LACK OF INHIBITION OF ULTRAVIOLET RADIATION-INDUCED ORNITHINE DECARBOXYLASE ACTIVITY BY RETINOIC ACID

Abstract— Normally present at low levels, ornithine decarboxylase (ODC) activity is induced to higher levels in animal skin by such disparate agents as tumor promoter 12–0-tetradecanoylphorbol-13-acetate (TPA), UV radiation, and hair plucking. Retinoids are known to inhibit induction by TPA. Repeated applications of retinoic acid (RA) in acetone have also been reported to inhibit UV-induced ODC in hairless mice. As a preliminary study, it was of interest to know whether RA in a cream vehicle would have the same effect. Groups of Skh-hairless-1 albino mice were irradiated once with Westinghouse FS-20 lamps (0.045 Joules/cm^/). Immediately post-irradiation, RA was applied to the dorsum in different concentrations (0.001%, 0.002%, 0.02%), vehicles (cream and acetone) and on various time schedules (1–5 times). Sacrifice was by cervical dislocation 24 h later. Epidermis was obtained by mild heat separation and two epidermal sheets were pooled for each extract. In all experiments, the 70-fold increase in UV-induced ODC activity was further increased by retinoic acid by a factor ∼ 1.6. Since ODC levels are usually elevated in proliferating systems, the results are in concordance with the fact that both UV radiation and RA induce epidermal hyperplasia.     

RECOVERY FROM INHIBITION BY UV-IRRADIATION OF ORNITHINE DECARBOXYLASE INDUCTION IN HUMAN CELLS: IMPLICATION OF EXCISION REPAIR

Abstract— Exposure of stationary-phase human breast carcinoma(T–47D) cells to far-UV light (254 nm) inhibited the appearance of induced ornithine decarboxylase (ODC) activity. The fluence response curve had a shoulder (D₄= 2 J m_-2) followed by an exponential decline (D₀= 4.2 Jm_-2). The cells could recover from this inhibition when the stimulus of induction of ODC was delayed for20–24 h after irradiation. Hydroxyurea (HU) when present at 3 mM during the recovery period eliminated completely the ability of the cells to recover. This effect of HU on ODC induction was partially reversed by 50 nM of the four deoxyribonucleosides required for DNA synthesis. Neither HU nor the deoxyribonucleosides by themselves affected ODC induction in unirradiated cells. Since HU inhibited the recovery from potentially lethal UV damage and is a known inhibitor of excision repair, we interpret the above results to mean that recovery from UV-induced inhibition of ODC induction depends on excision-repair of DNA damage. This interpretation is strongly supported by the finding that specific photolysis of 5-bromodeoxyuridine, incorporated into DNA during the recovery period, inhibited recovery of ODC induction from inhibition by UV light.     

EVALUATION OF STRUCTURALLY-RELATED PHENOLS FOR ANTI-PHOTOCARCINOGENIC AND PHOTOPROTECTIVE PROPERTIES

Abstract— Butylated hydroxytoluene (BHT) has previously been shown to provide protection against UV-mediated erythema, carcinogenesis, and induction of epidermal ornithine decarboxylase (ODC). Butylated hydroxytoluene is but one compound of a class of organic chemicals, the phenols, that demonstrates antioxidant properties. On the basis of structural characteristics, the phenolic compounds butylated hydroxyanisole (BHA), propyl gallate, and vanillin were compared to BHT for antiphotocarci-nogenic activity. SKH-Hr-1 female mice were fed diets containing 0.5% (wt/wt) BHT, BHA, vanillin or 0.3% propyl gallate. The animals received constant, daily (5 days/week) suberythemic levels of UVB radiation from Westinghouse BZS-WLG lamps. Radiation was discontinued when 25 sunburn units (SBUs) had been delivered at week eleven. Animals were evaluated weekly for tumor latency and multiplicity. Of the four phenols tested, only BHT conveyed significant inhibition of both carcinogenic parameters—increasing latency by 5.2 weeks and reducing tumor multiplicity by 60%. Vanillin significantly reduced tumor multiplicity (48%). BHA and propyl gallate were without effect. Because ODC has been considered a requisite step in carcinogenesis, ODC activity was assayed to determine whether its induction might serve as a rapid screen for potential anti-carcinogenic phenols. Animals were fed the respective phenol-containing diets for two weeks, exposed to 0.45 J/cm^/ of UVB from FS-20 lamps, and epidermal extract assayed for ODC activity 28 h post-irradiation. Both BHT and BHA significantly inhibited ODC induction, the former being most effective. Vanillin and propyl gallate were without effect. These data indicate that neither antioxidant or lipophilic properties, nor the tert-butyl substituent common to both BHT and BHA, are sufficient determinants of anti-photocarcinogenic activity. Nor does it appear that inhibition of ODC induction is an adequate indicator of such activity.     

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.     

HYDROGEN PEROXIDE MEDIATES UV-INDUCED IMPAIRMENT OF ANTIGEN PRESENTATION IN A MURINE EPIDERMAL-DERIVED DENDRITIC CELL LINE

Abstract— Ultraviolet-B (290–320 nm) radiation is known to impair the antigen-presenting cell (APC) function of Langerhans cells (LC), skin-specific members of the dendritic cell (DC) family. We sought to address mechanisms of this effect, focusing on the role played by hydrogen peroxide. For this purpose, we used a newly established murine DC line, XS52, which resembles epidermal LC in several respects. The APC capacity of XS52 cells, using two different CD4* T cell clones as responders, was inhibited significantly (>50%) by exposure to UV radiation (unfiltered FS20 sunlamps) at relatively small fluences (50–100 J/m²). Ultraviolet radiation also inhibited growth factor-dependent proliferation of XS52 cells. On the other hand, cell surface phenotype was relatively well preserved after irradiation; expression levels of B7-1 and B7-2 were reduced slightly, while other molecules ( e.g. Ia, CD54, CD1 la and CD18) were not affected. With respect to the role played by hydrogen peroxide, pretreatment with purified catalase (900 U/mL) prevented UV-induced inhibition of APC function. Short-term exposure to 3 miM H₂0₂ or f-butyl H₂0₂ mimicked UV radiation by inhibiting APC function. Finally, intrinsic catalase activity was substantially lower in XS52 cells compared with Pam 212 keratinocytes. These results indicate that the generation of hydrogen peroxide alone is sufficient to produce some, but not all, of the deleterious effects of UV radiation on DC derived from the skin.     

Glutathione Isopropyl Ester Reduces UVB-lnduced Skin Damage in Hairless Mice

Abstract— The protective effect of administration of glutathione (GSH) isopropyl ester on photodamage, such as lipid peroxidation, inflammation and tumorigenesis induced by UV exposure (290–400 nm, max. 312 nm), was investigated using hairless mice. Pretreatment with 20 mg/kg GSH isopropyl ester prevented the increases of thiobarbituric acid-reactive substance (TBARS) formation in skin and serum sialic acid, indices of lipid peroxidation and inflammatory reaction, respectively, which were caused by a single dose (15 kj/m²) of UV irradiation. The level of epidermal GSH in skins of the GSH ester-treated mice was maintained within normal limits. When mice were exposed to UV at a dose of 2 kj/m², three times weekly, skin tumors developed in all of them after 25 weeks. The formation of skin tumors was significantly inhibited by administration of 10 mg/kg GSH ester prior to each UV irradiation for 25 weeks. Moreover, the increases of cutaneous TBARS and serum sialic acid in the tumor-bearing mice were also prevented by continuous pretreatment with GSH ester. Even after 24 weeks, the epidermal GSH content of the pretreated mice was mostly retained compared to nonirradiated mice. However, administration of GSH prior to acute or chronic UV irradiation had no effect on the UV-induced damage. The present results suggest that the protection from photo-damage afforded by pretreatment with GSH ester is due to maintenance of a normal GSH level.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED SISTER CHROMATID EXCHANGES IN POTOROUS CELLS

Abstract— Exposure to visible light after UV-irradiation showed a remarkable effect on UV-induced sister chromatid exchanges (SCEs). After 6-h exposure to visible light (3 × 10⁵ J/m²), two-thirds of the UV-induced SCEs were prevented, confirming Kato's findings. Exposure to visible light before UV irradiation had no effect. This effect of visible light on UV-induced SCEs was temperature dependent, suggesting the presence of enzymatic photoreactivation.     

Effects of butylated hydroxytoluene upon PUVA-tumorigenesis and induction of ornithine decarboxylase activity in the mouse

Psoralen photochemotherapy (PUVA) is widely used in the treatment of psoriasis. Some therapy regimen have been associated with increased risk of skin cancer. Free radical species are thought to play a role in psoralen phototoxicity and photocarcinogenesis. It has been reported that the antioxidant butylated hydroxytoluene (BHT) inhibits acute phototoxicity by PUVA but does not reduce therapeutic efficacy. It has also been shown that BHT inhibits UVB-induced erythema, tumorigenesis and induction of ornithine decarboxylase (ODC) activity--ODC activity is thought by some to be associated with tumor promotion. Therefore, we have investigated the effect of BHT on psoralen tumorigenesis and PUVA-induced epidermal ODC activity. SKH-Hr-1 hairless albino mice were treated with topically applied 8-MOP and exposed to UVA (3X weekly) for 31 weeks with and without BHT administered either in the diet or topically. Induction of ODC activity was determined in similar experimental groups 24 h after a single exposure to UVA. Neither route of BHT administration had any effect on 8-MOP phototumorigenesis. However, BHT when administered in the diet reduced induction of ODC activity by 40% (p less than 0.05). These data indicate different mechanisms for UVB- and PUVA-induced carcinogenesis and again bring into question the relationship between induction of ODC activity and photocarcinogenesis.     

PHOTOREVERSIBILITY OF UV-INDUCED THYMINE DIMERS AND ABNORMAL MORPHOGENESIS IN SEA-URCHIN EMBRYOS

Abstract— Irradiation of synchronously dividing 16-cell embryos of a sea-urchin ( Hemicentrotus pul-cherrimus ) with 200 J m⁻² of UV light (254 nm) resulted in the complete inhibition of normal pluteus-larva formation when the embryos were cultured in the dark after UV-irradiation. Illumination of the UV-irradiated embryos with visible light (11 W m⁻²) for 1 h immediately after the UV-irradiation reversed the abnormal morphogenesis. Measurement of thymine dimers indicates that the degree of UV-induced abnormal morphogenesis is greatly correlated with the amount of thymine dimers in the DNA of the embryos. The degree of the photoreversal decreased with an increase in the interval between UV-irradiation and exposure to visible light. Visible light was ineffective as to the reversibility of both thymine dimers and the abnormal morphogenesis at 60 min after the UV-irradiation, when the UV-irradiated 16-cell embryos entered the next cell cycle.     

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.     

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.     

EFFECT OF PHOTOREACTIVATING LIGHT ON LETHAL AND PRE-MUTATIONAL UV LESIONS IN ESCHERICHIA COLI WP2s CARRYING THE R46 MUTATOR PLASMID

Abstract—An excision-deficient E. coli strain carrying the R46 mutator plasmid showed a different response towards photo-reactivation after UV irradiation than the same strain without plasmid. While the photoreactivation of lethal lesions was comparable in both strains, the number of UV-induced mutants per 10⁶ survivors was slightly reduced for the plasmid bearing strain by photoreactivating light at UV fluences below 60 mJ/m² but increased at higher fluences. To explain this it is proposed that some UV photoproduct(s) of DNA other than cyclobutane dipyrimidine dimers are pre-mutational lesions for error-prone DNA repair by the plasmid, P-repair, but not for SOS-repair.     

UV-INDUCED ALKALINE LABILE DNA DAMAGE IN HUMAN ADENOVIRUS AND ITS REPAIR AFTER INFECTION OF HUMAN CELLS

Abstract— UV-induced alkaline labile viral DNA damage was detected following irradiation of adenovirus type 2 and found to be repaired following the infection of human KB cells. Human adenovirus type 2 was irradiated with various doses of UV and subsequently used to infect human KB cells in tissue culture at approximately 2 × 10³ particles per cell. Before, and at various times after infection, the viral DNA was examined on alkaline sucrose gradients. Irradiated free virus DNA showed a dose dependent decrease in molecular weight compared to unirradiated virus DNA, indicating the presence of UV-induced alkaline labile lesions. Furthermore, an increase in the molecular weight of the irradiated virus DNA was found after infection indicating that alkaline labile lesions were removed from the viral DNA by a host mediated repair mechanism. After infection, the molecular weight of the irradiated virus DNA reached a value similar to that of unirradiated virus DNA for all the UV doses studied.     

UV-ACTION SPECTRUM (254-405 nm) FOR INHIBITION OF A K+ -STIMULATED ADENOSINE TRIPHOSPHATASE FROM THE PLASMA MEMBRANE OF ROSA DAMASCENA

A K ⁺-stimulated ATPase from suspension-cultured rose cells was isolated and subjected to UV radiation. The characteristics of the ATPase resembled those of a plasma-membrane associated enzyme and not those of the mitochondrial enzyme. The ATPase required Mg²⁺ and was further stimulated up to 100% by K⁺. K⁺ stimulation was specific for ATP. The order of stimulation by monovalent cations was K⁺ > Na⁺ > Li⁺. The enzyme had a pH optimum of 6.5 in the presence of 50 mM K⁺. It was almost completely inhibited by diethylstilbestrol and partially inhibited by vanadate. but was not affected by azide or oligomycin. The inhibition of ATPase activity by various fluences of UV indicated that one fraction of the K⁺-stimulated activity was very sensitive to radiation, while another fraction was relatively insensitive. It is possible that UV distinguished between two enzymes. The action spectra for inhibition of both fractions showed maxima at 290 nm and significant but much lower action throughout the near-UV region, resembling spectra in the literature for the inhibition of transport processes in bacteria.     

EXCISION REPAIR OF UVR-INDUCED PYRIMIDINE DIMERS IN CORNEAL DNA

Abstract— We measured excision repair of ultraviolet radiation (UVR)-induced pyrimidine dimers in DNA of the corneal epithelium of the marsupial, Monodelphis domestica , using damage-specific nucleases from Micrococcus luteus in conjunction with agarose gel electrophoresis. We observed that 100 J ^-2 of UVR from aFS–40 sunlamp(280–400 nm) induced an average of 2.2 ± 0.2 times 10^-2 endonuclease-sensitive sites per kilobase (ESS/kb) (pyrimidine dimers) and that ∼ 50% of the dimers were repaired within 12 h after exposure. We also determined that an exposure of 400 J m^-2 was needed to induce comparable numbers of pyrimidine dimers (2.5 times 10^-2) in the DNA of skin of M. domestica in vivo . In addition, we found that 50% of the dimers were also removed from the epidermal cells of M. domestica within 12 h after exposure. A dose of 100 J m^-2 was necessary to induce similar levels of pyrimidine dimers (2.0 ± 0.2 times 10^-2) in the DNA of the cultured marsupial cell line Pt K2 ( Potorous tridactylus ).     

SUNSCREENS WITH BROAD-SPECTRUM ABSORPTION DECREASE THE trans TO cis PHOTOISOMERIZATION OF UROCANIC ACID IN THE HUMAN stratum corneum AFTER MULTIPLE UV LIGHT EXPOSURES

Abstract The trans to cis photoisomerization of urocanic acid (UCA) in skin is considered to play an important role in the mechanism of immunosuppression. We have investigated the effects of skin type and various sunscreens with low sun protection factor (SPF) on the UV-induced cis -UCA formation in human skin after exposure to artificial IJV light. The rate of cis -UCA formation depends little on the skin type and is reduced by topical application of sunscreens. The rate of cis -UCA formation decreases with increasing SPF and only broad-spectrum, highly protective sunscreens offer protection against the UV-induced formation of cis -UCA, which accumulates in the stratum corneum after multiple UV exposures. A theoretical approach to estimate the distribution of cis -UCA after irradiation indicates that this compound may diffuse into the deeper layers of the epidermis with D ∼ 10⁻¹⁷ m²/s, and that its elimination from the stratum corneum is mainly due to desquamation.     

QUANTITATIVE EVIDENCE FOR ENZYMATICALLY-INDUCED DNA DOUBLE-STRAND BREAKS AS LETHAL LESIONS IN UV IRRADIATED pol+ AND polAl STRAINS OF E. COLI K-12

Abstract— We have recently reported that DNA double-strand breaks arise enzymatically during the course of excision repair in uvr ⁺ strains of Escherichia coli K-12. Survival curves for ultraviolet (UV) irradiated E. coli K-12 pol⁺ (JG139) and polA1 (JG138) strains have a pronounced shoulder region. The regions of the survival curves at which killing approaches exponential correspond to the fiuences at which DNA double-strand breaks (assumed to be lethal events) accumulate linearly. Reducing the number of UV photoproducts either by photoreactivation or fluence fractionation results in an increase in survival and a decrease in the yield of DNA double-strand breaks in both strains. These data support the hypothesis that enzymatically-induced DNA double-strand breaks may be the lesion ultimately responsible for UV-induced cell killing in the pol⁺ strain of E. coli K-12. and perhaps also in the polA1 strain.