THE ACTION SPECTRUM AND DOSE RESPONSE STUDIES OF UNSCHEDULED DNA SYNTHESIS IN NORMAL HUMAN FIBROBLASTS

Abstract— Excision repair of DNA damage by UV has been assessed in normal human fibroblasts in culture by measuring unscheduled DNA synthesis. Dose response experiments indicated that the same chromophore was involved in UV-induced damage and excision repair at three different wavelengths between 260 and 300 nm. Action spectra for unscheduled DNA synthesis were determined at wavelengths between 260 and 320 nm 30 min after irradiation using 2 doses of UV, 100 J m^-2and 10Jm^-2. Experiments at the lower dose were carried out because it appeared that repair was saturated with the higher dose at 260 and 280 nm. To explore this part of the spectrum further, experiments were performed with different doses at 260 and 280 nm and unscheduled DNA synthesis assessed 30 min and 24 h after irradiation. At 24 hr after irradiation a significantly greater amount of unscheduled DNA synthesis occurred at 280 nm. It is suggested, therefore, that both DNA and protein are concerned in the absorption of UV which leads to DNA damage and excision repair.     

PRIOR EXPOSURE OF HUMAN CELLS TO NEAR UV-RADIATION GIVES A DECREASE IN THE AMOUNT OF THE UNSCHEDULED DNA-SYNTHESIS INDUCED BY FAR UV-RADIATION

Pretreatment of human cells with near UV radiation (UVA) in fluences exceeding 5 × 10⁴ Jm⁻² caused a decrease in the amount of the unscheduled DNA synthesis induced by far UV radiation (UVC). The DNA repair synthesis, as measured by the incorporation of [³H] -thymidine, is reduced by nearly a factor of 2 for a UVA radiation exposure of 1.5 × 10⁵ Jm⁻². Since solar UVA fluence rate is rather independent of latitude, this figure corresponds to a UVA exposure time of 50-60 min from noon sunlight in the summer time.     

POSTIRRADIATION PROPERTIES OF A UV-SENSITIVE VARIANT OF CHO

Abstract A UV-hypersensitive mutant of Chinese hamster ovary (CHO) cells, termed 43-3B, has been used in a comparative study with the wild type CHO in order to determine the involvement of repair in several postirradiation phenomena. 43-3B has the same growth rate and chromosome number as the wild type CHO-9. It is hypersensitive to UV irradiation (D₀ of 0.3 J/m² as compared to 3.2 J/m² for the wild type). 43-3B shows only about 17% of the UV-stimulated unscheduled DNA repair synthesis of CHO-9 as measured by autoradiography. When breaks in supercoiled chromatin are measured after UV by the nucleoid sedimentation method, the mutant appears to be capable of carrying out only limited incision. A much reduced ability to recover control rates of semiconservative DNA synthesis after UV irradiation was observed in the repair-deficient 43-3B cell line, suggesting that the removal of UV-induced replication blocks by excision repair is the most important factor in allowing recovery of UV-inhibited DNA synthesis. Recovery of colony-forming ability between fractionated UV exposures was observed in the wild type CHO-9, but little recovery was seen in 43-3B. This indicates that excision repair capability can also be important in split-fluence recovery.     

THE ACTION OF ULTRAVIOLET RADIATION ON THE CELL PROLIFERATION OF TWO-CELL MOUSE EMBRYOS

Abstract— The two-cell mouse embryo has a unique cell cycle of a short DNA synthesis (S) phase and an extremely long post-DNA synthesis (G₂) phase. An attempt was made to investigate the radiation biology of the long G₂ phase using UV radiation as a probe. Two cell mouse embryos, at various positions in the cell cycle, were UV-irradiated in phosphate-buffered saline. The embryos were cultured for a few hours to 3 days to assay for their cell proliferative characteristics. The embryos were most sensitive to the killing action of UV radiation in the late G₂ phase. The embryos divided more than two times after low UV fluences before dying and experienced G₂ phase delays.
These results can be contrasted to the situation in somatic cells, in which the action of UV radiation is S phase selective. One possibility is that the target for the action of UV radiation is different in two-cell mouse embryos from that in somatic cells and that the target is similar to that for X-ray effects.     

EFFECTS OF 8-METHOXYPSORALEN-INDUCED PHOTOTOXIC EFFECTS ON MAMMALIAN EPIDERMAL MACROMOLECULE SYNTHESIS IN VIVO

Abstract— The influence of 8-methoxypsoralen (8-MOP) and ultraviolet (UVA; 315–400 nm) radiation-induced phototoxic responses on DNA, RNA and protein synthesis and the DNA repair phenomenon were investigated utilizing the hairless mouse epidermis in vivo . The radioactive tracers TdR-³H, cytidine-³H and histidine-³H were used to examine changes in these macromolecules. Using these techniques, we found that the 8-MOP-UVA phototoxic injury inhibited premitotic semiconservative DNA synthesis in the germanitive layer of the epidermis within the first few hours post-irradiation. Recovery occurred by 24 h, followed by a progressive acceleration of this function over the next 7 days. No depression in RNA or protein formation was noted through 36 h. By 48 h the cells in the upper 1/2 to 1/3 of the epidermis lost their normal appearance and discontinued synthesizing these macromolecules. At 72 h RNA and protein synthesis was again active throughout the epidermis and the apparently dead cells had desquamated. At this time the epidermis was notably acanthotic and the epidermal cells were markedly enlarged. Examination for the dark repair response revealed no evidence of unscheduled DNA synthesis following irradiation indicating that the excision repair process was not demonstrable within the first 15min after the phototoxic injury. These responses differ in a number of parameters from the phototoxic reactions induced by UV rays shorter than 320 nm.     

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 ROLE OF 4-THIOURIDINE IN LETHAL EFFECTS AND IN DNA BACKBONE BREAKAGE CAUSED BY 334 nm ULTRAVIOLET LIGHT IN Escherichia coli

Strains of Escherichia coli that lack 4-thiouridine (⁴Srd) are killed by monochromatic 334 nm UV light (UV) less efficiently than their wild-type parents, which contain ⁴Srd. Oxygen enhancement ratios (OER) at 10% survival are 3.3 for a strain that possesses ⁴Srd, and 2.6 for one that lacks ⁴Srd. Single-strand breaks in DNA caused by 334 nm UV accumulate more than twice as fast in the wild-type strains than in the strains lacking ⁴Srd. The results suggest that ⁴Srd is an important chromophore in some near-UV lethal effects. The results also suggest that the excitation energy from 334 nm UV light may be passed from RNA to DNA, resulting in single-strand breaks.     

INTERACTION OF RESTORATION PROCESSES IN UV IRRADIATED ESCHERICHIA COLZ CELLS

Abstract— An attempt was performed to estimate survival and course of DNA synthesis in Escherichia coli B/r hcr' and hcr^- cells in relation to the amount of unexcised dimers.
In exponential growing hcr⁺ cells irradiated with 30 Jm^-2, dimers were almost completely excised and survival of cells was equal to about 3%. In the hcr⁺ cells prestarved for amino acid and thymine and irradiated by the same fluence, survival of cells was almost equal while two thirds of dimers remained unexcised and could be detected in the hybrid DNA consisting of parental and daughter chains. In exponentially growing hcr^+- cells irradiated with 20Jm^-, the same amount of dimers was produced which remained unexcised in the prestarved hcr⁺ cells. However, their survival was equal to about 0.02%.
Despite the great differences in dimer contents, about one third of DNA was replicated after UV in both exponentially growing and prestarved hcr+ cells producing well defined HL-hybrid peak, and the newly synthesized DNA was normal-sized. In hcr⁺ cells which contained approximately the same amount of dimers as in hcr⁺ prestarved cells, the amount of replicated DNA was too low to form a detectable density labelled hybrid peak, and the newly synthesized DNA was in short pieces.
Thus, when hcr⁺ and hcr^+- cells contain the same number of residual dimers, they have different levels of tolerance to these 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.     

CHANGES IN TRANSMISSION CHARACTERISTICS OF POLYMETHYLMETHACRYLATE AND CELLULOSE (III) ACETATE DURING EXPOSURE TO ULTRAVIOLET LIGHT

Abstract— Ultraviolet-transparent polymethylmethacrylate (PMMA) and cellulose (III) acetate (CA) (often used as a cut-off filter in UVB [280–320 nm] biological effect studies) were exposed to a 20 W Philips TL 12 lamp to examine changes in transmission characteristics due to UVB exposure. Transmission of UVB and biologically weighted UVB (UV_BE(DNA)) through PMMA were similar, 88.3 and 83.5%, respectively. The absorption characteristics of PMMA did not change with time at any of the UV irradiance levels applied. However, transmission of UVB and UVB_BE(DNA)) through new CA differed considerably: 59% versus only 11%, respectively. Also, spectral absorption characteristics changed with time due to degradation of CA, at a rate that was dependent on the incident UVB irradiance. The decrease in transmission through CA of both UVB and UV_BE(DNA) can be described by exponential functions. The CA that was wrapped around the UV lamp showed dramatic changes in UV absorption over the first few hours of use. However, when CA was placed at a longer distance from the light source initial degradation was less. It is concluded that PMMA can be applied in UV effect studies as a reasonable alternative for quartz. The CA should, however, be used with care, because the large transmission decreases that were observed strongly hamper an accurate calculation of (biologically weighted) UVB dose rates.     

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.     

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [³H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5 μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15-0.90 J/cm² of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non-competitive, i.e. decrease in both the apparent K^m value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm² of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy-induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.     

PHOTOCHEMICAL ALTERATIONS IN DNA REVEALED BY DNA-BASED LIQUID CRYSTALS

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

LASER PULSE EXCITATION STUDIES OF THE FLUORESCENCE OF CHLOROPLASTS

Abstract. The fluorescence yield, φ, as a function of single picosecond laser pulse intensity was experimentally studied in spinach chloroplasts and for chlorophyll a in ethyl ether solution. The progressive decrease in φ with increasing incident intensity for in vivo chlorophyll was found to be adequately explained within the context of continuum bimolecular kinetics with a singlet-singlet fusion rate constant of γ=5×^-9cm^-3s^-1 at room temperature. We discuss qualitatively how the fluorescence quantum yield depends on the duration and intensity of the incident pulse. The identity of φ vs l (the number of absorbed quanta) curves at the emission maxima of 685 nm and 735 nm for single picosecond pulse mode of excitation is explained within the context of Butler's tripartite model of the fluorescence of chloroplasts at 77 K. Various models relating γ to the singlet exciton diffusion coefficient and the Förster energy transfer rate are used to infer lower bounds to these physical parameters. Predictions and supporting experimental evidence for the tripartite model are discussed.     

TOLUIDINE BLUE: THE MODE OF PHOTODYNAMIC ACTION IN YEAST CELLS

Abstract— Toluidine blue, a thiazine dye, was shown to have in vivo photodynamic activity through singlet oxygen (O₂¹Δ _g ) production. This was based mainly on the effective protection by N^-₃ and the marked enhancement in D₂O for the sensitized inactivation of yeast cells. The mode of the in vivo activity was, however, quite different from that of acridine orange, for which the singlet oxygen mechanism has also been proposed. The most characteristic feature in the toluidine blue-sensitization was the total lack of the induction of gene conversion (at trp 5), while the survival went down below 10%. The non-induction of genetic changes was confirmed at several pH's in the neutral region, whereas the inactivation was seen in parallel to the reported pH dependence of singlet oxygen production in vitro . Direct measurements by microspectrophotometry showed none of the toluidine blue was accumulated in the cell. It was also ascertained from acridine-sensitized induction of gene conversion that toluidine blue never interfered with the binding of acridine orange to cellular DNA. These findings suggested that the unique mode of photodynamic activity of toluidine blue is attributable to its action from outside of the cell. Furthermore, comparisons between the photodynamically treated cells (with toluidine blue) and non-treated cells with respect to the response to UV irradiation excluded certain cell functions relating to the expression of gene conversion from the possible damage sites. The photo-reactivation process of UV induced gene conversion was not disturbed by the pre-toluidine blue sensitition. In view of the foregoing results, the plasma membrane was tentatively suggested as the most likely site of damage.     

CONSTANCY OF THE UV SENSITIVITY OF E. COLI DURING THE CELL CYCLE*

Abstract— Cells from slowly growing glucose-limited chemostat cultures of two different strains of Escherichia coli were separated by size on sucrose gradients, and 254-nm ultraviolet (UV) survival curves were obtained for samples of the different cell sizes. With the repair-deficient strain B_s-1 there was no observable change in survival curve during the cell cycle. With the repair-proficient strain 15 THU, there was no observable change in the final slope, but y -axis intercepts increased slightly for the older cells. Our results provide further evidence for the approximate independence of UV sensitivity, k , upon cell age within the division cycle and upon the presence or absence of DNA synthesis. They are consistent with a model in which UV lesions in newly replicated strands of DNA do not contribute to lethality.     

NEAR ULTRAVIOLET AND POSTIRRADIATION DNA DEGRADATION: EFFECTS ON THE INDUCIBLE INHIBITOR OF IONIZING RADIATION-INDUCED DNA DEGRADATION

Abstract— Possible effects of near ultraviolet radiation (near UV) on DNA degradation were examined. Postirradiation DNA degradation induced by ionizing radiation in strain B_s-l ( uur-, lex- ) is shown to be inhibited by carbon monoxide (CO) and potassium cyanide (KCN) if the cells are grown on glycerol. Presumably the blockage of respiration by these agents lowers the amount of ATP in the cell. 50 kJ/m² near UV did not simulate the action of CO and KCN, indicating that at this Ruence the supply of ATP remains adequate for postirradiation DNA degradation. Near UV did not, itself, produce DNA degradation. In a strain (B/r) in which an inhibitor of postirradiation DNA degradation can be induced by both UV and ionizing radiation, near UV affects the inhibitor formation, whether administered before or after induction.     

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.     

RANDOM DISTRIBUTION OF HIGHLY REPETITIVE AND INTERMEDIATE FREQUENCY MOUSE L-929 CELL DNA SEQUENCES SYNTHESIZED AFTER UV LIGHT EXPOSURE

Abstract— The DNA precursor ³H-thymidine incorporation rate (dpm/pg DNA) in mouse L-929 cells decreases immediately after exposure to UV light. This decrease is initially dose dependent, but at exposures greater than 22.5 J/m² appears to be radio-resistant. This was not explained by measurements of uptake of ³H-thymidine into the acid soluble pool (dpm/pg DNA). The radio-resistant incorporation amounted to approximately 35% of the control rate. DNA reassociation ("C₀T") studies were performed with DNA labeled with ³H-thymidine immediately after exposure of L-929 cells with a dose of UV resulting in radio-resistant incorporation to determine whether this radio-resistant incorporation was occurring in sequences of a particular repetitious frequency. These studies, performed in the highly repetitive to intermediate range, showed that the radio-resistant incorporation was occurring in DNA of all classes of repetitious frequency. DNA synthesized at different times after UV exposure, during the period when post-replication repair can occur, was similarly labeled for short intervals and isolated. The DNA reassociation studies showed that this DNA synthesis was also of all classes of repetitious frequency.