PHOTOBINDING OF CHLORPROMAZINE AND ITS SULFOXIDE IN VITRO AND IN VIVO

Abstract— Photosensitivity as observed after chlorpromazine (CPZ) treatment is enhanced in the UVA- rather than the UVB region, whereas CPZ has its absorption maximum at 305 nm. This long wavelength sensitivity has sometimes been ascribed to CPZ-sulfoxide (CPZSO) which has an absorption maximum at 340 nm. We compared the photobinding properties of CPZSO and CPZ under both in vitro and in vivo conditions.
With 310 and 370 nm lamps CPZSO absorbs twice as much light as CPZ but still binds less efficiently to HSA in vitro. At wavelengths longer than 380 nm CPZSO does not absorb nor photobind to HSA (420 nm lamps) in contrast to CPZ.
In vivo the bioavailability of CPZ and CPZSO in ears, eyes and skin of the back of Wistar rats is comparable, yet irradiation with 370 nm light caused more CPZ-photobinding in these tissues. Chlorpromazine binds relatively more compared to CPZSO, to constituents of deeper lying tissues (dermis). This corresponds with the observation that both the ratio of in vitro CPZ photobinding to CPZSO photobinding, and the penetrating ability of light in the skin increase with wavelength.
In the eyes, where the lens efficiently filters out light with wavelengths shorter than 370 nm, no CPZSO photobinding was observed, in contrast to CPZ; this also corresponds with the in vitro experiments. Therefore it seems more likely that the observed wavelength maximum in the photosensitivity action spectrum after CPZ treatment should be attributed to the non-sulfoxidated drug rather than to the sulfoxidated compound.     

PHOTO-INDUCED BINDING OF SULFANILAMIDE TO CELLULAR MACROMOLECULES

Abstract— Ultraviolet light (A > 295 nm) induced binding of sulfanilamide to cellular macromoleculcs has been examined. It was found that the drug bound irreversibly to native DNA, and complexes containing one drug molecule per 80 nucleotides were obtained after 60 min of irradiation under anaerobic conditions. Oxygen reduced this binding significantly. More drug was bound to RNA and heat denatured DNA under identical conditions. The binding of sulfanilamide to DNA was found to induce nicking of circular closed plasmid DNA and cross-linking of calf thymus DNA. Oxygen significantly decreased nicking and cross-linking of DNA. Irradiation of sulfanilamide and human serum albumin resulted in covalent binding of the drug to the protein and a concomitant increase in protein crosslink-ing. While oxygen decreased covalent binding, crosslinking increased under aerobic conditions. These reactions may be important in the photosensitization caused by sulfanilamide.     

Study of the degradation of polyurethanes. III. Mechanism of the photodegradation of polyurethane

The effect of triplet sensitizers, benzophenone and anthraquinone and metal acetylacetonates [Co(II, III), Cu, Sn, and Ni] on the photodegradation of polyurethane was examined. Ultravioletvisible (UV-V) absorption spectra, gel formation, and luminescence emission of the polymer before and after irradiation were measured. Changes in UV-V absorption of the polymer and the formation of an insoluble fraction in the polymer were accelerated in the presence of the triplet sensitizers, and (Co(II, III)), Cu, and Sn acetylacetonates. Unirradiated polyurethane was excited by irradiation at 290 and 346 nm, and emitted light at 310 and 420 nm. After 1/2 hr irradiation emission of luminescence was observed at 430 nm, excitation at 290 and 346 nm; after 2hr irradiation at 530 nm, excitation at 420 nm was observed. The results suggest that photodegradation of the polyurethane proceeds via excited triplet states forming excimer between the polymers at the initial stage and exciplexes between the polymer and degradation products or intermediates after a certain irradiation.     

Photobinding of 8-methoxypsoralen, 4,6,4'-trimethylangelicin and chlorpromazine to Wistar rat epidermal biomacromolecules in vivo.

Photoinduced binding of drugs to endogenous biomacromolecules may cause both toxic and therapeutic effects. For example, photobinding of certain phenothiazines to biomolecules possibly underlies their phototoxic and photoallergic potential, whereas photobinding of furocoumarins to epidermal DNA is held responsible for their advantageous effects in the photochemotherapy of psoriasis. Usually, the in vitro photobinding of drugs is investigated. However, under in vivo conditions, the metabolism and distribution of the drug and the light absorption by endogenous compounds will significantly affect the photobinding of drugs to biomolecules. Therefore, in the present study, the photobinding of 8-methoxypsoralen (8-MOP), 4,6,4'-trimethylangelicin (TMA) (two therapeutically used furocoumarins) and chlorpromazine (CPZ) (a member of the phenothiazines) was investigated in vivo. The compounds were applied topically on the shaven skin of Wistar rats; one group was exposed to UVA and the other was kept in a dimly lit environment. Immediately, and at certain time intervals after UVA exposure, members of the two groups were sacrificed. By separating epidermal lipids, DNA/RNA and proteins by a selective extraction method, irreversible binding of 8-MOP, TMA or CPZ to each of these biomacromolecules was determined. In contrast with in vitro experiments, photobinding of CPZ to epidermal DNA/RNA was not found in vivo; apparently the bioavailability in the nucleus is very low. Compared with TMA, 8-MOP was observed to bind more extensively to epidermal DNA/RNA (again in contrast with findings from in vitro experiments) and proteins, but less extensively to lipids. The rates of removal of photobound 8-MOP and TMA were comparable. Photobound CPZ was more slowly removed from epidermal proteins and lipids than the furocoumarins. The observed in vivo photobinding is discussed with respect to the UVA-induced (side) effects of these drugs.     

Wavelength-specific activation of MAP kinase family proteins by monochromatic UV irradiation

The depletion of stratospheric ozone causes related increase in UV light below about 310 nm, which significantly affects biological and ecological systems. To understand the wavelength-specific effects of UV light, Molt4 cells (human T lymphoma cells) were irradiated with a series of monochromatic UV lights and the activities of three members of the mitogen-activated protein (MAP) kinase group were examined. Extracellular signal-regulated kinase was specifically activated within 1 min after UV irradiation in the range 320-360 nm. In contrast, P38 kinase was activated by 270-280 nm light with a peak at 1 min after irradiation. c-Jun N-terminal kinase activation was observed in a narrow range of UV light with a sharp peak at 280 nm occurring in 10 min. JNK translocated from the cytosol to the nucleus upon irradiation, while P38 remained in the cytosol even after UV irradiation. The activation of three MAP kinases was prevented by antioxidant reagents, suggesting that an oxidative signal initiates these responses. These results confirm that UV light affects various cellular functions through the activation of intracellular signaling systems including MAP kinase family proteins. However, the UV-induced activities of the separate MAP kinases show distinctly different dose, time and wavelength dependencies.     

Alterations of hairless mouse skin exposed to chronic UV irradiation and its prevention by hydrocortisone

Ultraviolet-induced alterations of skin were investigated in a murine animal model. Groups of hairless mice were exposed to UV (black light, lambda max 352 nm; UV distribution: 300-310 nm, 0.9%; 310-320 nm, 2.0%; 320-420 nm, 97.1%) for 20 weeks at a dose of 16.3 J/cm2 five times weekly on weekdays. At the end of 20 weeks irradiation, the dorsal skins were biochemically and histologically examined. Ultraviolet caused remarkable increases in amounts of hyaluronan, chondroitin sulfates and dermatan sulfates in skin (microgram/cm2). Interestingly, a significant change in a collagen content (hydroxyproline, microgram/g of dry powder) caused by UV irradiation was not observed, whereas the amount of collagen (hydroxyproline, microgram/cm2) increased remarkably. Histologically, no distinguishable thickening was observed in both upper dermis and lower dermis, but thickening of the epidermis was observed. Furthermore, the histological study indicated that UV irradiation caused a disappearance of crowds of adipocytes, alternative appearance of numerous fibroblasts and accumulation of collagen bundles and hyaluronan in lower dermis. Hydrocortisone, an anti-inflammatory agent, prevented both the fibrosis of lower dermis and the accumulation of the extracellular matrix components. Based on these results, it seems reasonable that UV penetrates into the lower dermis and causes fibrosis there, resulting from the inflammatory responses.     

Absorption spectra of viral components of Sendai virus in the wavelength region from 130 to 320 nm.

Using synchrotron radiation as a light source, the absorption spectra of purified viral components of the Sendai virus, i.e. messenger RNA, lipids, spike (envelope) proteins, reconstructed envelopes, core proteins and whole virions, were obtained in the wavelength region 130-320 nm by measuring the transmission of thin films. Viral (messenger) RNA two peaks at 260 and 190 nm, and a large increase below 160 nm. The absorption spectrum of lipids exhibited a broad peak at 190 nm and a very sharp increase below 160 nm. With spike proteins, a slight peak at 280 nm and a shoulder at 230 nm were observed in addition to a sharper peak at 190 nm and a rather slow increasing absorption below 160 nm. Reconstructed envelopes showed the features of a combination of lipids and proteins. The absorption spectra of core proteins and whole virions exhibited similar characteristics to spike proteins. Conventional UV data were also obtained in the wavelength range 210-320 nm with RNA and lipids. The UV and synchrotron radiation data were in good agreement in terms of the mass absorption coefficients. The molecular splitting of spike proteins was also examined. Proteins gave more diffuse reflection than their subunits, causing a reduction in absorption. This was explained by a loss of transparency with increasing molecular weight.     

Somatic-cell mutation induced by UVA and monochromatic UV radiation in repair-proficient and -deficient Drosophila melanogaster

Near-ultraviolet light (UVA: 320-400 nm) constitutes a major part of sunlight UV. It is important to know the effect of UVA on the biological activities of organisms on the earth. We have previously reported that black light induces somatic-cell mutation in Drosophila larvae. To investigate which wavelength of the UVA is responsible for the mutation we have now carried out a series of monochromatic irradiations (310, 320, 330, 340, 360, 380 and 400 nm) on Drosophila larvae, using the large spectrograph of the National Institute for Basic Biology (Okazaki National Research Institutes, Okazaki, Japan). Mutagenic activity was examined by the Drosophila wing-spot test in which we observe mutant wing hair colonies (spots) on the wings of adult flies obtained from the treated larvae. The induction of mutation was highest by irradiation at 310 nm and decreased as the wavelength became longer. Neither the 380 nor the 400 nm light was mutagenic. Excision repair is known to protect cells from UV damage. In the excision-repair-deficient Drosophila, the mutagenic response induced by 310 nm irradiation was 24-fold higher than that of the wild-type (7.2 +/- 1.5 spots/wing/kJ vs 0.3 +/- 0.08 spots/wing/kJ), and at 320 nm the difference of the response was 14-fold (0.21 vs 0.015 +/- 0.005). In the case of irradiation at 330 and 340 nm the difference of the response was only two-fold (at 330 nm, 6.9 +/- 2.9 x 10(-3) vs 3.1 +/- 1.1 x 10(-3) spots/wing/kJ; at 340 nm, 3.5 +/- 0.9 x 10(-3) vs 2.0 +/- 0.7 x 10(-3). These results suggest that the lesion caused in the larvae by 320 nm irradiation may be similar to the damage induced by 310 nm and that the lights of 330 and 340 nm may induce damage different from the lesions induced by shorter-wavelength lights.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—II. DAMAGE TO DNA

Abstract— HeLa cell suspensions, prelabeled with specific [¹⁴C]-nucleosides, were treated with proflavine and irradiated with visible light (400–500 nm). The DNA was isolated from the cells (as well as from the appropriate control cells) and examined for macromolecular and molecular changes. Although the UV absorbance spectrum of DNA from irradiated HeLa cells showed no discernible change, a fluorescence spectrum (excitation/emission: 305/405) indicated a molecular change in the DNA. Isolated DNA samples were hydrolyzed with 90% formic acid and chromatographed. There were no detectable differences between the irradiated and non-irradiated profile (R _f and radioactivity) for both guanine and adenine. However, the chromatograms of thymine and cytosine showed distinct changes. There was a loss of radioactivity in the [¹⁴C]-thymidine labeled samples, while the [¹⁴C]-cytidine labeled samples indicated the formation of a new compound, containing 10% of the radioactivity, running just ahead of cytosine. These data strongly suggest the formation of a new compound resulting from the photooxidation of cytosine when nuclear DNA was sensitized by proflavine.     

HOECHST 33258 DYE GENERATES DNA-PROTEIN CROSS-LINKS DURING ULTRAVIOLET LIGHT-INDUCED PHOTOLYSIS OF BROMODEOXYURIDINE IN REPLICATED AND REPAIRED DNA

Abstract— Substitution of bromodeoxyuridine for thymidine in the DNA of mammalian cells sensitizes them to a range of wavelengths of ultraviolet light. Cells are also sensitized to photochemical reactions involving dyes such as Hoechst 33258, which is used to produce differential staining of chromatids according to their bromodeoxyuridine content. Irradiation with 313 nm light of human and hamster cells containing bromodeoxyuridine in their DNA produced single-strand breaks, detectable by alkaline elution, but no DNA-protein cross-links. Irradiation with 360 nm light in the presence of Hoechst 33258 produced extensive DNA-protein cross-linkage as well as single-strand breaks. These cross-links were observed in DNA containing bromodeoxyuridine incorporated by either semiconservative or repair replication, and may provide a method for identification of proteins in close proximity to replication forks or repair sites. When the protein was removed with proteinase K, bromodeoxyuridine in repair patches after irradiation by doses of ultraviolet (254 nm) light as low as 0.26 J/m² could readily be detected. Hoechst 33258-mediated photolysis, therefore, provides a sensitive new technique for measuring repair replication after ultraviolet light irradiation.     

Reversible Covalent Stabilization of Stacking Contacts in DNA Assemblies

Stacking bonds formed between two blunt‐ended DNA double helices can be used to reversibly stabilize higher‐order complexes that are assembled from rigid DNA components. Typically, at low cation concentrations, stacking bonds break and thus higher‐order complexes disassemble. Herein, we present a site‐specific photochemical mechanism for the reversible covalent stabilization of stacking bonds in DNA assemblies. To this end, we modified one blunt end with the 3‐cyanovinylcarbazole (^cnvK) moiety and positioned a thymine residue (T) at the other blunt end. In the bound state, the two blunt‐ended helices are stacked together, resulting in a co‐localization of ^cnvK and T. Such a configuration induces the formation of a covalent bond across the stacking contact upon irradiation with 365 nm light. This bond can also be cleaved upon irradiation with 310 nm light, allowing repeated formation and cleavage of the same covalent bond on the timescale of seconds. Our system will expand the range of conditions under which stacking‐bond‐stabilized objects may be utilized.     

Photochemistry and Reactivity of the Phenyl Radical–Water System: A Matrix Isolation and Computational Study

The reaction of the phenyl radical 1 with water has been investigated by using matrix isolation spectroscopy and quantum chemical calculations. The primary thermal product of the reaction between 1 and water is a weakly bound complex stabilized by an OH???π interaction. This complex is photolabile, and visible‐light irradiation (λ>420 nm) results in hydrogen atom transfer from water to radical 1 and the formation of a highly labile complex between benzene and the OH radical. This complex is stable under the conditions of matrix isolation, however, continuous irradiation with λ>420 nm light results in the complete destruction of the aromatic system and formation of an acylic unsaturated ketene. The mechanisms of all reaction steps are discussed in the light of ab initio and DFT calculations.     

Optimal irradiation wavelength in iniferter‐based photocontrolled radical polymerization

In iniferter‐based photocontrolled radical polymerization optimal irradiation wavelength was examined for precise molecular design of star vectors, which can function as high‐performance gene carriers. Monochromatic light in the range from 330 to 400 nm with the interval of about 10 nm was irradiated to tetrafunctional iniferter, 1,2,4,5‐tetrakis(N,N‐diethyldithiocarbamylmethyl)benzene, in the presence of N,N‐dimethyaminopropylacrylamide (DMAPAAm), as a model monomer, in toluene. In all wavelengths tetrabranched polymers were produced except for 400‐nm‐irradiation. The highest conversion reaching to about 35% was observed at the wavelength of 370 nm after only 40 min of irradiation. There was no further increase in conversion by combination with another two wavelengths. HPLC analysis and NMR spectra showed that the polymerization at the optimal wavelength of 370 nm was occurred in a living manner with the ability to control the chain length (from about 5000 to 40,000) having very narrow polydispersity (about 1.6) by changing of the irradiation time, the intensity, and the monomer concentration. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 4505–4512, 2008     

PHOTOCHEMICAL BINDING OF PHENOTHIAZINES ON BIOLOGICAL MEMBRANE PROTEINS

Abstract— The photobinding of phenothiazine derivatives (chlorpromazine, fluphenazine, promazine and promethazine) was studied on four different types of biological membranes (microsomes, myelin and synaptosomes from rat brain as well as human erythrocytes). The photoreaction was performed by ultraviolet irradiation of the tritiated compounds in their long wavelength absorption band (313 nm) and bound photoproducts were analysed by autoradiography of the proteins separated by polyacrylamide gel electrophoresis. The specificity of binding is low, however, a 34000 dalton band is intensely labeled on synaptic membranes with chlorpromazine and fluphenazine. All the phenothiazines bind on erythrocyte membrane proteins and specially on band 4.2 and on a peptide located before actin on the electrophoresis gel. These results show the generality of the phenothiazine photobinding on membrane proteins. These photobinding properties can be used for the identification and localization of some of these proteins.     

Photoactivated Psoralens Elicit Defense Genes and Phytoalexin Production in the Pea Plant

In the pea plant ( Pisum sativum ), compounds that intercalate into DNA induce the production of ∼20 major proteins similar to the pattern induced during nonhost disease resistance to the bean fungal pathogen, Fusarium solani f.sp. phaseoli . The pea phytoalexin, pisatin, as well as RNA homologous to several disease-resistance response (DRR) genes accumulate following treatment with these compounds. Psoralen was chosen to characterize this interaction further because it intercalates into DNA and, following irradiation with 365 nm UV light (UV₃₆₅), forms covalent bonds with pyrimidines on either or both strands of DNA. This produces monoadducts or cross-links, respectively. Dose experiments showed that 60 μg/mL 4'-aminomethyl-4,5',8-trimethylpsoralen followed by 18 J/cm² UV₃₆₅ was sufficient to produce an accumulation of pisatin similar to that produced in response to the fungus. Under these inducing conditions, there was an average of 0.19 adducts per kb of pea genomic DNA. The accumulation of pisatin and the RNA of several DRR genes by psoralen required photoactivation, which suggests that covalent binding to DNA was necessary for induction. As the promoters of several putative fungal-induced pea genes contain long stretches of d(AT)_n, which is the preferred psoralen photobinding site, restriction fragments spanning DRR genes were examined after in vivo psoralen treatment. The rate of crosslinking was compared between fungal-induced and noninduced genes using a modified Southern blot analysis. Implications of the induction of the DRR due to psoralen binding are discussed.     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

Abstract
I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (< 290 nm) and UV-B irradiation (290–320 nm) in the 2'-deoxy-[³H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m^-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol . 52 , 519–524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10–20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

A polymeric photobase generator containing oxime–urethane groups: Crosslinking reaction and application to negative photoresist

A polymeric photobase generator containing oxime–urethane groups was prepared by copolymerization of methyl methacrylate and methacryloxyethyl benzophenoneoxime urethane, and its photo and thermal crosslinking reaction after irradiation was examined from the measurement of UV and IR absorption spectral changes, insoluble fraction, and molecular weight changes. The photo‐crosslinking reaction of the copolymer film was more efficient when irradiations were carried out with 310 nm UV light in the presence of benzophenone than with 254 nm UV light without the addition of benzophenone. The crosslinking reaction increased after postexposure baking (PEB), and this thermal crosslinking reaction mechanism was studied from the identification of the photolysis products of a model compound, benzophenoneoxime phenylurethane, by a high‐performance liquid chromatography. The results indicate that the thermal crosslinking reaction of the copolymer after PEB is due to the formation of urea‐type chemical bonds. Resist properties of the copolymer were examined from the measurement of normalized thickness and micropattern development. A negative tone image with a resolution of 2 μm was obtained with this copolymer, having a sensitivity (D) of 1200 mJ/cm² and contrast (γ_n) of 1.31, when irradiation was carried out with 310 nm UV light in the presence of benzophenone following chloroform development. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 975–984, 2004     

DETERMINATION OF CYTOSINE-CYTOSINE PHOTODIMERS IN THE DNA OF CLOUDMAN S91 MELANOMA CELLS USING HIGH PRESSURE LIQUID CHROMATOGRAPHY

I measured the induction of cytosine-cytosine dimer (C-C) densities after UV-C (less than 290 nm) and UV-B irradiation (290-320 nm) in the 2'-deoxy-[3H]cytidine labeled DNA of Cloudman S91 mouse melanoma cells using a new, sensitive high pressure liquid chromatography procedure. UV-B exposure resulted in 0.000034% C-C/J m-2 of the total cytosine radioactivity which is 10 times less than the rate during UV-C irradiation. Previous work with these melanoma cells showed a 4-fold lower rate of induction of thymine-containing pyrimidine dimers by UV-B than UV-C light (Niggli Photochem. Photobiol. 52, 519-524, 1990). Based on these results, the calculated ratios for the pyrimidine dimer subspecies showed no significant difference following UV-C and UV-B exposure. However, UV-C and UV-B light induce 10-20 times more thymine-containing pyrimidine dimers than C-C in the DNA of S91 cells.     

LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

Study on the interaction between a fluorine-containing amphiphilic cationic copolymer and nucleic acid by resonance light scattering technique

A novel fluorine-containing amphiphilic cationic copolymer P(HFMA-St-MOTAC)-g-PEG was synthesized as a new probe to detect DNA based on the RLS technique. The aggregation of P(HFMA-St-MOTAC)-g-PEG on the molecular surface of DNA occurred under pH 4.0-7.0 resulted in an enhanced resonance light-scattering (RLS) peaks at 370 nm, 400 nm, 420 nm and 470 nm. The intensity of resonance light-scattering was found to be proportional to the concentration of DNA. The detection limit was 5.8 μg L⁻¹. It was found that the P(HFMA-St-MOTAC)-g-PEG has strong interaction with DNA as confirmed by the resonance light scattering (RLS) spectroscopy, UV spectra, IR spectra, and TEM.