GERMICIDAL ULTRAVIOLET LIGHT INDUCES ORNITHINE DECARBOXYLASE IN MOUSE EPIDERMAL CELLS AND MODIFIES THE INDUCTION CAUSED BY PHORBOL ESTER TUMOR PROMOTERS

Germicidal ultraviolet light (UVC. 8–10 J/m²) induces ornithine decarboxylase (ODC) in mouse epidermal cells in vitro in a biphasic manner with maxima of 2–3 fold induction at 4–6 h and of 10–20 fold induction at 15–18 h after irradiation. At this dose of UVC overall protein synthesis is inhibited by 10–30% and RNA synthesis by 40–50%. Induction of both ODC peaks is prevented by actinomycin D or cycloheximide. Similar culture factors appear to influence the extent of ODC induction by UVC and by the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), since the ratio of peak activities is approximately constant at 2, whereas absolute values vary considerably between experiments. If cells are irradiated with UVC and then exposed to TPA, the effects are additive at 10 J/m², less than additive at higher and enhanced at lower doses of UVC.     

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.     

THE EFFECT OF ULTRAVIOLET RADIATION ON WHEAT ROOT VESICLES ENRICHED IN PLASMA MEMBRANE

Abstract— The irradiation of plant cells with UV radiation (254nm) causes various solutes to leak from the cells. Vesicles enriched in plasma membranes were prepared from wheat roots. These were used to determine whether UV radiation alters membrane function by direct action on the membranes and to distinguish between the chemical effects produced by high and low fluences of UV. The plasma membrane-associated K⁺-stimulated ATPase was very sensitive to UV radiation (100% inhibition with 1.35kJ/m²). ATPase activity measured in the absence of K⁺ and K⁺-stimulated ATPase activity measured in the presence of diethylstilbestrol were much less sensitive. Lipid breakdown, as measured by malondialdehyde production, occurred only at UV fluences greater than 1.8 kJ/m².     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

SATURATION OF DNA REPAIR IN MAMMALIAN CELLS*

Abstract—Excision repair seems to reach a plateau in normal human cells at a 254 nm dose near 20J/m². We measured excision repair in normal human fibroblasts up to 80J/m². The four techniques used (unscheduled DNA synthesis, photolysis of BrdUrd incorporated during repair, loss of sites sensitive to a UV endonuclease from Micrococcus luteus , and loss of pyrimidine dimers from DNA) showed little difference between the two doses. Moreover, the loss of endonuclease sites in 24 h following two 20J/m² doses separated by 24 h was similar to the loss observed following one dose. Hence, we concluded that the observed plateau in excision repair is real and does not represent some inhibitory process at high doses but a true saturation of one of the, rate limiting steps in repair.     

THE PHOTODYNAMIC EFFECT OF HEMATOPORPHYRIN ON DNA

Abstract— Breakage of DNA in vitro and inside E. colt cells has been determined after exposure to monochromatic 365 nm light in the presence of 10 µM hematoporphyrin. When measured by alkaline sucrose sedimentation, the yields of breaks were 1.4 × 10^-12 per dalton and per J/m² for Col El-DNA in vitro and 5.9 × 10^-3 per dalton and per J/m² for superinfecting phage Λ DNA inside E. coli cells made permeable by toluene. No breaks were found by neutral sucrose sedimentation, demonstrating that the lesions represent alkali-labile bonds. The majority of the alkali-labile bonds were induced by singlet oxygen, as evidenced by the several-fold higher yield obtained in D₂O-containing buffer.     

INTERACTION OF 8-METHOXYPSORALEN AND NEAR-UV LIGHT CAUSES MUTATION AND CYTOTOXICITY IN MAMMALIAN CELLS

Abstract The interaction of near-UV light and a photosensitizer, 8-methoxypsoralen (8-MOP), was studied in the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase system; cell survival (cloning efficiency) and mutation induction (resistance to 6-thioguanine) were quantified. Exposure of cells to either 8-MOP up to 20 μg/m l (93 μ M ) or near–UV light up to 40000 J/m² had no effect on either survival or mutation frequency. Preincubation of cells with 8–MOP from 5 to 120 min prior to irradiation with various fluences did not affect cell survival or mutation frequency. Survival decreased and mutation frequency increased linearly when either the 8-MOP concentration or fluence was increased while the other factor was held as a constant. Mutation frequency appears to show reciprocity relative to the product of 8-MOP concentration times fluence of near–UV light [(μg/m l )·(J/m²)] throughout a range apparently limited by high cell lethality. The observed pooled data on mutation, f (x), as a function of (μg/m l )·(J/m²), x , fit a linear dose–response line, f (x) = (34.2 + 0.05 x ) × 10^-6. Cell survival, however, does not appear to exhibit such reciprocity.     

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.     

ULTRAVIOLET LIGHT-INDUCED INHIBITION OF CELL DIVISION AND DNA SYNTHESIS IN AXENICALLY GROWN REPAIR MUTANTS OF DICTYOSTELIUM DISCOIDEUM

Cell division and DNA synthesis were studied during axenic growth following 254 nm ultraviolet light (UV) irradiation of a repair-proficient parental strain ( rad⁺ , D₁₀ colony formation = 195 J/m²) and two repair mutants ( rad C. D₁₀= 50 J/m²; rad B. D₁₀= 5 J/m²) of Dictyostelium discoideum. Isopycnic CsCI gradients were used to distinguish uptake of labeled precursors into nuclear (n) and mitochondrial (m) DNA, using Netropsin to enhance the density resolution. In all strains, m-DNA synthesis was inhibited to a lesser extent than was n-DNA synthesis. For rad C, which has been shown in other experiments to be slow in incision and dimer removal, the UV-induced lags in division and n-DNA synthesis were longer than for rad⁺. However, rad B showed a more complex response. Although brief division lags were observed for < 10 J/m², little immediate division lag was detected at greater fluences. Instead, a brief period of cell multiplication of up to but not exceeding two-fold occurred, followed by a cessation of division, and then by lysis. Fluences that yielded extensive lags in n-DNA synthesis in rad^- and rad C resulted in little detectable immediate postirradiation lag in n-DNA synthesis in rad B. However, later in the postirradiation period, when DNA synthesis had resumed in rad⁺ and rad C. it gradually declined to near zero in rad B. We conclude: (1) that the more extended lag in division and n-DNA synthesis in rad C is consistent with its slower rate of excision repair, and (2) that rad B contains a defect resulting in less initial blockage of DNA replication by UV lesions.     

USE OF METABOLIC INHIBITORS TO INVESTIGATE THE EXCISION REPAIR OF PYRIMIDINE DIMERS AND NON-DIMER DNA DAMAGES INDUCED IN HUMAN AND ICR 2A FROG CELLS BY SOLAR ULTRAVIOLET RADIATION

Abstract— ICR 2A frog and normal human skin fibroblasts were exposed to either 5 J/m² of 254 nm UV or 50 kJ/m² of the Mylar-filtered solar UV wavelengths produced by a fluorescent sunlamp. Following these approximately equitoxic treatments, cells were incubated in medium containing the DNA synthesis inhibitors hydroxyurea (HU) and 1–β-D-arabinofuranosyl cytosine (ara C) for 0–20 min (human fibroblasts) or 0–4 h (frog cells) to accumulate DNA breaks resulting from enzymatic incision during excision repair. It was found that breaks were formed in human cells at about a 200-f-old higher rate compared with the ICR 2A cells indicating a relatively low capacity for excision repair in the frog cells. In addition, the rate of DNA break formation in solar UV-irradiated cells was only one-third of the level detected in 254 nm-irradiated cells. This result is consistent with the conclusion that the pathway(s) involved in the repair of solar UV-induced DNA damages differs from the repair of lesions produced in cells exposed to 254 nm UV.     

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.     

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.     

AN FMN-PHOTOSYSTEM I PHOTOVOLTAIC CELL

Abstract— We have made a photovoltaic cell using Photosystem I subchloroplast particles isolated according to the method of Shiozawa et al. (1974). The particles were placed on a filter between two compartments one of which contained the electron donor, K₄Fe(CN)₆ and the other the electron acceptor, FMN. Upon illumination with white light ( I = 80 W/m²) a potential of 300 mV was observed across a 3000 Ω load resistance. Both Photosystem I photochemistry and direct photoreactions of FMN contribute to the process. A power output of 20 μW was observed for a 2 cm² filter containing 60 μg chlorophyll. This corresponds to 0.1 W/m². The power efficiency was 0.13%. The short circuit current was 108 μA.     

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.     

EFFECTS OF INTENSITY AND FLUENCE UPON DNA SINGLE-STRAND BREAKS INDUCED BY EXCIMER LASER RADIATION

Abstract— A Xenon-chloride excimer laser emitting energy at 308 nm was used to induce single-strand breaks (SSBs, frank breaks plus alkali-labile lesions as assayed by alkaline sucrose sedimentation techniques) in purified DNA from Bacillus subtilis . A fluence response study and a peak pulse intensity study were performed. At a pulse energy of 0.1 mJ/pulse, the radiation induced SSBs in a linear fashion (91 SSB/10⁸ Da per MJ/m²) to a maximum exprimental fluence of 1.28 MJ/m². The pulse intensity study showed that there were no significant changes in DNA breakage (105 SSB/10⁸ Da) between 2.93 times 10⁹ and 5.86 times 10¹¹ W/m² (0.11 and 22.0 mJ/pulse) at a constant total fluence of 1.1 MJ/m² (27000 mJ dose). This study has verified and extended previous work by quantifying the yield of SSBs induced in DNA by this laser radiation.     

ULTRAVIOLET IRRADIATION OF NUCLEIC ACIDS COMPLEXED WITH HEAVY ATOMS—III. INFLUENCE OF Ag + AND Hg 2+ ON THE SENSITIVITY OF PHAGE AND OF TRANSFORMING DNA TO ULTRAVIOLET RADIATION

Abstract— We have studied the influence of the heavy metal ions Ag⁺ and Hg²⁺ on the photoinactivation and photodimerization of transforming DNA and of bacteriophage. The rate of inactivation of Haemophilus influenzae transforming DNA by ultraviolet (UV) radiation was enhanced by a factor of 30 when it was complexed with Ag⁺. This enhancement was correlated with a comparable increase in the rate of thymine dimerization. In contrast, mercuric ions led to a reduction in the rates of both inactivation and dimerization. When we examined the effects of these metal ions on the photobiology of bacteriophage, we again found that Ag⁺ enhanced and Hg²⁺ reduced the rate of ultraviolet inactivation. These results demonstrate that heavy metals may be useful tools for studying the photochemistry and photobiology of nucleic acids.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

THE RATE OF REMOVAL OF SUNLIGHT-INDUCED PYRIMIDINE DIMERS FROM THE DNA OF CULTURED HUMAN DIPLOID FIBROBLASTS

Abstract The rate of excision of sunlight-induced pyrimidine dimers in DNA of exposed human cells was determined. Two normal excision repair-proficient human diploid fibroblast strains (WS-1 and KD) and a repair-deficient strain (XP12BE, group A) maintained in a nondividing state were exposed to summer noon-time sunlight for times (5 and 20 min) that induced numbers of dimers equivalent to far UV (254 nm) exposures of 1 and 4 J/m². Pyrimidine dimers were quantified in extracted DNA using a U V-endonuclease-alkaline sedimentation assay. The excision rates of these dimers were similar to those observed for the excision of UV-induced pyrimidine dimers. No sunlight-induced inhibition or stimulation of DNA repair was observed in either strain at these low exposures.     

EFFECT OF PARAMAGNETIC SPECIES ON 8-METHOXYPSORALEN-SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE LAMBDA

Abstract. 8-Methoxypsoralen-sensitized photoinactivation of bacteriophage lambda (Λ_vir) was examined in the presence of various metallic ions. Paramagnetic ions, Co²⁺, Ni²⁺ or Mn²⁺, strongly repressed the reaction, whereas diamagnetic ions, Ca²⁺ or Zn²⁺, exerted no influence. Significant repression was also observed in the sample solution equilibrated with air.