Photoreactivation in Paramecium tetraurelia under conditions of various degrees of ozone layer depletion

Photoreactivation (PR) is an efficient survival mechanism that helps protect cells against the harmful effects of solar-ultraviolet (UV) radiation. The PR mechanism involves photolyase, just one enzyme, and can repair DNA damage, such as cyclobutane-pyrimidine dimers (CPD) induced by near-UV/blue light, a component of sunlight. Although the balance of near-UV/blue light and far-UV light reaching the Earth's surface could be altered by the atmospheric ozone layer's depletion, experiments simulating this environmental change and its possible effects on life have not yet been performed. To quantify the strength of UVB in sunlight reaching the Earth's surface, we measured the number of CPD generated in plasmid DNA after UVB irradiation or exposure to sunlight. To simulate the increase of solar-UV radiation resulting from the ozone layer depletion, Paramecium tetraurelia was exposed to UVB and/or sunlight in clear summer weather. PR recovery after exposure to sunlight was complete at a low dose rate of 0.2 J/m2 x s, but was less efficient when the dose rate was increased by a factor of 2.5 to 0.5 J/m2 x s. It is suggested that solar-UV radiation would not influence the cell growth of P. tetraurelia for the reason of high PR activity even when the ozone concentration was decreased 30% from the present levels.     

XERODERMA PIGMENTOSUM FIBROBLASTS INCLUDING CELLS FROM XP VARIANTS ARE ABNORMALLY SENSITIVE TO THE MUTAGENIC AND CYTOTOXIC ACTION OF BROAD SPECTRUM SIMULATED SUNLIGHT

Abstract— The cytotoxic and mutagenic effects of broad spectrum simulated sunlight, as delivered by a Westinghouse Sun Lamp FS 20 filtered to eliminate wavelengths below 290 nm, were determined in diploid human skin fibroblasts which differ in their ability to repair pyrimidine dimers, and compared with results obtained with UV 254 nm radiation. The cell strains tested included normal fibroblasts; excision repair-deficient xeroderma pigmentosum (XP) cells from patients XP12BE (complementation group A). XP7BE (group D). and XP2BI (group G): and an XP variant patient (XP4BE) whose cells excise pyrimidinc dimers at a normal rate, but exhibit abnormal replication of DNA containing unexcised lesions. Cytotoxicity was assayed from loss of colony-forming ability. The group A cells were most sensitive to the killing effect of the Sun Lamp; the group D and G cells were slightly less sensitive; the XP variant cells showed intermediate sensitivity; and normal cells were most resistant. When the Sun Lamp survival curves for the group A, group D, the XP variant and normal cells were compared with their respective UV 254 nm survival curves, the relationships between the strains were virtually identical (i. e. the curves were related by a constant fluence modification factor). suggesting a common lesion for cell killing. The marker for mutagenesis was resistance to 6-thioguanine. The group A XP cells proved most sensitive to mutations induced by the simulated sunlight: the variant cells were intermediate; and the normal cells were the most resistant. Again, when the curves for mutations induced in these cell strains by simulated sunlight were compared with their respective 254 nm UV mutation curves, these were related by a constant fluence modification factor. suggesting a common lesion for mutagenesis. These results. taken together with published data indicating that at equicytotoxic levels of UV254 nm radiation and the filtered Sun Lamp. the number of pyrimidine dimers in the DNA of XP12BE cells was equal. support the hypothesis that the dimer is the lesion principally involved in both effects. Our data also support the hypothesis that mutations are involved in the sunlight-induced skin cancer of XP patients.     

TOXICITY, DNA DAMAGE AND INHIBITION OF DNA REPAIR SYNTHESIS IN HUMAN MELANOMA CELLS BY CONCENTRATED SUNLIGHT

A 1 m diameter water lens was used to focus solar radiation, giving an 8-fold concentration of the total spectrum and a cytocidal flux similar to that of laboratory UV sources. Survival curves for human melanoma cells were similar for sunlight and 254 nm UV, in that D _q, was usually larger than D _o. An xeroderma pigmentosum lymphoblastoid line was equally sensitive to both agents and human cell lines sensitive to ionizing radiation (lymphoblastoid lines), crosslinking agents or monofunctional alkylating agents (melanoma lines) had the same 254 nm UV and solar survival responses as appropriate control lines. Two melanoma sublines derived separately by 16 cycles of treatment with sunlight or 254 nm UV were crossresistant to both agents. In one melanoma cell line used for further studies, DNA strand breaks and DNA-protein crosslinking were induced in melanoma cells by sunlight but pyrimidine dimers (paper chromatography) and DNA interstrand crosslinking (alkaline elution) could not be detected. The solar fiuence response of DNA repair synthesis was much less than that from equitoxic 254 nm UV, reaching a maximum near the D _o value and then declining; semiconservative DNA synthesis on the other hand remained high. These effects were not due to changes in thymidine pool sizes. Solar exposure did not have a major effect on 254 nm UV-induced repair synthesis.     

PHOTODYNAMIC AND SUNLIGHT INACTIVATION OF ESCHERICHIA COLI STRAINS DIFFERING IN NEAR-UV SENSITIVITY AND RECOMBINATION PROFICIENCY

Abstract— Stationary cells of four Escherichia coli strains exhibiting all four possible combinations of genes controlling near-UV sensitivity ( nur vs nur ⁺) and recombination proficiency (far-UV sensitivity; recA1 us recA ⁺) have been inactivated by visible light in the presence of acridine orange (AO, 10µg/m l ) and sunlight. The results demonstrate that strains sensitive to near-UV inactivation are also sensitive to inactivation by visible light in the presence of AO and sunlight irrespective of the recA allele carried by the strain. These results may be interpreted to mean that major mechanisms of inactivation of stationary E. coli cells by near-UV, visible light in the presence of AO and sunlight are similar and not closely related to the mechanism of inactivation by far-UV.     

DEPENDENCE ON STAGE OF GROWTH OF MAMMALIAN CELL SENSITIVITY TO NEAR-UV IRRADIATION*

Abstract The variable sensitivity of exponentially growing mouse myeloma cells (66.2 subclone of MPC11) to monochromatic near-UV (365 nm) radiation was studied by determination of the relative cloning efficiency (i.e. survival) of cells in soft agar. Maximum sensitivity of the cells to near-UV light was found during the middle and late stages of exponential growth when the survival was only one-tenth of that during the early stage of growth. In addition, the shapes of near-UV survival curves changed with the stage of the cells irradiated. These data indicate that stages of growth can substantially alter the response of mammalian cells to near-UV radiation. These data further suggest the importance of testing the response of the cells throughout the growth employed in the experimental procedure.     

CORRELATION BETWEEN INACTIVATION OF HUMAN CELLS and NUMBERS OF PYRIMIDINE DIMERS INDUCED BY A SUN LAMP and 254 nm RADIATION

Abstract— Nondividing human fibroblasts are inactivated by radiation from a source (a Westinghouse sun lamp) that simulates the UV spectrum of sunlight. Survival curves determined for a DNA excision repair-proficient and a repair-deficient strain (XP12BE) are related to those determined using germicidal light (254 nm) by constant fluence modification factors. In addition, the same fraction of XP12BE cells are killed per pyrimidine dimer by 254 nm and sun lamp light. These results, when related to other survival and photoreactivation studies, suggest that the mechanism for inactivation of nondividing human cells by sun lamp light is the same as that by 254 nm and that pyrimidine dimers are the major responsible photolesion. Repair reverses some of the lethal effects of this light. We suggest that these conclusions apply to sunlight-irradiated skin cells in vivo.     

EARLY EFFECTS OF NEAR-ULTRAVIOLET RADIATION ON YEAST CELLS: CHANGES IN CELL SIZE

Exponentially growing yeast cells showed a drastic shrinkage when irradiated in water suspension (but not in buffer) with broad-band near-UV radiation. This was clearly seen soon after irradiation in size-distribution curves measured by a Coulter counter. This early shrinkage was observed at a near-UV fluence where the survival enters a region of exponential decline after a large shoulder. We further observed the formation of a distinct band in a density-gradient-centrifugation profile, presumably corresponding to the near-UV-affected cells. A survival test showed that the cells making up this band were dead.     

USE OF THE DYE HOECHST 33258 TO INCREASE THE PHOTOSENSITIVITY OF BROMODEOXYURIDINE-CONTAINING CELLULAR SLIME MOLDS

The photosensitivity of amoebae of Dictyostelium discoideum to near-ultraviolet light (near-UV; 365 nm peak) was investigated following growth in 5-bromodeoxyuridine (BrdUrd) and treatment with the dye Hoeschst 33258. Cell killing was studied as a function of dye-treatment time and concentration, and BrdUrd incorporation time and concentration. For example, cells grown for 13 h in 50 μg BrdUrd/ml and then treated for 1 h in 10 μg dye/ml were reduced to 10% survival by a near-UV exposure of 0.8 min. Combinations of any two of the three factors (BrdUrd, dye, near-UV) were ineffective for killing. The sensitivity of the cells to near-UV decreased rapidly as the holding time between the end of the dye treatment and the start of the UV exposure was increased.     

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.     

Inactivation and mutation induction in Saccharomyces cerevisiae exposed to simulated sunlight: evaluation of action spectra.

The effectiveness of polychromatic light irradiation was investigated for haploid yeast cells. Inactivation and mutation induction were measured in both a RAD-wildtype strain and an excision-repair defective strain. The behaviour of vegetative "wet" cells was compared to that of dehydrated cells. The aim of the study was to assess the interaction of UVC with other wavelengths in cells of different states of humidity. The irradiation procedure was therefore carried out using a solar simulator either with full spectrum or with a UVC-blocking filter (modified sunlight) added. The results were analysed on the basis of separately determined action spectra. The summation of the efficiency of individual wavelengths was compared to the values obtained from polychromatic irradiation. It is shown that the effects caused by the whole-spectrum irradiation in wet cells can be predicted sufficiently from the calculation, while dried wildtype cells exhibit higher mutation rates. Thus it can be assumed that drying-specific damage leads to lethal and mutagenic lesions which are processed in different ways, causing a synergistic behaviour in mutation induction. Irradiation of vegetative cells with modified sunlight (UVC-) results in less inactivation and lower mutation rates than were calculated. From these results it can be concluded that this antagonistic behaviour is caused by the interaction of near-UV photoproducts.     

Sunlight-induced killing of nondividing human cells in culture

Nondividing populations of human diploid fibroblasts that are DNA excision repair proficient (WS-1, KD. SSCW) and repair deficient (XP12BE) were exposed to mid-day summer sunlight for a determination of survival based on an ability of cells to remain attached to a culture vessel surface. Whereas mid- and far-UV wavelengths and radiation emitted from a sunlamp cause a gradual degeneraton and detachment of cells in a dose-dependent manner, sunlight does not promote cell killing that is evidenced by these criteria in repair proficient cells. Detachment of repair deficient cells is promoted to a limited extent but only at sunlight exposure times that are low with respect to the amount of DNA damage (pyrimidine dimers) induced. Repair proficient and deficient cells exposed to sunlight for longer times do not detach but are incapable of excuding a viable stain several days after exposure and appear as histologically fixed cells. Pyrimidine dimer levels in these sunlight irradiated cells were great enough to have promoted detachment had these levels been induced by UV (254 nm) alone. Other photodamage induced by these exposures evidently inhibits the dimer-induced cell degeneration that leads to cell detachment. We conclude that pyrimidine dimers are responsible for cell killing at short sunlight exposure times (< 40 min) but that at longer exposures (> 80 min) cells arc killed by a different mechanism that is independent of dimer-caused death.     

Alteration of uracil-DNA glycosylase activity by uracil dimers in DNA

Abstract The formation of colonies in solid medium was used as a criterion of viability to determine the effect of ultraviolet radiation on Trichomonas vaginalis. Both viability (colony) counts and total cell (hemocytometer) counts were used to estimate physiological ages of cell populations to be irradiated. Washed-cell suspensions in 0.6% saline were exposed to far- (254 nm) and near-UV (300–400 nm) radiation and dose-response survival curves were constructed from colony counts. The effect of far-UV was found to be independent of growth phase with the D₀ for exponential, early stationary, and late stationary cells 2.6, 2.7, and 2.7 J/m², respectively. Survival to near-UV increased with the age of cells with the estimated D₅₀ being 216 J/m² for exponential cells, 1360 J/m² for early stationary cells, and 4200 J/m² for late stationary cells. Exponential cells of Trichomonas gallinae irradiated with near-UV had a D₅₀ of 340 J/m². T. vaginalis is highly sensitive to far-UV relative to protozoa. T. vaginalis and T. gallinae are highly sensitive to near-UV relative to other microorganisms.     

INHIBITION OF PHOTOTAXIS IN Volvox aureus BY NATURAL AND SIMULATED SOLAR ULTRAVIOLET LIGHT

Exposure to artificial UV wavelengths and the UV component of sunlight delays positive phototaxis in the green alga Volvox aureus. Broad band wavelength filters were used to modify the output from UV-B sources (280-320 nm) and natural sunlight. The delay in phototaxis by artificial UV is increased with exposure to shorter UV-B wavelengths. Natural sunlight experiments were performed with exposure to full sunlight and to its UV component only. The UV component present in summer sunlight produced long periods of inhibition in phototaxis and even lethality, while exposure to the total spectrum of sunlight had no significant effects on movement or survival. The data indicate that although this species of alga is well equipped to deal with present levels of UV exposure, increases in the short UV-B wavelengths in sunlight may force an alteration in patterns of photomovement.     

OXYGEN EFFECT IN SURVIVAL OF Dictyostelium discoideum TREATED WITH NEAR ULTRAVIOLET RADIATION

Abstract— Fluence-response survival curves have been measured for the cellular slime mold Dictyostelium discoideum exposed to near ultraviolet radiation. Data were obtained for a wild type strain and three UV-sensitive mutant strains in exponential growth phase. Fluences for 10% survival (F₁₀) are about 1 MJ m⁻² for cells irradiated in saline solution saturated with nitrogen. When air is bubbled through the saline, the Fm values are only one third as large. Strain HPS50, which is the strain most sensitive to gamma radiation and to 254 nm UV, also exhibits the greatest sensitivity to near UV. However, the difference in sensitivity to near UV between wild type and mutant strains is small compared to other physical and chemical agents known to damage DNA.     

INHIBITION OF PHOTOTAXIS IN Volvox aureus BY NATURAL AND SIMULATED SOLAR ULTRAVIOLET LIGHT

Abstract
Exposure to artificial UV wavelengths and the UV component of sunlight delays positive phototaxis in the green alga Volvox aureus. Broad band wavelength filters were used to modify the output from UV-B sources (280–320 nm) and natural sunlight. The delay in phototaxis by artificial UV is increased with exposure to shorter UV-B wavelengths. Natural sunlight experiments were performed with exposure to full sunlight and to its UV component only. The UV component present in summer sunlight produced long periods of inhibition in phototaxis and even lethality, while exposure to the total spectrum of sunlight had no significant effects on movement or survival. The data indicate that although this species of alga is well equipped to deal with present levels of UV exposure, increases in the short UV-B wavelengths in sunlight may force an alteration in patterns of photomovement.     

THE EFFECT OF CHANGES IN CELL GEOMETRY ON THE SENSITIVITY TO ULTRAVIOLET RADIATION OF MAMMALIAN CELLULAR CAPACITY

Abstract— We have measured a calcium and magnesium dependent change in cell shape when mammalian cell monolayers are being prepared for irradiation by replacing their growth medium with certain buffers. In some cases, flattened cells (umbonate) assumed a spherical configuration. In order to assume a centrally located target molecule, we used a DNA-dependent cellular function–pacity for herpes viral growth–as the parameter to measure ultraviolet (UV) sensitivity of cells irradiated while in either of the two shapes. Umbonate cells were more sensitive to UV than were spherical cells. Exposures to the cell that lowered the cellular capacity of umbonate cells to the 10% survival level only lowered spherical cells to the 50% level. Twenty-seven per cent additional UV exposure to spherical cells was required to get the same effect as with umbonate cells. Included in the text are photographs of both cell types, survival curves for cellular capacity, a measure of the absorbance of cell homogenates, and a calculation of the relative number of photons absorbed by each cell nucleus.     

HOST CELL REACTIVATION IN MAMMALIAN CELLS-V. PHOTOREACTIVATION STUDIES WITH HERPES VIRUS IN MARSUPIAL AND HUMAN CELLS

Abstract— The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promote photoreactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7-0.8 for ovan cells and 0.5-0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more efficient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survhal curves for herpes virus in Potoroo cells indicated a high level of "dark" host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (Λ > 600 nm) and human cells with normal repair and with ceils deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreacti-vating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

ANOMALOUS DOSE-RESPONSE CHARACTERISTICS INDUCED BY CAFFEINE IN ULTRAVIOLET-IRRADIATED V79–79 CHINESE HAMSTER CELLS

Abstract— Cultured Chinese hamster cell line V79–79 exhibits an increase in survival with increasing UV fluence after a sharp decrease when exposed to 2.5 mM caffeine for 44 h after far-UV irradiation resulting in an anomalous maximum in the survival curve. No survival maximum is evident when either 0 or 1 mM caffeine is administered under the same conditions. The UV survival curve for 2.5 mM caffeine crosses the corresponding 1 mM curve and apparently becomes asymptotic to the OmM curve as UV fluence is increased. Chinese hamster cell lines V79–753B (related to V79–79 by derivation from the same parental line) and M3–1F3 (unrelated) exhibit only potentiation of post-UV lethality by the same concentration of caffeine and have no caffeine-induced anomalies in their survival curves. Xanthine. used alone or in combination with caffeine, only potentiated a slight amount of lethality and appears not to be a major causative factor of the anomaly.     

Daily and circadian variation in survival from ultraviolet radiation in Chlamydomonas reinhardtii

The survival of organisms depends on their ability to adapt to their environment, one important aspect of which is the daily cycle of day and night. During the day, organisms use a variety of strategies to protect themselves from deleterious ultraviolet (UV) wavelengths of sunlight. Among those strategies could be timing of UV-sensitive cellular processes to occur at night to avoid UV-induced damage. We tested whether the unicellular alga Chlamydomonas reinhardtii uses this strategy by measuring the survival of cells following exposure to UV radiation at different phases of the day. Chlamydomonas cells displayed a rhythm of survival from UV radiation where the most sensitive phases occurred during the end of the day and at the beginning of the night. This phase of sensitivity corresponds to the time of nuclear division. The rhythm continues in constant light indicating control by a circadian clock. The results presented here suggest a hypothesis of how circadian clocks may have evolved; a temporal program whereby light-sensitive processes are timed to avoid sunlight-induced damage would be advantageous and therefore selected.