 m depth had only 17% of the pyrimidine dimers found at the surface. Bacteriophage phi X174 DNA, while reduced 96% in plaque-forming ability by a one day exposure to solar UV at the surface of the water, showed no effect on plaque formation after a similar exposure at 3 m. The data collected at the water's surface showed a "surface-enhanced dose" in that DNA damages at the real surface were greater than at the imaginary surface, which was obtained by extrapolating the data at depth to the surface. These results show the sensitivity of both the biochemical (dimers) and biological (phage plaques) DNA dosimeters. DNA dosimeters offer a sensitive, convenient and relatively inexpensive monitoring system, having both biochemical and biological endpoints for monitoring the biologically effective UV-B flux in the marine environment. Unlike physical dosimeters, DNA dosimeters do not have to be adjusted for biological effectiveness since they are sensitive only to DNA-mediated biologically effective UV-B radiation. Results of pyrimidine dimer induction in DNA by solar UV accurately predicted UV doses to the phage DNA.     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.