UV photolysis of catalase revisited: a spectral study of photolytic intermediates

The 365-nm irradiation of 4.6 microM (approximately equal to 1.1 mg/ml) catalase solutions in pH 7.4 phosphate buffer induces spectral modifications. Difference spectra show maxima at 434, 555, 584 nm at the beginning of the irradiation, then a final spectrum with a maximum at 568 nm and a shoulder at 530 nm is observed. These results suggest the formation of compound III (oxyferrous catalase) and compound II, respectively. In deaerated 0.1 M, pH 8.7 borate buffer, the ferrous catalase is characterized by maxima at 563 and 594 nm. Hydrogen donors such as ethyl alcohol, formate and p-cresol inhibit, but citrate ions enhance the formation of these intermediates. A mechanism involving Fe(III) reduction according to an internal electron transfer is proposed.     

An insight into the mechanisms of the phototoxic response induced by cyamemazine in cultured fibroblasts and keratinocytes

The phototoxicity of cyamemazine (CMZ, Tercian), a neuroleptic of the phenothiazine family, has recently been reported in humans. CMZ has an absorbance maximum at 267 nm (molar absorptivity, 25,800 M(-1) cm(-1)) but a weaker molar absorptivity in the ultraviolet A (UV-A) region. CMZ exhibits a fluorescence with maximum emission at 535 nm and a quantum yield of 0.11. CMZ is a powerful photosensitizing agent toward HS 68 human skin fibroblasts and NCTC 2544 keratinocytes. At a UV-A radiation dose of 10 J/cm2, innocuous to cells in the absence of CMZ, the LD50 (lethal dose corresponding to 50% killing) are 0.5 and 1 microM for the fibroblasts and the keratinocytes, respectively, after overnight incubation with the drug. Short incubation times do not significantly alter the LD50. The CMZ-induced phototoxicity is accompanied by lipid membrane peroxidation consistent with the amphiphilic character of this photosensitizer. Keratinocytes are an order of magnitude less sensitive to the photosensitized lipid peroxidation than fibroblasts. Microspectrofluorometry reveals that lysosomal membranes are major sites of CMZ incorporation into the two cell lines because a Forster-type resonance energy transfer process occurs from CMZ to LysoTracker Red DND99 (LTR), a specific fluorescent probe of lysosomal membranes. The CMZ-photosensitized destruction of LTR demonstrates that CMZ retains its photosensitizing capacity after its lysosomal uptake.     

One-electron reduction of superoxide radical-anions by 3-alkylpolyhydroxyflavones in micelles. Effect of antioxidant alkyl chain length on micellar structure and reactivity

In micellar solutions, one-electron reduction of (*)O 2 (-) radical-anions by 3-alkylpolyhydroxyflavones (FnH) with alkyl chains of n = 1, 4, 6, 10 carbons produces phenoxyl radicals ( (*)Fn) identical to those obtained by one-electron oxidation by (*)Br 2 (-) radical-anions or by repair of tryptophan radicals. In cetyltrimethylammonium bromide (CTAB), F1H localizes in the Stern layer, and alkyl chains of other FnH solubilize in the hydrophobic interior, interacting with cetyl tails. This interaction produces more compact micelles with lower intramicellar fluidity, as suggested by the increase in the pseudo-first-order rate constant of (*)Fn formation ( k 1) from approximately 390 s (-1) for n = 1 to 610 s (-1) for n = 10, leading to an intramicellar bimolecular rate constant of 1 x 10 (5) M (-1) s (-1). Additionally, (*)F1 and (*)F4 decay by intermicellar bimolecular reaction (2 k = 20 and 2 x 10 (5) M (-1) s (-1), respectively) whereas other (*)Fn radicals are stable over seconds due to increased localization with regards to the Stern layer. In contrast, the thick uncharged hydrophilic palisade layer and the compact hydrophobic core of Triton X100 micelles are responsible for a much higher microviscosity resulting in a decrease in k 1 from approximately 15.6 s (-1) for n = 1 to 9.6 s (-1) for n = 10.     

Synthesis, cellular uptake of, and cell photosensitization by a porphyrin bearing a quinoline group

A tetraphenyl porphine linked to a 7-chloroquinoline (5,10,15,20-tetraphenyl-1-3-[4-(4-aminobutyl)7-chloroquinoline] propioamidoporphine, TPPQ) was synthesized and examined as a potential photosensitizer for photodynamic therapy (PDT) of proliferative diseases. With respect to haematoporphyrin, TPPQ is a good in vitro photodynamic sensitizer producing singlet oxygen in 1% Triton X100 solutions. As with other hydrophobic porphyrins used in PDT, blood lipoproteins strongly bind TPPQ. Thus one low density lipoprotein (LDL) can incorporate 25 TPPQ molecules and 17 TPPQ molecules are taken up by one high density lipoprotein (HDL). Cell delivery of TPPQ using HDL or human serum albumin (HSA) as carrier is rather weak. However, an efficient TPPQ delivery to human skin fibroblasts is observed, partly aided by receptor-mediated endocytosis of LDL. Fluorescence spectroscopy shows that the cellular localization of TPPQ is both carrier and time dependent. During its delivery with LDL, TPPQ does not significantly impair the endocytosis of LDL-receptor complexes. After delivery with LDL, TPPQ is as efficient as other haematoporphyrin derivatives used in the PDT of cancers in photosensitizing human skin fibroblasts.     

Alteration of the endocytotic pathway by photosensitization with fluoroquinolones

The endocytotic pathway is profoundly altered by the UVA-induced photosensitization of HS 68 fibroblasts by the fluoroquinolone (FQ) antibiotics lomefloxacin, BAYy 3118, norfloxacin and ciprofloxacin, which preferentially localize in lysosomes. The endocytosis of low-density lipoproteins (LDL) loaded with two carbocyanine dyes compatible for effective Forster-type resonance energy transfer (FRET), namely 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) as the donor and 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as the acceptor, has been used as a model system. Binding of LDL to their cell surface receptors is impaired by irradiation with 10 J cm(-2) of UVA and/or treatment with 250 microM BAYy 3118 during 2 h. Perturbation of the plasma membrane by the FQ is revealed by the change in the rate of exchange of DiO from the LDL to the cell membrane as compared to untreated cells. The lysosomal degradation of LDL, demonstrated by the disappearance of FRET between DiO and DiI, is partly inhibited by the FQ. The actin filament network, involved in the fusion of mature endosomes with lysosomes, is readily destroyed upon photosensitization with the four FQ. However, actin depolymerization can be avoided by incubation of the cells with trans-epoxysuccinyl-1-leucylamido-(4-guanidino)butane, an inhibitor of lysosomal cathepsins prior to FQ photosensitization. All these data suggest that several components of the endocytotic pathway are impaired by photosensitization with these FQ.     

Structure-activity relationships in hydroxy-2,3-diarylxanthone antioxidants. Fast kinetics spectroscopy as a tool to evaluate the potential for antioxidant activity in biological systems

A structure-activity relationship has been established for eight hydroxy-2,3-diarylxanthones (XH) bearing hydroxy groups on the two aryl rings. One-electron oxidation by superoxide radical-anions (˙O(2)(-)) and ˙Trp radicals as well as reaction with ˙CCl(3)O(2) and ˙CHCl(2)O(2) radicals demonstrates that two OH groups are required for efficient antioxidant reactivity in cetyltrimethylammonium bromide micelles. Hydroxy groups at the meta and para positions on either of the two phenyl rings confer enhanced reactivity, but XH bearing an OH at the para position of either phenyl ring is unreactive. While oxidation is favoured by OH in both meta and para positions of 2-aryl xanthone substituents, addition of a third and/or fourth OH enhances electron-donating capacity. In Cu(2+)-induced lipid peroxidation of human LDL, the lag period preceding the commencement of lipid peroxidation in the presence of XH bearing OH at meta and para positions on the 3-phenyl ring is extended to twice that observed with a comparable concentration of quercetin, a reference antioxidant. These antioxidants are also superior to quercetin in protecting human skin keratinocytes against tert-butylhydroperoxide-induced oxidative stress. While XH antioxidant activity in model biological systems is consistent with the structure-activity relationship, their response is also modulated by the localization of XH and by structural factors.     

The role of the low density lipoprotein receptor pathway in the delivery of lipophilic photosensitizers in the photodynamic therapy of tumours

Lipoproteins are now recognized as major blood carriers of many hydrophobic porphyrins and related chromophores which are being investigated as possible photosensitizers in the photodynamic therapy of tumours. In vitro and in vivo studies have demonstrated the role of the low density lipoprotein (LDL) receptor pathway in the delivery of photosensitizers to tumour cells and its importance in porphyrin accumulation by tumours. Lysosomes, which are involved in the cellular processing of LDL, are important intracellular targets in the LDL-porphyrin-induced phototoxicity. The use of the LDL receptor pathway as a tool for enhancing the selectivity of photosensitizer delivery to tumour cells appears to be a promising field of research in the photodynamic therapy of tumours.     

Riboflavin photodegradation and photosensitizing effects are highly dependent on oxygen and ascorbate concentrations

Riboflavin (RF) is a normal component of the eye lens which triggers a strong photosensitizing activity when exposed to light. Upon irradiation with short wavelength radiations below 400 nm, RF-photosensitized damage may occur. However, vitamin C is present at high concentrations in the normal lens and plays an important role in inhibiting these photosensitization processes. An in vitro simple model was used with the objective of understanding better the relationships between vitamin C and oxygen concentrations on the mechanisms of RF-mediated photodegradation of tryptophan (Trp), a target particularly sensitive to photo-oxidation. Under nitrogen, the RF decomposition reached its maximal value, and vitamin C and Trp photo-oxidation was negligible. When increasing oxygen pressure, RF photodegradation dropped and vitamin C photo-oxidation strongly increased and was maximal at 100% O2. RF-induced photodegradation of Trp first increased with oxygen concentration, up to 40 microM O2, and then decreased. RF and Trp degradation were significantly protected by vitamin C so that no more than 20% of the substrates concentration were oxidized in the presence of vitamin C higher than 0.8 mM. From our results we conclude that in the specific conditions of the normal lens, the high vitamin C concentration (2 mM) is compatible with the UVA radiation hazard, despite the presence of RF. However, if lenticular vitamin C decreases below 0.8 mM, photodegradation of RF may occur and Trp may therefore be photo-oxidized by a Type-I mechanism.     

Phototoxic properties of perfumes containing bergamot oil on human skin: photoprotective effect of UVA and UVB sunscreens

As part of an international cooperative study of the photophysical, photomutagenic and photocarcinogenic properties of bergamot oil and the effect of UVA and UVB sunscreens, the phototoxic properties of model perfumes containing 5, 15 and 50 ppm 5-methoxypsoralen (5-MOP) in bergamot oil with and without a sunscreen have been investigated on human skin. It has been confirmed that the photosensitivity of human skin is maximal 2 h after perfume application. Interestingly the addition of a UVA sunscreen is more efficient for decreasing the phototoxic properties of bergamot oil than is a UVB sunscreen. The addition of sunscreens in a model perfume containing 50 ppm 5-MOP in bergamot oil can reduce the phototoxic properties of this perfume to a toxicity equivalent to that produced by the application of a model perfume containing 15 ppm 5-MOP without sunscreens. However, despite their promising protective effect in vitro, UVB and UVA sunscreens at low concentration (0.5%-1%) in perfumes cannot suppress the phototoxicity of bergamot oil on human skin.     

Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: evaluation and modification of risk of human use in an albino mouse skin model

The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle (i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations. Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations. Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.