Topical delivery of retinoids counteracts the UVB-induced epidermal vitamin A depletion in hairless mouse

UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.     

Effect of visible light on normal and P23H-3 transgenic rat retinas: characterization of a novel retinoic acid derivative present in the P23H-3 retina

Transgenic rats with the P23H mutation in rhodopsin exhibit increased susceptibility to light damage, compared with normal animals. It is known that light-induced retinal damage requires repetitive bleaching of rhodopsin and that photoreceptor cell loss is by apoptosis; however, the underlying molecular mechanism(s) leading to photoreceptor cell death are still unknown. Photoproducts, such as all-trans retinal or other retinoid metabolites, released by the extensive bleaching of rhodopsin could lead to activation of degenerative processes, especially in animals genetically predisposed to retinal degenerations. Using wild-type and transgenic rats carrying the P23H opsin mutation, we evaluated the effects of acute intense visible light on retinoid content, type and distribution in ocular tissues. Rats were exposed to green light (480-590 nm) for 0, 5, 10, 30 and 120 min. Following light treatment, rats were sacrificed and neural retinas were dissected free of the retinal pigment epithelium. Retinoids were extracted from retinal tissues and then subjected to HPLC and mass spectral analysis. We found that the light exposure affected relative levels of retinoids in the neural retina and retinal pigment epithelium of wild-type and P23H rat eyes similarly. In the P23H rat retina but not the wild-type rat retina, we found a retinoic acid-like compound with an absorbance maximum of 357 nm and a mass of 304 daltons. Production of this retinoic acid-like compound in transgenic rats is influenced by the age of the animals and the duration of light exposure. It is possible that this unique retinoid may be involved in the process of light-induced retinal degeneration.     

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age-related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time-resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV-visible absorption of longer-lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady-state and time-resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time-resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age-related decline of RPE function and blue light damage.     

Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells

Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.     

Retinal photodamage.

The retina represents a paradox, in that, while light and oxygen are essential for vision, these conditions also favour the formation of reactive oxygen species leading to photochemical damage to the retina. Such light damage seems to be multi-factorial and is dependent on the photoreactivity of a variety of chromophores (e.g., vitamin A metabolites, lipofuscin, melanin, flavins, porphyrins, carotenoids) endogenous to the retina. The aim of this article is to provide a detailed review of our current understanding of the photochemistry and photobiology of these chromophores and to consider how they may contribute to retinal ageing and pathology.     

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.     

Photodecomposition of vitamin A and photobiological implications for the skin

Vitamin A (retinol), an essential human nutrient, plays an important role in cellular differentiation, regulation of epidermal cell growth and normal cell maintenance. In addition to these physiological roles, vitamin A has a rich photochemistry. Photoisomerization of vitamin A, involved in signal transduction for vision, has been extensively investigated. The biological effects of light-induced degradation of vitamin A and formation of reactive species are less understood and may be important for light-exposed tissues, such as the skin. Photochemical studies have demonstrated that excitation of retinol or its esters with UV light generates a number of reactive species including singlet oxygen and superoxide radical anion. These reactive oxygen species have been shown to damage a number of cellular targets, including lipids and DNA. Consistent with the potential for damaging DNA, retinyl palmitate has been shown to be photomutagenic in an in vitro test system. The results of mechanistic studies were consistent with mutagenesis through oxidative damage. Vitamin A in the skin resides in a complex environment that in many ways is very different from the chemical environment in solution and in in vitro test systems. Relevant clinical studies or studies in animal models are therefore needed to establish whether the pro-oxidant activity of photoexcited vitamin A is observed in vivo, and to assess the related risks.     

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photo-oxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.     

Studies of all-trans-retinal as a photooxidizing agent

The photophysical properties of all-trans-retinal (RAL) have been extensively studied because of the importance of the retinoids in the visual process. However, little information is available regarding the participation of RAL in photochemical transformations such as photoxidation. RAL is one of several native chromophores that have been suggested to act as photosensitizers of damage in the human retina, and this damage would likely occur through oxidative pathways. Time-resolved and steady state techniques have been used to examine the photoreactivity of RAL toward several suitable substrates. The lifetime of the RAL triplet excited state is observed to decrease with increasing concentration of the well-known electron and hydrogen atom donors, 2,3,5,6-tetramethyl-1,4-phenylenediamine (DAD), hydroquinone (HQ), methylhydroquinone (MHQ), 2,3-dimethylhydroquinone (DMHQ) and trimethylhydroquinone (TMHQ), although the bimolecular rate constants for the reaction are much less than that of diffusion controlled (2.9 x 10(7) M-1 s-1, 1.2 x 10(5) M-1 s-1, 1.2 x 10(5) M-1 s-1, 1.5 x 10(5) M-1 s-1 and 1.6 x 10(6) M-1 s-1, for DAD, HQ, MHQ, DMHQ and TMHQ, respectively). In the presence of the donors, new absorptions grow concomitant with the decay of the triplet excited state, and for DAD and TMHQ, the observed spectra are similar to the spectra of p-phenylenediamine and TMHQ radicals. Irradiation of RAL in argon-saturated methanol results in fairly efficient photobleaching of RAL and in the formation of two new compounds having absorption spectra that are shifted below 300 nm. Irradiation of RAL in argon-saturated acetonitrile also results in photobleaching of RAL, but the reaction proceeds at a slower rate.     

THE PHOTOCHEMISTRY OF HUMAN RETINAL LIPOFUSCIN AS STUDIED BY EPR

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light-induced contribution of these fluorophores to various diseases including age-related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen-free methanol led to the formation of a 5,5-dimethylpyrroline-N-oxide (DMPO) spin-trapped carbon-centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform-soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age-related decline of RPE function and blue light damage in the retina.     

Cytotoxicity of All‐Trans‐Retinal Increases Upon Photodegradation†

All‐trans‐retinal (AtRal) can accumulate in the retina as a result of excessive exposure to light. The purpose of this study was to compare cytotoxicity of AtRal and photodegraded AtRal (dAtRal) on cultured human retinal pigment epithelial cells in dark and upon exposure to visible light. AtRal was degraded by exposure to visible light. Cytotoxicity was monitored by imaging of cell morphology, propidium iodide staining of cells with permeable plasma membrane and measurements of reductive activity of cells. Generation of singlet oxygen photosensitized by AtRal and dAtRal was monitored by time‐resolved measurements of characteristic singlet oxygen phosphorescence. Photodegradation of AtRal resulted in a decrease in absorption of visible light and accumulation of the degradation products with absorption maximum at ～330 nm. Toxicity of dAtRal was concentration‐dependent and was greater during irradiation with visible light than in dark. DAtRal was more cytotoxic than AtRal both in dark and during exposure to visible light. Photochemical properties of dAtRal indicate that it may be responsible for the maximum in the action spectra of retinal photodamage recorded in animals. In conclusion, photodegradation products of AtRal may impose a significant threat to the retina and therefore their roles in retinal pathology need to be explored.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS. XVII. BENZANTHRONE

The photochemistry of benzanthrone (7H-benz[de]-anthracene-7-one) has been studied using electron paramagnetic resonance (EPR) in conjunction with the spin trapping technique and the direct detection of singlet molecular oxygen luminescence. Irradiation (lambda ex = 394 nm) of benzanthrone (BA) in aerated ethanol, dimethylsulfoxide or benzene resulted in the generation of superoxide (O2-.) which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The ethoxy radical was also detected in ethanol. Photolysis of BA in deaerated basic ethanol led to the formation of BA anion radical, BA-., which was detected directly by ESR. This radical anion decayed back to BA with a unimolecular rate constant of 1.5 x 10(-3) s-1. The 1O2 quantum yields (lambda ex greater than 345 nm) for BA in ethanol, 90% ethanol and basic ethanol (0.1N NaOH) were 0.89, 0.88 and 0.28 respectively relative to Rose Bengal. The lower yield of 1O2 in basic ethanol may be attributable to the reaction of oxygen with BA-. (which is generated in higher yield at alkaline pH) to give O2-.. These findings suggest that on exposure to light BA can generate active oxygen species which may be responsible for the photocontact dermatitis caused by BA in industrial workers exposed to this chemical.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—1. THE MOLECULAR MECHANISMS*

Abstract— DNA and RNA syntheses were inhibited immediately after proflavine treated HeLa cells were irradiated with visible light (400–500 nm). The molecular mechanism for this photooxidation may be either a free radical-mediated (Type I) or singlet oxygen-mediated (Type II) reaction. Non-toxic free radical and singlet oxygen quenchers were added to cells and sensitizer before irradiation to quench the appropriate excited state intermediate. Photooxidative damage (the inhibition of incorporation of [¹⁴C]-thymidine) in this system was greatly reduced in the presence of free radical quenchers (glutathione, penicillamine) and not significantly affected by the presence of singlet oxygen quenchers (α-tocopherol, β-carotene, DABCO). This suggests that at least part of the photodynamic damage in HeLa cells is via a Type I mechanism.     

Photooxidation Mechanism of Levomepromazine in Different Solvents

Unwanted photoinduced responses are well‐known adverse effects of most promazine drugs, including levomepromazine (LPZ, Levoprome^® or Nozinan^®). This drug is indicated in psychiatry primarily for the treatment of schizophrenia and other schizoaffective disorders. Levomepromazine's particular sedative properties make it especially fit for use in psychiatric intensive care. Nevertheless, it is photolabile under UV‐A and UV‐B light in aerobic conditions resulting in the formation of its sulfoxide. The LPZ photochemistry in acetonitrile (MeCN) is completely different from that in methanol (MeOH) and phosphate buffer solutions (PBS, pH = 7.4). The major photoproduct in PBS and MeOH under aerobic conditions is levomepromazine sulfoxide (LPZSO), although the amount is considerably higher in the aqueous environment. The corresponding main photoproduct in MeCN could not be characterized. The destruction quantum yields of LPZ in PBS, MeOH and MeCN are 0.13, 0.02 and <10^?3, respectively. It is further demonstrated that LPZSO does not form by the reaction of singlet oxygen with ground‐state LPZ. This oxidation product is actually produced by the reaction of the cation radical of LPZ (LPZ·⁺) with molecular oxygen. This cation radical in turn, is produced by an electron transfer process between the ³LPZ* and ground‐state molecular oxygen.     

AN ESR STUDY OF THE VISIBLE LIGHT PHOTOCHEMISTRY OF GILVOCARCIN V

Photolysis of gilvocarcin (GV) at 405 nm in argon saturated dimethylsulfoxide (DMSO) or 50% DMSO-water solutions in the presence of the sodium salt of 3,5-dibromo-2,6-dideutero-4-nitrosobenzene sulfonic acid (DBNBS-d2) generates the CH3-DBNBS-d2.spin adduct. It is postulated that this spin adduct is produced by photoreduction of DMSO by GV and the consequent formation and trapping of the generated methyl radicals. Gilvocarcin V also photoreduces oxygen and methyl viologen with quantum yields of 0.019 and 0.0012 respectively. The quantum yield for singlet oxygen formation by GV in DMSO, determined by measuring the rate of production of the nitroxyl radical produced by the reaction of 2,2,6,6-tetramethylpiperidinol with singlet oxygen, was found to be 0.15. Thus, GV photochemistry proceeds by both Type I and Type II pathways which could contribute to the reported GV phototoxicity in biological systems.     

PHOTOINDUCED REDUCTION OF CYTOCHROME c IN THE PRESENCE OF HYDROPEROXIDE DERIVATIVE OF BIPHENYL

Abstract The photosensitive hydroperoxide derivative of biphenyl (BPP) was synthesized by the ozonolysis of phenanthrene in methanol. When cytochrome c (cyt c ) was illuminated by UVB light in the presence of BPP (BPPUV), it was reduced both under aerobic and anaerobic conditions. The action spectrum of the reduction was consistent with that of photolytic decomposition of BPP. Both gave maximum reactions at wavelengths around 300–310 nm. Electron spin resonance studies, using 5,5-dimethyl-1-pyrroline N -oxide as a spin-trapping reagent, revealed the generation of hydroxyl radicals in the BPPUV system. Product analysis of adamantane oxidation by BPPW also suggested the generation of hydroxyl radicals rather than singlet oxygen. However, the effects of scavengers were complicated. Singlet oxygen scavengers significantly inhibit the reaction while none of the hydroxyl radical scavengers tested was effective in inhibiting the BPPUV-mediated cyt c reduction. Deuterium oxide, which extends the lifetime of singlet oxygen, inhibited rather than enhanced the reaction. Reduction of cyt c was inhibited by salts, and their activities were correlated to the electron-donating nature of the anions. These results suggest that reduction of cyt c is mediated by electron transfer from a light-induced product of BPP, rather than by free hydroxyl radicals or singlet oxygen.     

Reaction of singlet oxygen with trans-4-propenylanisole. Formation Of

We report the effects of added acid in the reaction of singlet oxygen with trans-4-propenylanisole (1). We provide evidence that solvent acidity modifies the behavior of the transient intermediates. Relative to reactions in aprotic solvent, enhanced dioxetane concentrations are observed in MeOH and in nonprotic solvents with acid. We suggest a new mechanism that invokes a proton transfer from MeOH and benzoic acid to perepoxide (2) and zwitterion (3) intermediates.     

The role of A2E in prevention or enhancement of light damage in human retinal pigment epithelial cells

The process of sight (photostasis) produces, as a by-product, a chromophore called 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E, 5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E, 5E-hexatrienyl]-pyridinium (A2E), whose function in the eye has not been defined as yet. In youth and adulthood, A2E is removed from human retinal pigment epithelial (h-RPE) cells as it is made, and so it is present in very low concentrations, but with advanced age, it accumulates to concentrations reaching 20 microM. In the present study we have used photophysical techniques and in vitro cellular measurements to explore the role of A2E in h-RPE cells. We have found that A2E has both pro- and antioxidant properties. It generated singlet oxygen (phiso = 0.004) much less efficiently than its precursor trans-retinal (phiso = 0.24). It also quenched singlet oxygen at a rate (10(8) M(-1) s(-1)) equivalent to two other endogenous quenchers of reactive oxygen species in the eye: alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C). The endogenous singlet oxygen quencher lutein, whose quenching rate is two orders of magnitude greater than that of A2E, completely prevented light damage in vitro, suggesting that singlet oxygen does indeed play a role in light-induced damage to aged human retinas. We have used multiphoton confocal microscopy and the comet assay to measure the toxic, phototoxic and protective capacity of A2E in h-RPE cells. At 1-5 microM, A2E protected these cells from UV-induced breaks in DNA; at 20 microM, A2E no longer exerted this protective effect. These results imply that the role of A2E is not simple and may change over the course of a lifetime. A2E itself may play a protective role in the young eye but a toxic role in older eyes.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

Photochemical and Photophysical Studies of 3-Amino-6-lodoacridine and the Inactivation of λ Phage

Abstract— The photochemistry and photophysics of 3-amino-6-io-doacridine (Acr-I) was studied. Photolysis (350 nm) of Acr-I (free base) generates products consistent with a free radical intermediate in methanol, benzene and carbon tetrachloride. The Acr-I hydrochloride is shown to bind to calf thymus DNA and to the self-complementary dinucleotide cytidylyl-(3′-5′)-guanosine (CpG) minidu-plex in a manner similar to that of proflavine (Acr-NH₂), a known DNA intercalator. The Acr-I is shown to more efficiently nick supercoiled plasmid DNA pBR322 upon 350 nm or 420 nm photolysis than Acr-NH₂. The efficiency of Acr-I-sensitized DNA nicking is not oxygen dependent. Photolysis of the Acr-I/(CpG)₂ complex leads to cleavage of the dinucleotide and to cytidine base release by selective damage to a specific ribose moiety. Dinucleotide cleavage occurs equally well in the presence or absence of oxygen, thereby eliminating a singlet oxygen- or peroxyl radical-mediated process. Photolysis of Acr-I in the presence of a mononucleotide (GMP) or a non-self-complementary dinucleotide (uridylyl-[3′-5′]-cytidine– UpC) does not lead to fragmentation and base release. Similarly, photolysis of the Acr-NH₂/(CpG)₂ complex does not lead to fragmentation and base release. The data indicate that photolysis of an iodinated intercalator bound to CpG or plasmid DNA generates an intercalated aryl radical and that the reactive intermediate initiates a sequence of reactions that efficiently nick nucleic acids. The inactivation of Λ phage sensitized by Acr-I with UV (350 nm) light is oxygen independent but with visible (420 nm) light is strongly oxygen dependent. The Acr-I fluoresces more intensely when excited at 446 than at 376 nm. Thus, UV photolysis may lead to C-I bond homolysis and free radical formation, a process that is not energetically feasible with visible light. The results demonstrate the difficulty of extrapolating model studies involving simple molecules and DNA to understanding the mechanism of viral inactivation with a particular sensitizer.