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.     

HERPES VIRUS PRODUCTION IN MONKEY KIDNEY AND HUMAN SKIN CELLS TREATED WITH ANGELICIN OR 8-METHOXYPSORALEN PLUS 365 nm LIGHT

Abstract—At an cquimolar concentration of 50 μM the bifunctional furocoumarin, 8-methoxypsoralen (8-MOP), is about 36 times more efficient in inhibiting the colony forming ability of CV-I monkey kidney cells than the monofunctional furocoumarin angelicin. In contrast 8-MOP is only 7.5 times more efficient than angelicin for the inhibition of herpes simplex virus (HSV) production in CV-1 cells. This latter factor seems to reflect differences in photoreactivity of the two compounds with host cell DNA.
A substantial recovery of HSV production was seen when cells were infected at different time intervals after treatment with angelicin-plus-light, whereas recovery was very limited after 8-MOP plus light treatment. The recovery process was slow as compared to that observed after UV (254 nm)-irradiation.
The repair capacities of treated normal and xeroderma pigmentosum (group A) skin fibroblasts were estimated by measuring HSV production and unscheduled DNA synthesis. XP-A cells repaired angelicin induced damage less efficiently than did normal cells. Neither cell type showed any repair activity after 8-MOP plus light treatment.     

MEASUREMENT OF DNA CROSSLINKS BY S1 NUCLEASE: INDUCTION AND REPAIR IN PSORALEN-PLUS-360 nm LIGHT TREATED ESCHERICHIA COLI

Abstract—DNA crosslinks in Escherichia coli cells. exposed to 4.5',8-trimethylpsoralen plus 360 nm light, were measured using a rapid and sensitive new approach. The assay is based on the specificity of S₁ nuclease from Aspergillus oryzae to single-stranded DNA. Bacterial cells were lysed and the DNA denatured by alkali. Following acid neutralization. crosslinked DNA undergoes spontaneous renaturation and is rendered S₁-nuclease resistant and therefore acid-precipitable. The single-stranded fraction after denaturation by alkali decreases with increasing near UV light exposure in the presence of TMP following first order kinetics. The kinetics were faster when exposure was at 4°C rather than at 20°C. This suggests that excision of crosslinks occurs during exposure at the higher temperature. Indeed. since the rate of DNA crosslinking in a uvr B mutant which is excision-deficient was higher than in wild type bacteria at 4°C, some excision must have occurred even in the cold. DNA from excision-proficient cells incubated at 37°C following exposure to TMP-plus-near UV at 4° showed a greater single stranded fraction than that from non-incubated cells. This indicates repair of DNA crosslinks. which proceeded with a half-time of 8 min at 37°C and was unaffected by substitution of thymine in DNA by 5-bromouracil.     

POSSIBLE INVOLVEMENT OF MEMBRANE DAMAGE IN THE IN ACTIVATION BY BROAD-BAND NEAR-UV RADIATION IN Saccharomyces cerevisiae CELLS

Multiple cellular effects of near-UV radiation (300-380 nm) on inactivation, disruption of the permeability barrier and induction of gene conversion at the trp 5 locus were simultaneously measured in the same culture of a diploid strain of the yeast Saccharomyces cerevisiae in order to assess the critical lethal damage. Inactivation of exponential phase cells in water appeared to be closely related to the disruption of the permeability barrier. Inactivation and membrane damage were remarkably oxygen dependent, whereas the induction of genetic changes was very low and dependent much less on oxygen. The dependence on the temperature for inactivation and membrane damage was both low, conforming with the expectation that the processes are mainly photochemical and not enzymatic. These features are very contrasted with the characteristics of far-UV radiation effects. Possible involvement of membrane damage in near-UV inactivation of exponential phase yeast cells is discussed.     

MEMBRANE DAMAGE AND RECOVERY ASSOCIATED WITH GROWTH DELAY INDUCED BY NEAR-UV RADIATION IN Escherichia coliK–12

Abstract— The growth delay induced by near-UV radiation has been largely attributed to injured tRNA's and to the stringent response. We report an associated membrane perturbation whose recovery determines substantial modifications in the behavior of log phase Escherichia coli K–12 exposed to sublethal doses of near-UV radiation (366 nm). When incubated at 37°C in plain nutrient broth, cells suffered a growth delay of about 100 min with parallel inhibition of several membrane functions. Conversely, when grown in conditions known to influence membrane activities, these were slightly inhibited and the growth delay lasted about 50 min. All the above conditions triggered the stringent response, characterized by an equivalent post-irradiation burst of intracellular guanosine 5'3' tetra and pentaphosphate and by a similar decay rate of the nucleotides accumulated at time 0 of the growth lag. According to our data the polyphosphates' half decay time in irradiated cells remains practically constant and close to 15 min. But, while cells from unsupplemented broth at 37°C resumed normal growth in around 100 min those with recovered membranes were rescued from growth inhibition in about one half of that time.     

FACTORS INFLUENCING THE NEAR-UV EFFECT ON DNA IN THE PRESENCE OF 8-METHOXYPSORALEN: RENATURABILITY OF DNA.

Factors affecting the near-UV induced reaction of calf thymus DNA in the presence of 8-methoxypsoralen were examined. DNA became more renaturable with increasing concentration of 8-methoxypsoralen and fluence. Existence of paramagnetic ions, Mn²⁺, Co²⁺, or Ni²⁺, largely repressed the photoreaction. It was ascertained that the highest renaturability was attained by the illumination with light between 310 and 345 nm.     

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.     

FAILURE OF UVR DOSE RECIPROCITY FOR SKIN TUMORIGENESIS IN HAIRLESS MICE TREATED WITH 8-METHOXYPSORALEN

Abstract— 8-Methoxypsoralen (8-MOP) phototumorigenesis was studied in hairless albino mice irradiated with solar simulated radiation (SSR). Animals were exposed to three different daily doses of SSR after application of an oily vehicle with or without 8-MOP. Tumor production was dependent on daily SSR dose irrespective of topical treatment. The phototumorigenic potency of SSR was increased by the vehicle and to a much greater extent by inclusion of 8-MOP. Irrespective of topical treatment, a failure in dose reciprocity for tumorigenesis was demonstrated; in terms of cumulative SSR dose, the lower daily doses were more tumorigenic than the higher doses. This reciprocity failure in 8-MOP photosensitized mice is similar to that previously observed for UV-B phototumorigenesis. The relevance of these findings to therapeutic use of psoralens is discussed.     

DNA REPAIR IN ULTRAVIOLET LIGHT IRRADIATED HeLa CELLS AND ITS REVERSIBLE INHIBITION BY HYDROXYUREA

Abstract— –The repair of u.v. damaged DNA in HeLa cells can be detected using the alkaline sucrose gradient technique. As a result of pyrimidine dimer excision single strand breaks are produced in DNA of irradiated cells. Rejoining of these breaks occurs during an 8 hr post-irradiation incubation period and is prevented by hydroxyurea and acriflavine. The inhibition of repair by hydroxyurea can be reversed by a mixture of all 4 deoxyribonucleosides at a concentration that does not reverse the inhibition of total DNA synthesis.     

SITES SENSITIVE TO S1 NUCLEASE and DISCONTINUITIES IN DNA NASCENT STRANDS OF ULTRAVIOLET IRRADIATED MOUSE CELLS

Abstract Mouse 3T3 cells irradiated with ultraviolet light synthesize DNA containing sites sensitive to the single-strand specific SI nuclease. The appearance of these sites correlates well with the presence of discontinuities in nascent strands, detected by the methodology of sedimentation in alkaline sucrose gradient. Thus, both the sites sensitive to SI nuclease and the discontinuities in nascent strands (i) are stabilized by caffeine; (ii) are no longer formed late after irradiation and (iii) disappear faster when a certain UV fluence is split into two fluences whose sum equals the single fluence. Moreover, the recovery in synthesizing DNA without SI sensitive sites is not dependent on excision repair of pyrimidine dimers or on continuous DNA synthesis. These SI sensitive sites are exclusive of replicative structures of irradiated cells and should correspond to stretches of single-strand DNA (gaps) formed during replication.     

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.     

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.     

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.     

HPLC ANALYSIS OF 4'',5''-MONOADDUCT FORMATION IN CALF THYMUS DNA and SYNTHETIC POLYNUCLEOTIDES TREATED WITH UVA and 8-METHOXYPSORALEN

Abstract— 8-methoxypsoralen monoadduct formation in calf thymus DNA irradiated with subbands of ultraviolet A light has been quantitated by HPLC analysis of the enzymatic hydrolysates of the DNA. Normalization of the yield of monoadducts for the variation in source output and the absorptivity of 8-MOP at each of the irradiating wavelengths showed that the 4',5'-furan monoadduct was the principal photoproduct and the efficiency of its formation was independent of irradiating wavelength. Synthetic polynucleotides irradiated with ultraviolet A light demonstrated a base composition and sequence dependence for 8-MOP photoreactivity: (poly(dAdT.dAdT) > poly(dA.dT) > poly(dGdC.dGdC) in both the B and Z forms > pofy(dT).     

PHOTOCHEMICAL DAMAGE TO DNA TREATED WITH CHLORPROMAZINE AND NEAR UV RADIATION UNDER AEROBIC AND ANAEROBIC CONDITIONS

Double stranded salmon sperm DNA in a chlorpromazine (CPZ) solution is damaged when irradiated with near UV light. The damage of irradiated DNA can be estimated by measuring the increase in extinction at 260 nm following incubation at 60°C of the DNA with formaldehyde. Moreover, DNA irradiated in the presence of CPZ or kept in the dark separate quite differently in an aqueous polymer two-phase system. DNA irradiated in the presence of CPZ seemed to be susceptible to digestion by endonuclease S1, while the endonuclease of Neurospora crassa could not digest this DNA. Irradiation under aerobic conditions seemed to be less disastrous for DNA than under anaerobic conditions.     

INACTIVATION BY MONOCHROMATIC NEAR-UV RADIATION OF AN Escherichia coli hemA8 MUTANT GROWN WITH AND WITHOUT δ-AMINOLEVULINIC ACID: THE ROLE OF DNA vs MEMBRANE DAMAGE

Abstract— Escherichia coli strain RT8 hemA8 [blocked in biosynthesis of δ-aminolevulinic acid (δ-ALA), and unable to manufacture porphyrins unless exogenously supplied with δ-ALA] is inactivated more efficiently by monochromatic 334- and 405-nm radiations if the cells are grown with δ-ALA supplementation. The fiuence enhancement factor for δ-ALA sensitization is larger for light at 405 nm than at 334 nm. Both irradiation conditions (plus or minus δ-ALA) showed prominent oxygen enhancement ratios, which were also larger at 405 nm than at 334 nm. At 334 nm, δ-ALA supplementation did not affect the accumulation of DNA breaks, while at 405 nm, the induction of DNA breaks doubled for cells supplemented with δ-ALA. Rubidium leakage caused by 405-nm radiation occurred at a smaller fiuence in cells supplemented with higher concentrations of δ-ALA than in cells supplemented with a lower concentration. The results suggest that (1) porphyrin derivatives may have a role in cell killing by near-UV radiations, and (2) damage to cytomembranes may be a critical lesion produced by these events, whereas DNA breakage may not.     

PRIMARY DNA DAMAGE, HPRT MUTATION AND CELL INACTIVATION PHOTOINDUCED WITH VARIOUS SENSITIZERS IN V79 CELLS

DNA strand breaks and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants were measured in parallel in photochemically treated (PCT) cells and compared at the same level of cell survival. Chinese hamster fibroblasts (V79 cells) were either incubated with the lipophilic dyes tetra(3—hydroxyphenyl)porphyrin (3THPP) and Photofrin II (PII), the anionic dye meso -tetra(4—sulfonatophenyl)porphine (TPPS₄) or the cationic dye meso -tetra( N -methyl-4-pyridyl)porphine ( p -TMPyPH₂ before light exposure. In the cells, the lipophilic dyes were localized in membranes, including the nuclear membrane, while the hydrophilic dyes were taken up primarily into spots in the cytoplasm. In addition, the hydrophilic TPPS₄ was distributed homogeneously throughout the whole cytoplasm and nucleoplasm. According to the HPRT mutation test, the mutagenicity of light doses survived by 10% of the cells was a factor of six higher in the presence of 3THPP than of PII, whereas for X-rays it was a factor of three higher than for PCT with 3THPP. Light exposure in the presence of the hydrophilic dyes TPPS₄ and p -TMPyPH₂ was not significantly mutagenic. There was no correlation between the induced rates of HPRT mutants and of DNA strand breaks. Thus, TPPS₄ was the most efficient sensitizer with regard to DNA strand breaks when compared at the same level of cell survival, followed by 3THPP, PII and p -TMPyPH₂. Hence, the rate of DNA strand breaks cannot be used to predict the mutagenicity of PCT.     

EFFECT OF INTENSITY AND WAVELENGTH OF FLUORESCENT LIGHT ON CHROMOSOME DAMAGE IN CULTURED MOUSE CELLS

Abstract—A single 3- to 20-hr exposure of line NCTC 9266 mouse cells to cool-white fluorescent light (4.6 W/m²) produces chromatid breaks and exchanges. The effective wavelength is in the visible range and coincides with the mercury emission peak at 405 nm. Increasing light intensity from 4.6 W to 15.3 W/m² for 20 h causes a concomitant increase both in production of chromosome damage and formation of hydrogen peroxide (H₂O₂) in the serum-free medium. Cells washed free of medium and illuminated in saline for 3 h show chromosome damage to the same extent as cells illuminated in culture medium. Addition of catalase during the exposure period of 3 h eliminates the light-induced damage. We conclude that the light-induced chromatid breaks and exchanges result from H₂O₂ production within the cell and that exogenous catalase can enter the cell and prevent the damage.     

FORMATION OF PHOTOPRODUCTS LETHAL FOR HUMAN CELLS IN CULTURE BY DAYLIGHT FLUORESCENT LIGHT AND BILIRUBIN LIGHT

Abstract. Irradiation of Dulbecco's modified Eagle's tissue culture medium with "Daylight,""Special Blue," or "Bilirubin" fluorescent light produces photoproducts lethal to human cells. Killing is abolished when (1) riboflavin, (2) tryptophan and tyrosine, or (3) riboflavin, tryptophan and tyrosine are deleted from medium prior to irradiation with any of the above fluorescent lamps. Toxic photoproducts are also formed when buffered salt solutions containing (a) riboflavin and tryptophan, (b) riboflavin and tyrosine, or (c) riboflavin, tryptophan and tyrosine are exposed to any of these light sources.