INCREASED LEVELS OF UNSCHEDULED DNA SYNTHESIS IN UV-IRRADIATED HUMAN FIBROBLASTS PRETREATED WITH SODIUM BUTYRATE

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 m M results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m². The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine.     

UNSCHEDULED DNA SYNTHESIS: A QUANTITATIVE INDICATOR OF RESIDUAL IMMUNODETECTABLE PYRIMIDINE DIMERS IN HUMAN FIBROBLASTS AFTER ULTRAVIOLET-B IRRADIATION

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.     

THE DOSE RESPONSE CURVE IN PHYTOCHROME-MEDIATED ANTHOCYANIN SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —The dose response curve for light (phytochrome)-induced anthocyanin synthesis was determined in the mustard seedling. The curve gives the amount of anthocyanin (A) synthesized within 24 h as a function of the amount of P_fr* produced by a brief light pulse. The [P_fr] response curve is composed of two linear parts with very different slopes ( a _1,2) connected by a relatively narrow transient range (curved segment). The [P_fr] response curve extrapolates precisely through zero [P_fr]. The reciprocity law is valid over the whole range investigated (up to 320 s of irradiation). It is concluded that the initial (or primary) reaction of P_fr (P_fr+ X → P_frX) does not involve any significant cooperativity in the case of phytochrome-mediated anthocyanin synthesis. It is speculated that the linear parts of the [P_fr] response curve truly reflect the mode of phytochrome action ( A = a _1,2 [P_fr]; X does not come into play since it is not rate limiting) whereas the curved segment represents a transition of the reaction matrix of P_fr. The large difference between a₁ and a₂ seems to indicate that the physiological effectiveness of a given amount of P_fr (or P_frX) is determined by [P_fr] through a P_fr-induced change in the reaction matrix.     

THE ACTION SPECTRUM OF AN IN VITRO DNA PHOTOREACTIVATION SYSTEM

Abstract— The wavelength-dependence of in vitro photoreactivation of transforming DNA by yeast extract has been determined. There is an intensity-dependent lag at the beginning of the biological reaction. There is a similar lag in the splitting of thymine dimers by the yeast extract in the light, a process known to account for most or all of the increase in transforming activity of photoreactivated DNA. The most efficient wavelengths for photoreactivation are around 3550 and 3850 Å. Although the action spectrum is not very similar to flavin absorption, riboflavin at very low concentration inhibits photoreactivation, as it also inhibits a number of flavoenzymes, suggesting that the photoreactivating enzyme might be a flavoprotein.     

ACTION SPECTRUM AND ELECTROPHYSIOLOGICAL RESPONSES CORRELATED WITH THE PHOTOPHOBIC RESPONSE OF STENTOR COERULEUS

Abstract— The blue-green ciliate. Stentor coeruleus , is found predominantly in shady places. This concentration occurs because stentor responds when swimming from a shaded area to a lighted area by reversing the direction of its ciliary beat and reorienting its swimming direction until it once again is in the shaded area. A graded receptor potential is recorded from microelectrodes in vacuoles of stentor when the animal is photically stimulated. For all but very weak stimuli this receptor potential is sufficient to elicit a regenerative transmembrane response of variable amplitude in a swimming animal. Suprathreshold electrical stimuli also elicit this regenerative response. In turn the regenerative response is coupled to ciliary reversal. Thus ciliary reversal appears to be produced whenever the photic receptor potential crosses the threshold for elicitation of the regenerative response.
Using the threshold for production of ciliary reversal as a criterion response, an action spectrum was obtained. This action spectrum correlates well with the absorption spectrum of the major pigment of S. coeruleus , stentorin. Stentor bleached of pigment also have an elevated threshold for ciliary reversal. Thus stentorin seems to be the photosensitive pigment in stentor responsible for its photophobic behavior.     

CORRELATION OF UVC AND UVB CYTOTOXICITY WITH THE INDUCTION OF SPECIFIC PHOTOPRODUCTS IN T-LYMPHOCYTES AND FIBROBLASTS FROM NORMAL HUMAN DONORS

Abstract— By using specific monoclonal antibodies in situ and a computer-assisted image analysis system we have determined the relative induction of cyclobutane dimers, (6–4) photoproducts and Dewar isomers in human mononuclear cells and fibroblasts following irradiation with UVC, broad-spectrum UVB and narrow-spectrum UVB. The lamps produced these lesions in different proportions, with broad-spectrum UVB inducing a greater combined yield of (6–4) photoproducts and Dewar isomers per cyclobutane dimer than UVC or narrow-spectrum UVB. The relative induction ratios of (6–4) photoproducts compared to cyclobutane dimers were 0.15, 0.21 and 0.10 following irradiation with UVC, broad- or narrow-spectrum UVB, respectively. Although Dewar isomers were induced by UVC, their relative rate of formation compared to cyclobutane dimers was significantly greater after irradiation with either broad-spectrum or narrow-spectrum UVB. These values were 0.001, 0.07 and 0.07, respectively. With each lamp source, we have determined the survival of normal human T-lymphocytes and fibroblasts at fiuences, which induce equivalent yields of cyclobutane dimers, (6–4) photoproducts or (6–4) photoproducts plus Dewar isomers. Killing of fibroblasts appears to be associated with (6–4) photoproduct formation, whereas killing of T-lymphocytes seems to be mediated by combined (6–4) plus Dewar yields. These results emphasize the need to study the biological effects of UVB because cellular responses may be different from those following UVC irradiation.     

ACTION SPECTRUM STUDIES FOR INDUCTION OF IMMUNOLOGIC UNRESPONSIVENESS TO DINITROFLUOROBENZENE FOLLOWING IN VIVO LOW DOSE ULTRAVIOLET RADIATION

Abstract— Topical application of the contact sensitizer dinitrofluorobenzene to the skin of C3H mice previously exposed in vivo to low doses of UVB radiation from FS20 sun lamps resulted in the acquisition of antigen-specific unresponsiveness to that hapten. When narrow band radiation at various wavelengths between 260 and 320 nm was employed, increasing doses of radiation were found to produce increasing amounts of immunosuppression. Construction of an action spectrum curve revealed that radiation between 260 and 300 nm was most efficient in producing unresponsiveness. Wavelengths above 300 nm were much less efficient than those below 300 nm. These results indicate that there are major differences in the effectiveness of various components of the short and middle wavelength UV spectrum in eliciting immunologic unresponsiveness to a topically administered hapten. Potential chromophores for this UVB-induced immunosuppressive effect include DNA and urocanic acid.     

INDUCTION OF DNA STRAND BREAKS IN NORMAL HUMAN FIBROBLASTS EXPOSED TO MONOCHROMATIC ULTRAVIOLET AND VISIBLE WAVELENGTHS IN THE 240–546 nm RANGE

Abstract— The induction of DNA single-strand breaks in normal human fibroblasts exposed to monochromatic wavelengths from 240–546 nm was measured by the alkaline elution assay. The cells were irradiated at 1°C to prevent both repair of induced breaks and formation of enzymatically induced breaks through excision repair. The cultures were also washed with and irradiated while suspended in phosphate buffered saline to prevent the formation of DNA damaging photoproducts from medium components. The action spectrum for DNA strand breakage was found to exhibit one peak at 265 nm, consistent with DNA absorption, and a second peak at 450 nm. The normalized action spectrum in the visible is similar to the normalized absorption spectrum for riboflavin, a known photosensitizing agent, implicating this molecule as the absorbing chromophore.     

LIGHT-INDUCED SYNTHESIS OF ANTHOCYANIN IN CARROT CELLS IN SUSPENSION—IV. THE ACTION SPECTRUM

Abstract— Using carrot cell suspension in 2,4-dichlorophenoxyacetic acid (2,4-D)-depleted culture medium, fluence-response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence-response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence-dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV-B photoreceptor.
Although red (R)-light alone had a minor effect on anthocyanin accumulation, it modulated the action of UV-B light. That is, when carrot cells were irradiated with R-light either before or after UV-B irradiation, anthocyanin formation was greatly enhanced above the level enhanced by UV-B light alone. The most effective wavelength for this enhancement was 660 nm. The effect of R-light on the anthocyanin formation of the UV-B irradiated cells was reversed by immediately following it with far-red light, suggesting the involvement of phytochrome in the R-effect.     

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.     

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.     

ACTION SPECTRUM FOR ANTHRACENE-INDUCED PHOTOSENSITIZATION OF HUMAN SKIN

Abstract— The action spectrum for photosensitization by topically applied anthracene was determined in human volunteers. Spectral reactivity was demonstrated in the range between 320 and 380 nm, with peak activity at around 360 nm. Three distinct inflammatory responses viz. immediate transient erythema, delayed erythema, and wealing were evoked following exposure to effective wavelengths. The action spectra for these responses were similar but the threshold doses were different. Prior treatment with a mast cell degranulating agent (codeine) abolished anthracene-UVA induced wealing but did not influence the erythema response. These findings suggest that photosensitized damage to cutaneous mast cells may be partially responsible for some of the observed inflammatory responses, but other sites of photochemical injury are also involved.     

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.     

INHIBITION OF DNA SYNTHESIS BY PSORALEN-INDUCED LESIONS IN XERODERMA PIGMENTOSUM AND FANCONI''S ANEMIA FIBROBLASTS

Abstract— Exposure of human cells to psoralens and near-UV light produces a mixture of monoadducts and crosslinks in DNA, which inhibit DNA synthesis by blocking replicon initiation and chain elongation. 8-Methoxypsoralen (8-MOP) has a greater effect than angelicin in normal, xeroderma pigmentosum, and Fanconi's anemia cells. Recovery of DNA synthesis is not detectable up to 8 h after exposure. The average distance between lesions that block replication in individual replicons was measured by means of bromodeoxyuridine photolysis. After exposure to 10 μg/mℓ of 8-MOP and 7500 J/m² of near-UV light, blocks were formed every 20 μm. Replicon initiation was inhibited by exposure to near-UV light alone in normal and xeroderma pigmentosum. Exposure to low concentrations of angelicin or 8-MOP plus near-UV light inhibited replicon initiation in normal and Fanconi's anemia cells, but not in xeroderma pigmentosum cells. Inhibition of initiation was not obvious after treatment with high concentrations of 8-MOP or angelicin because of the dominant effect of crosslinks in blocking chain elongation.     

ENDOGENOUS GLUTATHIONE PROTECTS HUMAN SKIN FIBROBLASTS AGAINST THE CYTOTOXIC ACTION OF UVB, UVA AND NEAR-VISIBLE RADIATIONS

Both the UVB (290-320 nm) and UVA (320-380 nm) regions of sunlight damage human skin cells but, particularly at the longer wavelengths, information is scant concerning the mechanism(s) of damage induction and the roles of cellular defense mechanisms. Following extensive glutathione depletion of cultured human skin fibroblasts, the cells become strongly sensitized to the cytotoxic action of near-visible (405 nm), UVA (334 nm, 365 nm) and UVB (313 nm) but not UVC (254 nm) radiations. In the critical UVB region, the magnitude of the protection afforded by endogenous glutathione approaches that of the protection provided by excision repair. The results suggest that a significant fraction of even UVB damage can be mediated by free radical attack and that a major role of glutathione in human skin cells is to protect them from the cytotoxic action of sunlight.     

INDUCTION OF SLOWLY DEVELOPING ALKALI-LABILE SITES IN HUMAN P3 CELL DNA BY UVA AND BLUE- AND GREEN-LIGHT PHOTONS: ACTION SPECTRUM

Action spectra (365–520nm) for the formation of DNA single-strand breaks (SSB) and slowly developing alkali-labile sites (SDALS) in human teratocarcinoma P3 cells in culture were determined. Induction of SDALS results from the absorption of blue- and green-light photons. The spectrum has a broad peak that is maximal between 400 nm to 500 nm and declines sharply above and below these wavelength regions. Negligible yields of SDALS were produced by photons at wavelengths of 365 nm or shorter and at 520 nm or longer, whereas for SSB, the action increases with shorter wavelength throughout the whole spectral range studied. The configuration of the SDALS action spectrum suggests that the primary chromophore, and therefore possibly the photosensitizer, is a mixture of porphyrin and flavin residues.     

SUBSTRATE DEPENDENCE OF THE ACTION SPECTRUM FOR PHOTOENZYMATIC REPAIR OF DNA

Abstract— The absolute action spectrum has been determined for photoenzymatic splitting of cyclobutadipyrimidines ("pyrimidine dimers") from natural DNA, and from the synthetic polydeoxyribonucleotides poly(dA)·poly(dT) (forming only cyclobutadithymine) and poly(dG)·poly(dC) (forming only cyclobutadicytosine). These action spectra differ strikingly from each other, even when using the same enzyme preparations. On the other hand, the action spectrum for splitting cyclobutadithymine in natural DNA containing "dimers" of only this one type closely resembles the action spectrum for splitting the total mixture of "dimer" types in natural DNA, and is entirely different from the spectrum for splitting of the same photoproduct from poly(dA)·poly(dT). These results mean that the action spectrum is not simply the absorption spectrum of a chromophore carried by the photoreactivating enzyme, nor is it solely determined by the nature of the substrate photoproduct. It is at least partly determined by the over-all polynueleotide structure (viz. exact helical dimensions, pattern of neighboring bases to the "dimers," or both), affecting a ground state interaction between the enzyme and substrate in the enzyme-substrate complex.     

LASER-UV-MICROIRRADIATION OF CHINESE HAMSTER CELLS: THE INFLUENCE OF THE DISTRIBUTION OF PHOTOLESIONS ON UNSCHEDULED DNA SYNTHESIS

Abstract— Fibroblastoid Chinese hamster cells synchronized by mitotic selection were microirradiated in G1, using a low power laser-UV-microbeam (λ= 257 nm). The incident energy was either concentrated on a small part of the nucleus (mode 1) or distributed over the whole nucleus (mode 11). Using the same incident UV energy, the local UV fluences were estimated to differ by two orders of magnitude. Following microirradiation the cells were incubated with [³H]-thymidine for 2 h and thereafter processed for autoradiography. Silver grains were concentrated over the microirradiated part after mode 1 and distributed over the whole nucleus after mode 11 irradiation. To quantify the amount of unscheduled DNA synthesis, the number of grains per nucleus was determined. It increased with the total incident energy, but was not or only slightly affected by the mode of microirradiation, if appropriate autoradiographic conditions were used. The findings suggest that within the investigated range of energy densities (2.7–1000 J/m²), the total amount of unscheduled DNA synthesis depends on the total number of pyrimidine dimers but not on their distribution in nuclear DNA.     

PHOTOBEHAVIOR OF EUGLENA GRACILIS: ACTION SPECTRUM FOR THE STEP-DOWN PHOTOPHOBIC RESPONSE OF INDIVIDUAL CELLS

Abstract—Light-induced behavioral responses of Euglena gracilis have been investigated in single cells by means of a video system coupled to an optical microscope. Light intensity-effect curves at different wavelengths in the near UV and visible range have been determined. From these curves the action spectrum for the step-down photophobic response of Euglena has been calculated. From a comparison with the results obtained using a population method by means of a phototaxigraph, it is concluded that a single photomotile reaction is responsible for cell accumulation, brought about by trapping in the light spot and possibly by phototaxis towards scattered light from organisms already in the light field.     

DNA-PROTEIN CROSSLINKING IN NORMAL HUMAN SKIN FIBROBLASTS EXPOSED TO SOLAR ULTRAVIOLET WAVELENGTHS

Three normal human skin fibroblast cell lines were exposed to the simulated solar UV radiation produced by a fluorescent sunlamp under conditions in which the wavelength components shorter than either 295, 305 or 315 nm were excluded. The level of DNA-protein crosslinks (DPC) was then measured in those cells using the alkaline elution technique either immediately after irradiation or following a 24 h incubation. In each case, cells were exposed to fluences that induce similar levels of DPC. For cells exposed to 10 kJ m(-2) of sunlamp UV > 295 nm, the level of DPC exhibited a 2-5-fold increase following incubation. In contrast, 40-100% of the DPC were removed upon incubation of cells irradiated with either 100 kJ m(-2) of sunlamp UV > 305 nm or 150 kJ m(-2) of sunlamp UV > 315 nm. A major difference between the effects induced by these wavelength regions is that, in addition to DPC, a very high level of pyrimidine dimers is also produced by sunlamp UV > 295 nm, whereas much lower dimer yields result from treatment with either sunlamp UV > 305 nm or sunlamp UV > 315 nm. A potential role for type II DNA topoisomerase in the formation of these DPC resulting from either the change in conformational structure caused by the presence of a high level of dimers or an involvement of this enzyme in dimer excision repair is discussed.