NON-DIMER DNA DAMAGE IN CHINESE HAMSTER V-79 CELLS EXPOSED TO ULTRAVIOLET-B LIGHT

To understand and characterize non-dimer DNA damage and cytotoxicity induced by ultraviolet-B light (UV-B, 290-320 nm), an alkaline elution technique for analysis of DNA damage was used on Chinese hamster V-79 cells. Ultraviolet-B exposure produced a dose-dependent induction of DNA single strand breaks and DNA-protein crosslinks; however, there was an absence of DNA-DNA interstrand crosslinks. Neither of these types of DNA damage were repaired within a a 24 h incubation of the cells following a single UV-B exposure; rather the damage increased. Using a colony forming assay, we found that UV-B exposure resulted in an increase of cytotoxicity in a dose-dependent fashion. In addition, UV-B exposure inhibited DNA and RNA synthesis. The role of non-dimer DNA damage in the cytotoxicity induced by UV-B is discussed.     

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

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

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES-XL PHOTOTOXICITY OF SULFONATED ALUMINUM NAPHTHALOCYANINES TOWARDS V-79 CHINESE HAMSTER CELLS

The phototoxicity of sulfonated aluminum naphthalocyanines towards V-79 Chinese hamster cells is investigated. The disulfonated naphthalocyanine exhibits similar photostability, but better cell penetrating properties than the tetrasulfonated dyes. The capacity of the naphthalocyanines to generate singlet oxygen is comparable to that of the corresponding phthalocyanines. However, in contrast to the phthalocyanine dyes, the sulfonated aluminum naphthalocyanines show very little phototoxicity towards the V-79 cells, suggesting close association with non-vital cell constituents or extensive formation of photoinactive adducts and aggregates.     

EFFECT OF VITAMIN E ON CYTOTOXICITY, DNA SINGLE STRAND BREAKS, CHROMOSOMAL ABERRATIONS, AND MUTATION IN CHINESE HAMSTER V-79 CELLS EXPOSED TO ULTRAVIOLET-B LIGHT

The effect of pretreatment with vitamin E on cytotoxicity, DNA single strand breaks, and chromosomal aberrations as well as on mutation induced by ultraviolet-B light (UV-B) was investigated in Chinese hamster V-79 cells. Cellular pretreatment with non-toxic levels of 25 microM alpha-tocopherol succinate (vitamin E) for 24 h prior to exposure resulted in a 10-fold increase in cellular levels of alpha-tocopherol. Using a colony-forming assay, this pretreatment decreased the cytotoxicity of UV-B light. However, alkaline elution assays demonstrated that pretreatment with vitamin E did not affect the number of DNA single strand breaks caused by UV-B light. In addition, UV-B exposure produced a dose-dependent induction of chromosomal aberrations and mutations at the HGPRT locus, and neither of these actions of UV-B was influenced by pretreatment with the vitamin. These results suggest that vitamin E protects cells from UV-B-induced cytotoxicity, possibly through its ability to scavenge free radicals. The results also suggest that the extent of genotoxicity induced by UV-B light may not correlate directly with the cytotoxic action of this wavelength region in sunlight.     

INDUCTION OF DNA–PROTEIN CROSS-LINKING IN CHINESE HAMSTER CELLS BY MONOCHROMATIC 365 AND 405 NM ULTRAVIOLET LIGHT

Abstract— The survival, the induction of DNA-protein cross-linking, and the number of T4-endonuclease sensitive sites were measured in Chinese hamster cells that had been irradiated with 365 and 405 nm monochromatic light. The survival measurements show that cells are somewhat less sensitive to 405 nm light than to 365 nm light. The difference is expressed predominantly in the shoulder widths of the survival curves, whereas the slopes of the two curves are about the same. Induction of pyrimidine dimers, as indicated by the number of endonuclease-sensitive sites, after exposures that produce about 10% survival is very low at 365 nm (˜ 4 endonuclease sites per 2 × 10⁸ daltons), while no dimers are detected at 405 nm. In contrast, DNA-protein cross-links are induced rather effectively at either wavelength even after exposures that result in a relatively high survival (60-20%). Our measurements support the conclusion that lethality in mammalian cells after irradiations with 365 or 405 nm light is caused by a nondimer damage, possibly DNA-protein cross-links.     

AN ACTION SPECTRUM FOR CELL KILLING AND PYRIMIDINE DIMER FORMATION IN CHINESE HAMSTER V-79 CELLS

We have determined action spectra for pyrimidine dimer formation and loss of colony-forming ability in Chinese Hamster V-79 cells and have found a very strong correlation between the two. These data are consistent with the notion that damage to DNA is the principle cause of cell death and that the most important type of damage is the pyrimidine dimer. While the shape of the V-79 spectra mimics that of action spectra for bacteria. phage, and purified DNA, V-79 cells are about twice as sensitive to radiation at long wavelengths, relative to the sensitivity at 265 nm. However, if the action spectra are normalized to 297 nm. a wavelength included in the solar spectrum, the two sets of action spectra would coincide at wavelengths relevant to human skin-cancer. Thus an action spectrum based on microorganisms should be adequate for extrapolation to humans in terms of risk due to ozone depleteion.     

BIOLOGICAL ACTIVITIES OF PHTHALOCYANINES–VII. PHOTOINACTIVATION OF V-79 CHINESE HAMSTER CELLS BY SELECTIVELY SULFONATED GALLIUM PHTHALOCYANINES

Abstract Gallium chloride phthalocyanines sulfonated to different degrees were tested for their ability to inactivate V-79 Chinese hamster cells in the presence of red light. The mono- and disulfonated compounds were the most active whereas the tri- and tetrasulfonated complexes were completely void of photoactivity. In addition, large variations in photoactivity were observed among the four isomeric disulfonated derivatives with the most hydrophobic isomer exhibiting the highest photoactivity. Prolonged exposure to the disulfonated complex resulted in increased photosensitization. Complexing the dye with Al instead of Ga resulted in a slightly increased photosensitizing effect.     

DNA SINGLE-STRAND LESIONS DUE TO ''SUNLIGHT'' AND UV LIGHT: A COMPARISON OF THEIR INDUCTION IN CHINESE HAMSTER AND HUMAN CELLS, AND THEIR FATE IN CHINESE HAMSTER CELLS

Abstract— It has been shown that the lethal properties of germicidal UV light (254 nm) and sunlight-simulating near UV light are qualitatively different (Elkind et al ., 1978). Further to compare these two radiations, the induction of single-strand DNA breaks (i.e. frank breaks plus alkali-labile lesions) was measured in two cell lines. Equal numbers of breaks in Chinese hamster cells require a dose of UV 5.5% of a near UV dose but in HeLa cells a UV dose of 7.6% of a near-UV dose is required. The rate of break production by these radiations is about 1/10-th of that due to X-rays when a comparison is made on an equal killing dose basis. The inventory of breaks in Chinese hamster cells was also followed and was found to be characteristically different for UV compared to near UV light. These data indicate that significant differences exist, at a molecular level, in the effects produced by ultraviolet and sunlight-simulating light, and further emphasize the need for caution in attempting to extrapolate from observed molecular or biological effects due to the former to those to be expected from the latter.     

A STUDY OF THE RELATIONSHIP BETWEEN SURVIVAL AND REPAIR SYNTHESIS'' OF DNA AFTER ULTRAVIOLET LIGHT EXPOSURE

Abstract— The influence of amino acid prestarvation on both the resistance to u.v. and the postirradiation repair synthesis of E. coli 15 T^- 555-7 thy meth arg trp and E. coli B/r (HCR⁺) was followed. Prestarvation increased the number of survivors about 30–100 fold in both strains at doses 600-1200ergs/mm². In contrast to survival no increase in repair synthesis was observed. Thus, the increase in survival has to be brought about by a mechanism which seems to be independent of additional repair synthesis.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

THE FATE OF PYRIMIDINE DIMERS IN THE DNA OF ULTRAVIOLET-IRRADIATED CHINESE HAMSTER CELLS

Abstract —As an aid to understanding the relationship between dimer repair and cellular recovery, we have studied dimer removal and replication of dimer-containing DNA in Chinese hamster ovary (CHO) cells irradiated with ultraviolet light (254 nm). These investigations demonstrated that (1) dimers are not excised as polynucleotides of less than 500,000 mol. wt, (2) fractionation of the ultraviolet dose does not enhance dimer excision, (3) dimer-containing DNA is replicated in ultraviolet-irradiated CHO cells, and (4) the dimers are conserved in the replicated DNA. These findings support the proposed mechanism of bypass of photoproducts during DNA replication in mammalian cells.     

THE PHOTODYNAMIC EFFECTS ONV–79 CHINESE HAMSTER CELLS

Abstract— Holding of acriflavine sensitizedV–79 cells in growth medium before visible light exposure decreases inactivation by visible light. The decrease depended upon the period of holding, indicating that there was release of cellular dye during this period. Exposures to visible light were done in two conditions: (a) with no dye in the medium during visible light exposure (washed) and (b) with dye in the medium during exposure (unwashed). Caffeine was found to slightly increase the sensitivity of the cells to visible light in the washed condition, whereas, in the unwashed condition no such effect was observed. Interaction studies with far UV did not reveal any correlation between photodynamic damage and UV damage. Visible light exposure of acriflavine sensitized cells was found to be mutagenic, as studied from the induction of 8-azaguanine resistant mutants. Inhibition of singlet oxygen production by sodium azide suppressed the induction of mutants. All these, taken together, have been discussed with respect to the relative importance of DNA and non-DNA damage in the photodynamic action of acriflavine.     

INACTIVATION OF NUCLEOTIDE EXCISION REPAIR IN CHINESE HAMSTER OVARY CELLS BY EXPOSURE TO AN ALKYLATING AGENT

Abstract— Unscheduled DNA synthesis and excision of pyrimidine dimers in Chinese hamster ovary cells irradiated with UV light were inhibited by prior exposure to the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)(1–10 μ M ) Although the pathways for excision of pyrimidine dimers and alkylation damage are known to differ, alkylations from MNNG exposure appear to have a direct effect on the nucleotide excision repair system. These results indicate that the method of exposing cells to two DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.     

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.     

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.     

MEMBRANE LYSIS IN CHINESE HAMSTER OVARY CELLS TREATED WITH HEMATOPORPHYRIN DERIVATIVE PLUS LIGHT

Abstract Chinese hamster ovary cells in exponential growth were incubated with various concentrations of hematoporphyrin derivative (HpD). Cellular porphyrin content was determined after 2 h incubation at 37°C using [³H]-hematoporphyrin derivative. Photoactivation of cell-bound HpD by red light resulted in a family of survival curves with terminal slopes proportional to cellular HpD concentration. The degree of cellular lysis, assayed 1 h after illumination using a chromium-51 labeling technique, was also found to be related to cellular HpD concentration. The amount of 51Cr released increased with post-irradiation incubation to a level parallel to cell lethality as measured by colony formation. These data suggest that lysis of the cell membrane may be largely responsible for cellular inactivation following HpD photoirradiation.     

PHOTODYNAMIC INACTIVATION OF CHINESE HAMSTER CELLS

Abstract— Visible light exposures have been shown to kill acriflavine bound Chinese hamster cells. Such killing was enhanced when (a) dye was present in the medium during irradiation and (b) the pH of the medium was 8.5, instead of the normal 7.5 during the exposure. The induced killing could be suppressed by the presence of sodium azide during exposure. The results were taken to indicate that both DNA and non-DNA sites were involved in the cellular inactivation by visible light and that singlet oxygen was involved in the process.     

ENHANCED KILLING OF CHINESE HAMSTER CELLS FOLLOWING COMBINED EXPOSURE TO 'SUNLIGHT' AND X-RAYS*

Abstract— Enhanced cell killing due to combined exposure to sunlight-like near-UV light (Westinghouse Sun Lamps, FS20) and X-rays, indicative of damage interaction, is observed at all stages of the cell cycle. The greatest interaction is observed in the middle of the DNA synthetic phase. At equal survival, 'sunlight' damage is only partly additive to X-ray damage, and vice versa , whereas in earlier studies we found that far-UV light (254 nm) produces damage completely additive to X-ray damage. Loss of damage interaction between radiations is more rapid after a first dose of X-rays than after a first dose of 'sunlight'.     

INDUCTION OF DNA-PROTEIN CROSS-LINKS IN CHINESE HAMSTER CELLS BY THE PHOTODYNAMIC ACTION OF CHLOROALUMINUM PHTHALOCYANINE AND VISIBLE LIGHT

Abstract— Chloroaluminum phthalocyanine (CAPC) is an efficient photosensitizer for the inactivation of Chinese hamster V79 cells. In order to investigate possible molecular mechanisms in the photo-dynamic action of CAPC and visible light, the induction and repair rate of two classes of DNA lesions have been determined, i.e. DNA single-strand breaks and DNA-protein cross-links. In cells pretreated with 1 μ.M CAPC, a fluence of 12 kJ/m² of red light (>600 nm) kills approximately 50% of the cells and induces 3 to 3.5 Gy-equivalents of single-strand breaks. The repair of these breaks was slower than the repair of single-strand breaks induced by -irradiation. The photodynamic action of CAPC also induces a large number of DNA-protein cross-links which, in contrast to -radiation-induced DNA-protein cross-links, do not appear to be repaired during 4 h of post-treatment incubation in fresh medium. These studies suggest that DNA may be an important target for the cytotoxicity of CAPC + red light.     

DNA REPAIR IN V-79 CELLS TREATED WITH COMBINATIONS OF PHYSICAL AND CHEMICAL CARCINOGENS*

Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated into parental DNA during repair in Chinese hamster V-79 cells treated with 254 nm of ultraviolet radiation (UV), 7,12-dimethylbenz[a]anthracene 5,6-oxide (DMBA-epoxide), N-acetoxy-2-acetylaminofluorene (AAAF), 4-nitroquinoline 1-oxide (4NQO), 2-methoxy-6-chloro-9-[3(ethyl-2-chloroethyl)-aminopropylamino]acridine dihydrochloride (ICR-170), X-rays, ethylmethanesulfonate (EMS), methyl methanesulfonate (MMS) and combinations of these agents. Compared to normal human cells V-79 were defective in repair of UV lesions and the lesions induced by the UV-mimetic chemicals. The extent of the defects varied from 10 to 50% and was similar to those in Xeroderma pigmentosum group C cells (XP C). V-79 cells repaired X-ray damage and damage from the alkylating agents EMS and MMS to the same extent as human cells. Repair was additive after a combination of UV plus MMS indicating, as expected, that there are different rate-limiting steps for removal of the damages from these agents. Repair was less than additive in cells treated with UV plus ICR-170, AAAF plus ICR-170, AAAF plus 4NQO, and 4NQO plus ICR-170 and approximately equal to that observed for the higher of the two agents separately, indicating that there may be similar rate-limiting steps for removal of lesions. Although the results on repair after combinations of UV plus 4NQO, UV plus DMBA-epoxide or X-rays plus MMS were difficult to interpret, there was not any inhibition of repair in these combinations.