The photodegradation of quercetin: relation to oxidation.

The photostability of quercetin in alcoholic solutions was studied. Both UVA and UVB light induced degradation of quercetin, yielding a single product 1 deriving from oxidation and addition of an alcohol molecule to the 2,3 double bond. The same mechanism operated when quercetin was dissolved in alkaline solutions, and again a product 2 due to oxidation and addition of water was characterized. Comparison with quercetin analogs confirmed that, despite the presence of five hydroxy groups in quercetin, those in positions 3, 3', and 4' are mainly involved in the antioxidant activity of the compound , as well as in its photolability.

A novel tetrahydrobenzoangelicin with dark and photo biological activity.

The synthesis of 8,9,10,11-tetrahydro-5-(3-dimethylaminopropoxy)-4-methylbenzofuro[2,3-h]coumarin (5) is described. The new compound showed the ability to inhibit cell growth both upon UVA irradiation and in the dark. The investigation on the mechanism of action highlighted the capacity of 5 to covalently photoadd to thymine, as demonstrated by the isolation and characterization of the 4',5'-monoadduct. Furthermore, in the ground state 5 interferes with the topoisomerase II relaxation activity, suggesting that this enzyme could constitute a molecular target responsible for the dark antiproliferative effect.

Photoreactivity of 5-fluorouracil under UVB light: photolysis and cytotoxicity studies.

The photodegradation of the chemotherapeutic agent 5-fluorouracil (5-FU) under UVB light was studied both in aqueous and methanol solutions and in systemic and topical formulations. As monitored by HPLC, photodegradation in solution takes place in a concentration dependent manner; thus, the solution for parenteral administration (10(-1) M) showed negligible loss of the active principle. On the contrary, the commercial cream containing 5% of 5-FU showed low stability under UVB exposure. When dissolved either in water or methanol, 5-FU yields two photoproducts which have been characterized as two isomers coming from the addition of the solvent to the 5,6 double bond of the drug. As a consequence, photomodified 5-FU loses its antiproliferative activity on HCT-15 and HeLa cells. MS analysis showed that photoaddition occurred with nucleophilic amino acids, such as cysteine and serine, while susceptible amino acids (cysteine and methionine) were oxidized. In fact, high production of the superoxide anion under UVB light as well as photooxidation of BSA suggests protein photodamage as a mechanism of photosensitization. Indeed, some phototoxicity was shown in experiments on NCTC keratinocytes and MCF-7 resistant cells irradiated with UVB light. The interactions with these biological targets may contribute to skin phototoxicity and photoallergy induced by 5-FU in vivo.

Erythroid induction of chronic myelogenous leukemia K562 cells following treatment with a photoproduct derived from the UV-A irradiation of 5-methoxypsoralen.

Induction of terminal erythroid differentiation can be an efficient strategy to inhibit proliferation of chronic myelogenous leukemia cells. Psoralens, well-known photo-chemotherapeutic agents, were found to be efficient at inducing erythroid differentiation of K562 cells, an in vitro cell line isolated from the pleural effusion of a patient with chronic myelogenous leukemia in blast crisis. The effects of crude pre-irradiated solutions of 5-methoxypsoralen on erythroid differentiation of human leukemic K-562 cells were evaluated. The major photoproduct was characterized and analyzed, and it was found to induce erythroid differentiation of K562 cells and inhibit NF-kappaB/DNA interactions.

UVB photolysis of betamethasone and its esters: characterization of photoproducts in solution, in pig skin and in drug formulations.

The stability of the synthetic glucocorticosteroid betamethasone under UVB light was studied both in vitro (water and methanol solution and in topical and injectable commercial formulations) and ex vivo (pig skin). From irradiated methanol solutions three main photoproducts were isolated by HPLC and TLC and characterized by NMR/MS analyses. The modifications involve parts of the molecule peculiar for the therapeutic activity, that is, rearrangement of ring A ("lumi"- and "photolumiderivatives"), and Norrish Type I fragmentation of the ketolic chain ("androderivative"). Two clinically used esters of betamethasone were also studied, namely the 17-valerate and 21-phosphate, and their photoproducts identified. The HPLC method developed for the photolysis studies in solution was also applied to the analysis of commercial formulations. In a cream and a solution for parenteral use, betamethasone highly decomposed under UVB irradiation, even in the presence of the bactericidal agents chlorocresol and phenol, which are able to absorb part of the incoming radiation. As a model for the UV exposed skin to which the drug is applied, ex vivo pig skin was used; not only the yield of photodegradation was evaluated, but the photoproducts were also identified. A test on THP-1 cells demonstrated the loss of anti-inflammatory activity of betamethasone, when modified by UVB light.

UVB photolysis of hydrocortisone 21-acetate.

Hydrocortisone 21-acetate (HCA) in methanol solution undergoes photodegradation under UVB light, as monitored by HPLC. Five main photoproducts have been isolated and characterized by means of NMR and mass spectroscopy. One of them derives from a Norrish I photoreaction which cleaves the C17-C20 bond of the steroid yielding the andro-derivative, a second product comes from a Yang-type photorearrangement which links C18 to C20 yielding a cyclobutane adduct. The former photoproduct, in turn, undergoes further photolysis giving rise to various photoproducts, of which three have been characterized. The first is a stereoisomer of the andro-derivative, the others arise from the opening of the five-membered ring. HCA also proved photounstable in the solid state and in a commercial formulation for topical use, thus confirming the requirements of the Pharmacopeias for light protection of this drug. Indeed, experiments on LPS-stimulated THP-1 cells demonstrated the loss of anti-inflammatory activity when HCA was UVB-photodegraded. The radical mechanism involved in HCA photolysis seems also responsible for the in vitro photohemolytic effect and lipid peroxidation induced by HCA in combination with UVB light.

Photoaddition of fluphenazine to nucleophiles in peptides and proteins. Possible cause of immune side effects.

By the action of UVA light, fluphenazine reacted with nucleophiles through a mechanism involving defluorination of its trifluoromethyl group, giving rise to carboxylic acid derivatives that were easily detected by electrospray mass spectrometry. This photoreaction took place with alcohols, sulphydryls, and amines. When irradiation of fluphenazine was carried out in the presence of an amino acid at pH 7.4, the alpha-amino group was covalently bound to the drug. With amino acids possessing a further nucleophilic residue on the side chain, such as lysine, tyrosine, and cysteine--but not serine--both groups reacted, resulting in a fluphenazine-amino acid-fluphenazine diadduct. The same occurred with the physiological peptide glutathione (gamma-glutamylcysteinylglycine). By means of MALDI mass spectrometry, it was shown that fluphenazine also covalently bound to peptides and proteins such as calmodulin. This binding may result in the formation of antibodies, ultimately leading to the destruction of the granulocytes and thus suggesting that photoactivation of this drug may play a role in its clinical side effects, such as agranulocytosis.

The mitochondrial effects of novel apoptogenic molecules generated by psoralen photolysis as a crucial mechanism in PUVA therapy.

The generation of photoproducts of psoralen (POPs) might be relevant in cell death induced by psoralen plus UVA, namely PUVA, which is a recognized effective treatment for cutaneous T-cell lymphoma, chronic graft-versus-host disease, and psoriasis. We investigated the occurrence of POP-induced cell death and the underlying mechanisms. POPs were produced by irradiating a psoralen solution with UVA. Jurkat cells treated in the dark with these mixtures died mainly through an apoptotic mechanism. POPs were separated by high-performance liquid chromatography (HPLC), and cells were added with each of these fractions. A total of 2 dimers of psoralen and 6-formyl-7-hydroxycoumarin (FHC) were identified in the apoptogenic fractions. Apoptosis was preceded by mitochondrial dysfunction caused by the opening of the mitochondrial permeability transition pore (PTP). In fact, both mitochondrial depolarization and cell death were prevented by the PTP inhibitor cyclosporin A (CsA). PTP opening was also documented in isolated mitochondria added with POP, suggesting that apoptosis is caused by a direct effect of POP on mitochondria. In fact, FHC alone induced PTP opening and CsA-inhibitable cell death of Jurkat cells, whereas nontransformed T lymphocytes were resistant. Along with identifying novel apoptogenic molecules, the present results indicate that POP generation directs transformed cells to apoptosis.

Mechanism of action of 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one, a very active angular furocoumarin-like sensitizer.

The molecular structure of 1,4,6,8-tetramethylfuro[2,3-h]quinolin-2(1H)-one (FQ), a recent furocoumarin-like photosensitizer, has been modified with the aim of reducing its strong genotoxicity, by replacing the methyl group at 4 position with a hydroxymethyl one, and so obtaining 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ). This modification gave rise to a strong reduction of lipophilicity and dark interaction with DNA. The formation of monoadducts (MA) was deeply affected, whereas the induction of bifunctional adducts between DNA and proteins (DPC(L>0)) was replaced by an efficient production of DNA-protein cross-links at zero length (DPC(L=0)), probably via guanine damage. Because of its angular molecular structure, HOFQ does not form interstrand cross-links (ISC): therefore, DPC(L=0) and MA represent the main lesions induced by HOFQ in DNA. In comparison with FQ (which induces MA and DPC(L>0)) and 8-methoxypsoralen (8-MOP) (MA, ISC, DPC(L>0)), HOFQ seems to be a more selective agent. In fact, contrary to FQ and 8-MOP, HOFQ, together with a noticeable antiproliferative activity, shows low levels of point mutations in bacteria and of clastogenic effects in mammalian cells. HOFQ is also an efficient apoptosis inducer, especially in comparison with 8-MOP, when tested at equitoxic experimental conditions; this property might be correlated with the complete HOFQ inability of inducing skin erythemas, a well-known side effect of classic furocoumarin photosensitization.

Mitochondria and plasma membrane as targets of UVA-induced toxicity of neuroleptic drugs fluphenazine, perphenazine and thioridazine.

In order to gain insights into the mechanism of phototoxicity of the neuroleptic drugs fluphenazine, perphenazine and thioridazine in cultured cells, studies were performed with murine 3T3 fibroblasts, aimed at identifying some cellular targets responsible for photoinduced cell death and possible cytotoxic reactive species involved in the photosensitization process. 3T3 fibroblasts incubated with 5 microM drugs and irradiated with UVA light (up to 8 J/cm2) underwent cell death, the extent of which depended on light dose. Of the three drugs, fluphenazine exhibited the highest phototoxicity and 100% cell death was achieved with a light dose of 5 J/cm2. Superoxide dismutase and alpha-tocopherol exerted a dose-dependent protective effect against drug phototoxicity, whereas N-acetylcysteine failed to do so. These findings indicate that superoxide anion and other free radical intermediates, generated in lipophilic cellular environments, play a role in photoinduced toxicity. Phototreatment of drug-loaded cells induces release of the cytosolic enzyme lactate dehydrogenase and causes loss of activity of mitochondrial NADH dehydrogenase, indicating that plasma membrane and mitochondria are among the targets of the phototoxicity of these drugs.

Photochemistry and phototoxicity of fluocinolone 16,17-acetonide.

Fluocinolone 16,17-acetonide is a corticosteroid used topically to treat various inflammatory skin diseases. Its photoreactivity was studied under UV-A and UV-B light in aqueous buffer in the presence of oxygen. This drug is photolabile under UV-B light and, to a lesser extent, under UV-A light, which is absorbed far less. In phosphate buffer, approximately 80% of fluocinolone acetonide decomposes after 5 J/cm2 of UV-B irradiation, whereas under 30 J/cm2 of UV-A light approximately only 20% decomposes. Both the drug and its photoproducts have been evaluated through a battery of in vitro studies and found to cause photohemolysis and induce photodamage to proteins (erythrocyte ghosts, bovine serum albumin) and linoleic acid. In addition, one of the photoproducts (the 17-hydroperoxy derivative) is highly toxic in the dark. Therefore, both loss of therapeutic activity and light-induced adverse effects may be expected when patients expose themselves to sunlight after drug administration. A major mechanism for phototoxicity involves radicals forming from drug breakdown, at least under UV-B, although reactive oxygen species may play a role, particularly under UV-A.

1-Thioangelicin: crystal structure, computer-aided studies and photobiological activity.

1-Thioangelicin is a furocoumarin analog synthesized to investigate the role of the substitution of sulfur for oxygen in the parent compound angelicin. The compound was examined by X-ray diffraction, and its interaction with DNA, both in the dark and by UVA irradiation, studied by means of linear flow dichroism, chromatography and (1)H NMR. Further insight into the steric and electronic features of 1-thioangelicin has been reached through theoretical calculations, including molecular mechanics optimization, docking studies and frontier molecular orbital investigations. The experimental data indicate that thioangelicin is able to intercalate in the DNA helix and subsequent irradiation yields a cis-syn adduct, in agreement with theoretical calculations within the lower/higher singly occupied molecular orbital formalism. Antiproliferative activity has been assessed on Balb/c 3T3 cultured cells.

In vitro phototoxic properties of triamcinolone 16,17-acetonide and its main photoproducts.

The phototoxicity of triamcinolone 16,17-acetonide has been estimated through a panel of in vitro tests. The main target involved in phototoxicity induced by triamcinolone appeared to be the cell membrane. Oxygen-independent photohemolysis was observed. A photochemical study in water and buffered solutions supported the conclusion that this is related to the action of radicals formed upon UV irradiation (in particular UV-B) by Norrish Type-I fragmentation of the C-20 ketone group. Peroxy radicals were formed in the presence of oxygen and were the active species in that case. Three photoproducts, isolated from the photodegradation of the drug, were submitted to the same toxicity tests. Two of them were proved to possess toxic or phototoxic properties on erythrocytes, primarily induced by UV-B light, and may participate in the photosensitizing activity of triamcinolone 16,17-acetonide. Our in vitro results suggest that the drug can elicit weak photosensitizing properties in vivo.

Topically applied vitamin C and cysteine derivatives protect against UVA-induced photodegradation of suprofen in ex vivo pigskin.

Exposure of the nonsteroidal anti-inflammatory drug suprofen (SUP) to UV-radiation results in the formation of radicals, reactive oxygen species (ROS), photodecarboxylated products and photoadducts with biomacromolecules. Using an ex vivo pigskin explant model, we investigated whether topical coapplication of the water-soluble antioxidants vitamin C (Lascorbic acid, ASC), N-acetyl-L-cysteine (NAC) or L-cysteine ethylester (CYSET) with SUP reduced ultraviolet A (UVA)-induced decomposition of SUP. UVA-induced changes in antioxidant bioavailability in the stratum corneum and epidermis were also studied. Epidermal bioavailability of SUP in sham-irradiated pigskin increased 2.2- to 4.1-fold after the lowest antioxidant doses (P < 0.05). As compared with no applied antioxidant, increasing doses of all tested antioxidants resulted in increased levels of SUP and decreased levels of photoproducts (P < 0.05). A maximal protection against SUP photodegradation of 70% was found after an ASC dose of 1 micromol/cm2; these values were 60% for a NAC dose of 10 micromol/cm2 and 50% for a CYSET dose of 5 micromol/cm2. Skin antioxidant levels increased with increasing applied dose (P < 0.05); the bioavailability of CYSET was approximately three-fold lower than that of ASC and NAC. UVA exposure resulted in 30-50% consumption of the topically applied ASC or NAC in the stratum corneum, whereas CYSET was not consumed. In conclusion, the topically applied water-soluble antioxidants ASC, NAC and CYSET protect against UVA-induced decomposition of SUP by scavenging radicals and ROS. Coapplication of these antioxidants may therefore be an effective way to reduce or prevent the phototoxic effects of SUP in vivo.

Photoisomerization of fluvoxamine generates an isomer that has reduced activity on the 5-hydroxytryptamine transporter and does not affect cell proliferation.

Fluvoxamine, a selective serotonin re-uptake inhibitor, is used as antidepressant/anxiolytic. The presence of a C=N double bond in the structure of fluvoxamine implies the existence of two geometric isomers: E- (trans) and Z- (cis), and suggests the hypothetical susceptibility of the molecule to photoisomerization. Clinically effective fluvoxamine is in its trans form. UVB (ultraviolet light, class B, wavelength range 290-320 nm) irradiation of aqueous solutions of fluvoxamine generated a photoproduct, which was isolated and analyzed by nuclear magnetic resonance (NMR) and mass spectrometry (MS), and identified as the cis isomer of fluvoxamine. This cis-isomer lost capacity to inhibit serotonin uptake, suggesting that light exposure might reduce the clinical efficacy of fluvoxamine. Alternatively, the photoproduct could be used as an inactive isomer in the studies of antidepressant mechanisms. Recent proposal suggests that antidepressants increase neurogenesis in the adult brain, whereas either an inhibitory or a stimulatory action of antidepressants on [(3)H]thymidine uptake in vitro has been attributed to their interaction with serotonergic mechanisms. Lower concentrations (i.e., 2 microM) of fluvoxamine and fluoxetine (another selective serotonin re-uptake inhibitor) stimulated [(3)H]thymidine uptake in mature, but inhibited it in immature cultures of rat cerebellar granule cells; the photoproduct was ineffective. A high concentration of fluvoxamine (i.e., 20 microM) but not the photoproduct was toxic to both immature and mature cultures. We suggest that a mechanism sensitive to fluvoxamine photoisomerization might be involved in the action of antidepressants on cell proliferation.

PUVA-induced apoptosis involves mitochondrial dysfunction caused by the opening of the permeability transition pore.

The mechanism of cell death was investigated in Jurkat cells exposed to the combination of psoralen and UVA irradiation (PUVA). Apoptosis was by far prevailing over necrosis and involved mitochondrial dysfunction. The collapse of mitochondrial membrane potential, appears to be caused by the opening of the mitochondrial permeability transition pore since its inhibitor, cyclosporin A, prevented mitochondrial dysfunction and largely attenuated apoptosis. Apoptosis also occurred in cells treated with the photoproducts generated by irradiating psoralen in vitro with an oxygen-dependent process. Thus, the involvement of reactive oxygen species in the onset of PUVA-induced apoptosis appears mostly related to psoralen photooxidation.

Photobiological properties of hydroxy-substituted flavothiones.

Flavothione (FT) and a series of 18 hydroxy- and methoxy-substituted flavothiones were screened for photobiological activity. The 5-hydroxy-substituted compounds (group 3) and the methoxy-substituted flavothiones were inactive. FT and the remaining hydroxy-substituted compounds, all displayed photobiological activity. Among these, the 3-hydroxy-substituted compounds (group 2) were the most efficient photosensitizers overall in spite of their concurrent fast photodegradation. FT and all other hydroxyflavothiones, not substituted in the 3- or 5-positions (group 1), were inefficient compared with group 2. Detailed photobiological tests were carried out for four flavothiones of groups 1 and 2. The biological tests included fungi, several strains of Escherichia coli, Salmonella typhimurium and mammalian cells. In addition, the ability of these flavothiones to perform lipid peroxidation was evaluated. FT and 6-hydroxyflavothione (group 1) induce DNA damage via H-atom abstraction from the lowest n, pi* triplet state of the thione (oxygen independent). For 3-hydroxy and 3,6-dihydroxyflavothione (group 2), both DNA and the membrane are targets. The mechanism likely involves both energy transfer and electron transfer from the lowest pi, pi* triplet state to oxygen, to form singlet oxygen and the superoxide anion. Some of these compounds could be considered as models for environmentally safe photopesticides.

Furocoumarin photolysis: chemical and biological aspects.

Furocoumarins (psoralens) undergo photolysis when subjected to UVA radiation in solution. Several products are formed, depending on the molecular structure and experimental conditions. Some products are the result of anoxic mechanisms (cyclodimerisation, addition of solvent), others of oxic pathways, leading to oxidised products. The mechanisms thought to underlie photolysis are described and the possible biological relevance of the photoproducts and intermediates is discussed.