Photosensitisation by voriconazole-N-oxide results from a sequence of solvent and pH-dependent photochemical and thermal reactions.

The phototoxicity of voriconazole (VN) prescribed in the treatment of severe fungal infections is frequently reported. Its major metabolite, a N-oxide derivative (VNO), was suspected to be the photosensitizer because it shows a maximum absorbance at ~310 nm in aqueous solutions. It was reported that the VNO photoproduct (VNOP) was phototoxic to human keratinocytes. Steady state and laser flash photolyses were performed to shed light on the phototoxic properties of VNO and VNOP. The quantum yield of the VNOP production by UVB-UVA light in buffered or alcoholic solutions is 0.6. VNOP has been identified as (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-7-oxa-1,3-diazabicyclo[4.1.0]hepta-2,4-dien-4-yl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol. VNOP undergoes a marked thermal degradation and an efficient UVA photolysis with well differentiated kinetics and end-products. The temperature-dependent VNOP dark degradation produces a single product VNOPD identified as 6-[(2S,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-5-fluoropyrimidin-4-ol with absorbance maximum at 308 nm and ε = 2700 M-1 cm-1. Under UVB-UVA irradiation, VNOPD, the stable end-product, is a remarkable photodynamic photosensitizer towards Trp and His. The Trp photo-oxidation (Φox(Trp) = 0.13) mainly involves type I radical reactions whereas His is oxidized by 1O2 (Φox(His) = 0.012). These results force us to question the validity of the in vitro photosensitization of human keratinocytes by VNO and VNOP previously reported.

A molecular insight into the phototoxic reactions observed with vemurafenib, a first-line drug against metastatic melanoma.

The electronic properties of vemurafenib (VB) provide a rational basis for understanding its strong UVA-induced phototoxicity. Thus, solvation of hydrophobic VB by hydrogen bonding solvents controls its photophysical, photochemical and photosensitizing properties. Addition of phosphate buffered saline (PBS) to methanol (MeOH) induces a bathochromic shift of the VB absorbance spectrum and a fluorescence emission (λmax = 450 nm, quantum yield (Φ) = 0.011). Phosphorescence (λmax = 461 nm) is observed at 77 K in MeOH. 308 nm laser flash spectroscopy demonstrates that the lifetimes (τ) and quantum yields of the VB triplet state ((3)T(*)(1)) in deaerated MeOH (τMeOH = 0.41 µs, λmax ∼ 380 nm), MeOH-PBS and HSA solutions markedly depend on the microenvironment. A long-lived radical (half-life >200 µs) is also formed. The state (3)T(*)(1) is quenched by O2 and electron donors (Cys and 2'-deoxyguanosine) at a rate constant >1 × 10(9) M(-1) s(-1). UVA-irradiation of VB in air-saturated MeOH or MeOH-PBS solutions produces a UVA-absorbing photoproduct (Φ âˆ¼ 5 × 10(-4)). VB photosensitizes Trp destruction by type I (radical formation) and type II (singlet oxygen ((1)O2) formation) photodynamic reactions (Φ = 0.005). Singlet oxygen production is further demonstrated by the VB-photosensitized His oxidation (ΦMeOH = 0.006).

Chemistry of free radicals produced by oxidation of endogenous α-aminoketones. A study of 5-aminolevulinic acid and α-aminoacetone by fast kinetics spectroscopy.

BACKGROUND: Excess 5-aminolevulinic acid (ALA) and α-aminoacetone (AA) are implicated in ketosis, porphyrinpathies and diabetes. Pathologic manifestations involve O2⁻, H2O2, OH, enoyl radicals (ALA and AA) and their oxidation end products. METHODS: To characterize enoyl radicals resulting from reaction of OH radicals with ALA and AA, micromolar OH concentrations were produced by pulse radiolysis of ALA and AA in aqueous solutions. RESULTS: ALA and AA react with OH at k=1.5 × 109 M⁻¹s⁻¹. At pH7.4, the ALA absorbance spectrum has a maximum at 330 nm (ε=750 M⁻¹cm⁻¹). This band appears as a shoulder at pH8.3 where two ALA species are present: (NH3)⁺-CH2-CO-CH2-CH2-COO⁻ and NH2-CH2-CO-CH2-CH2-COO⁻ (pKa=8.3). At pH8.3, ALA reacts with oxygen (k=1.4 × 108 M⁻¹s⁻¹) but not with O2⁻. At pH8.3, AA oxidation produces two AA species characterized by an absorbance spectrum with maxima at 330 and 450 nm. ALA and AA are repaired by antioxidants (quercetin (QH), catechin, trolox, ascorbate) which are semi-oxidized (k>10(8)M⁻¹s⁻¹). QH bound to HSA or to apoferritin and ferritin repairs ALA and AA. In O2-saturated apoferritin solutions, Q, O2⁻, AA and reaction product(s) react with QH. CONCLUSIONS: The optical absorption properties and the time evolution of ALA and AA were established for the first time. These radicals and their reaction products may be neutralized by antioxidants free in solution or bound to proteins. GENERAL SIGNIFICANCE: Adjuvant antioxidant administration may be of interest in pathologies related to excess ALA or AA production.

On the repair of oxidative damage to apoferritin: a model study with the flavonoids quercetin and rutin in aerated and deaerated solutions.

Ferritin (Ft) impairment through (•)O2(-), H2O2, and (•)OH production occurs in the cases of ketoses, diabetes mellitus, acute intermittent porphyria and tyrosinemia. In addition to (•)Trp and TyrO(•) radical production, ferrous iron liberation and Ft synthesis stimulation, site-specific oxidation reactions are induced leading to toxic iron accumulation in organs with high Ft content, for example, liver and brain. To elucidate the potential pathways to Ft recovery, repair of oxidative damage to horse spleen apoferritin (apoFt) and Ft by quercetin (QH) or rutin (RH) was studied in the presence and absence of oxygen. (•)Trp and TyrO(•) radicals were produced in pulse radiolysis through apoFt oxidation by (•)Br2(-) radicals. QH and RH bind to apoFt on eight sites with binding constants of ˜80,000 and ˜32,000 M(-1), respectively. In deaerated solutions, a repair of apoFt radicals is observed involving both bound and free flavonoids. This repair occurs by a fast intra- and a slow inter-molecular electron transfer from bound and free flavonoids, respectively. With QH, the rate constants are 10(4) s(-1) and 3.5 × 10(7) M(-1) s(-1) for the intra- and intermolecular repair reactions, respectively. Oxygen does not interfere with repair of apoFt or Ft by bound QH but inhibits 90% of Ft repair by RH. These results taken together indicate that flavonoid antioxidants may help alleviate Ft impairment in diseases involving an oxidative stress.

Endoplasmic reticulum and Golgi apparatus are the preferential sites of Foscan localisation in cultured tumour cells.

Intracellular photosensitiser localisation significantly influences the mechanism of response to photodynamic therapy (PDT), since the primary sites of damage are closely related to the specific sensitiser distribution. Foscan subcellular localisation in the MCF-7 human adenocarcinoma cell line has been studied by means of confocal microscopy and microspectrofluorometry. The fluorescence topographic profiles recorded after cells costained with Foscan and organelle-specific fluorescent probes revealed that Foscan presents low localisation in lysosomes and a weak accumulation in mitochondria. Alternatively, the Foscan fluorescence topographic profile turned out to colocalise perfectly with that obtained for the endoplasmic reticulum (ER) and the Golgi apparatus. The patterns of fluorescence derived from confocal microscopy studies were consistent with predominant localisation of Foscan in these organelles. Furthermore, evaluation of enzymatic activity of selected organelles immediately after laser light irradiation (650 nm) indicated the Golgi apparatus and ER as the primary damaged sites resulting from Foscan-mediated PDT in the MCF-7 cell line. To our knowledge, this is the first study to demonstrate unambiguously that the ER and the Golgi apparatus are preferential sites of Foscan accumulation.

Redox reactions of the urate radical/urate couple with the superoxide radical anion, the tryptophan neutral radical and selected flavonoids in neutral aqueous solutions.

The kinetics of several processes involving the potential antioxidant role of urate in physiological systems have been investigated by pulse radiolysis. While the monoanionic urate radical, .UH-, can be produced directly by oxidation with .Br2- or .OH, it can also be generated by oxidation with the neutral tryptophan radical, .Trp, with a rate constant of 2 x 10(7) M-1s-1. This radical, .UH-, reacts with .O2- with a rate constant of 8 x 10(8) M-1s-1. Also, .UH- is reduced by flavonoids, quercetin and rutin in CTAB micelles at rate constants of 6 x 10(6) M-1s-1 and 1 x 10(6) M-1s-1, respectively. These results can be of value by providing reference data useful in further investigation of the antioxidant character of urate in more complex biological systems.

Flavonoids and urate antioxidant interplay in plasma oxidative stress.

Flavonoids are naturally occurring plant compounds with antioxidant properties. Their consumption has been associated with the protective effects of certain diets against some of the complications of atherosclerosis. Low-density lipoprotein (LDL) oxidative modification is currently thought to be a significant event in the atherogenic process. Most of the experiments concerning the inhibition of LDL oxidation used isolated LDL. We used diluted human whole plasma to study the influence of flavonoids on lipid peroxidation (LPO) promoted by copper, and their interaction with uric acid, one of the most important plasma antioxidants. Lipid peroxidation was evaluated by the formation of thiobarbituric acid reactive substances (TBARS) and of free malondialdehyde (MDA). The comparative capability of the assayed flavonoids on copper (II) reduction was tested using the neocuproine colorimetric test. In our assay system, urate disappears and free MDA and TBARS formation increase during the incubation of plasma with copper. Most of the tested flavonoids inhibited copper-induced LPO. The inhibition of LPO by flavonoids correlated positively with their capability to reduce copper (II). The urate consumption during the incubation of plasma with copper was inhibited by myricetin, quercetin and kaempferol. The inhibition of urate degradation by flavonoids correlated positively with the inhibition of LPO. Urate inhibited the copper-induced LPO in a concentration-dependent mode. Luteolin, rutin, catechin and quercetin had an antioxidant synergy with urate. Our results show that some flavonoids could protect endogenous urate from oxidative degradation, and demonstrate an antioxidant synergy between urate and some of the flavonoids.

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.

Interactions of superoxide anion with enzyme radicals: kinetics of reaction with lysozyme tryptophan radicals and corresponding effects on tyrosine electron transfer.

The kinetics of O2*- reaction with semi-oxidized tryptophan radicals in lysozyme, Trp*(Lyz) have been investigated at various pHs and conformational states by pulse radiolysis. The Trp*(Lyz) radicals were formed by Br2*- oxidation of the 3-4 exposed Trp residues in the protein. At pH lower than 6.2, the apparent bimolecular rate is about 2 x 10(8) M(-1) s(-1); but drops to 8 x 10(7) M(-1) s(-1) or less above pH 6.3 and in CTAC micelles. Similarly, the apparent bimolecular rate constant for the intermolecular Trp*(Lyz) + Trp*(Lyz) recombination reaction is about (4-7 x 10(6) M(-1) s(-1)) at/or below pH 6.2 then drops to 1.3-1.6 x 10(6) M(-1) s(-1) at higher pH or in micelles. This behavior suggests important conformational and/or microenvironmental rearrangement with pH, leading to less accessible semi-oxidized Trp* residues upon Br2*- reaction. The kinetics of Trp*(Lyz) with ascorbate, a reducing species rather larger than O2*- have been measured for comparison. The well-established long range intramolecular electron transfer from Tyr residues to Trp radicals--leading to the repair of the semi-oxidized Trp*(Lyz) and formation of the tyrosyl phenoxyl radical is inhibited by the Trp*(Lyz) + O2*- reaction, as is most of the Trp*(Lyz) + Trp*(Lyz) reaction. However, the kinetic behavior of Trp*(Lyz) suggests that not all oxidized Trp residues are involved in the intermolecular recombination or reaction with O2*-. As the kinetics are found to be quite pH sensitive, this study demonstrates the effect of the protein conformation on O2*- reactivity. To our knowledge, this is the first report on the kinetics of a protein-O2*- reaction not involving the detection of change in the redox state of a prosthetic group to probe the reactivity of the superoxide anion.

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.

Contrasting effects of excess ferritin expression on the iron-mediated oxidative stress induced by tert-butyl hydroperoxide or ultraviolet-A in human fibroblasts and keratinocytes.

Iron and/or ferritin accumulation are known to occur under pathological conditions in many inflammatory skin diseases or in human skin chronically exposed to UV light. Under such conditions, ferritin is believed to play an effective protective role in accommodating and 'deactivating' excess 'free' iron produced by the inflammatory process or the UV illumination. The present study compares the relationship between ferritin over-expression and effects of an oxidative stress induced chemically by tert-butyl hydroperoxide or photochemically by UV-A radiation. As shown by immunoassay, cultured MRC 5 and HS 68 fibroblasts treated for at least one day with transferrin or overnight with non-toxic concentrations of the ferric nitrilotriacetate complex express up to 10 times more ferritin than untreated cells, whereas a five-fold increase is obtained with NCTC 2544 keratinocytes. In all cases a parallel increase in soluble cellular iron is measured by inductive plasma emission spectroscopy. The superoxide dismutase and catalase activities and total glutathione levels are not modified by the iron treatment, whereas a transient increase in the Se-dependent glutathione peroxidase activity of keratinocytes is observed after a short incubation with the iron complex. In keratinocytes and fibroblasts, ferritin over-expression after iron treatment markedly inhibits lipid peroxidation but, paradoxically, not the mortality induced by tert-butyl hydroperoxide. In contrast, this excess ferritin does not protect cells from both the peroxidation and mortality induced by moderate doses (30 J/cm2) of UV-A radiation. As a consequence, protection against oxidative damage by excess ferritin synthesis clearly depends on the nature of the oxidative stress on cell targets and it seems to be of lesser importance in the case of photochemically induced oxidation.

Inactivation of iron responsive element-binding capacity and aconitase function of iron regulatory protein-1 of skin cells by ultraviolet A.

The ultraviolet-A (UVA) component of sunlight produces in cutaneous cells a highly toxic oxidative stress mediated by redox cycling reactions of Fe ions. A tight regulation of cell iron uptake and storage by iron regulatory proteins (IRP) of keratinocytes and fibroblasts avoids these damaging reactions. We report here that about 40 J/cm2 of UVA are required to inactivate half of the binding capacity of apo-IRP-1 to iron responsive elements (IRE) of RNA whereas 15 J/cm2 already inhibit half of the holo-IRP-1 aconitase activity. No increase in the holo-IRP-1 activity is observed during the apo-IRP-1 photoinactivation suggesting that UVA does not trigger a shift between these two forms. As opposed to holo-IRP-1, which contains a 4Fe-4S cluster, apo-IRP-1 has no UVA chromophore. Thus it should be inactivated indirectly by reactive oxygen species generated by the UVA-induced endogenous photo-oxidative stress. The apo-IRP-1 photoinactivation is weakly prevented by the lipophilic oxyradical scavenger vitamin E but not by the hydrophilic azide anion, a singlet oxygen quencher or by diethyldithiocarbamate, a superoxide dismutase inhibitor. However, full protection against photoinactivation of the apo form is observed after incubation with N-acetylcysteine but the latter only partially protects the aconitase function of the holo-IRP-1 from photoinactivation. The marked difference in the kinetics of photoinactivation of the apo and holo forms, the light dose-independent effect of the sulfhydril group reagent, 2-mercaptoethanol and the partial protection brought by the ferric ion complexing agent desferrioxamine suggest that the photochemistry of the 4Fe-4S cluster of the holo form plays little, if any, role in the photoinactivation of the apo-IRP-1/IRE interaction. It is concluded that the apo/holo equilibrium is irreversibly destroyed by UVA irradiation.

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.

Damage to mitochondria of cultured human skin fibroblasts photosensitized by fluoroquinolones.

The phototoxic fluoroquinolones ofloxacin, lomefloxacin, norfloxacin, ciprofloxacin and BAYy 3118 have ionizable groups with pKa values close to neutrality. Different ionic species of these fluoroquinolones, therefore, partition in various compartments and organelles of living cells according to their ionic equilibria. While all these fluoroquinolones accumulate in lysosomes, they more or less stain the rest of the cytoplasm of living HS 68 fibroblasts. As a result, photosensitized damage to other cytoplasmic sites than lysosomes can also be expected. Using microfluorometry and rhodamine 123 (Rh 123) as a specific fluorescent probe which is released from mitochondria by light absorption, we show that under ultraviolet A (UVA) irradiation norfloxacin and ciprofloxacin readily damage mitochondrial membranes. as evidenced by the UVA dose-dependent strongly accelerated release of Rh 123 from mitochondria in cells treated with norfloxacin and ciprofloxacin. Damages are already noticeable at UVA doses as low as 2 J/cm2. By contrast, no such photoinduced damage can be observed with ofloxacin, lomefloxacin and BAYy 3118, the latter being the most phototoxic derivative towards HS 68 fibroblasts. The initial photodamage induced by norfloxacin and ciprofloxacin can then propagate after the irradiation as shown by the strongly increased rate of release of Rh 123 from mitochondria of cells that have been incubated with these two fluoroquinolones and left in the dark after a pre-irradiation with 18 J/cm2 of UVA. Interestingly, the same pre-irradiation after cells have been treated with BAYy 3118 and lomefloxacin induces similar post-irradiation effects, although they have no apparent immediate photosensitizing action on mitochondria.

Role of nuclear factor-kappa B in colon cancer cell apoptosis mediated by aminopyropheophorbide photosensitization.

Aminopyropheophorbide (APP) is a second generation of photosensitizer for photodynamic therapy (PDT). We demonstrated that APP strongly absorbed red light and, after being taken up by colon cancer cells (HCT-116 cells), was localized in cytoplasmic and internal membranes but not in mitochondria. The APP-mediated photosensitization was cytotoxic for HCT-116 cells through an induction of apoptosis. Indeed, DNA fragmentation (DNA laddering and terminal deoxyuridine nick-end labeling) and chromatin condensation (4',6-diamidine-2'-phenylindole staining) could be visualized soon after photosensitization. Because nuclear factor (NF)-kappa B is involved in the response to many photosensitizers, we also demonstrated its nuclear translocation in two waves: a rapid and transient one, followed by a slow and sustained phase. The NF-kappa B turned out to be involved in an antiapoptotic response to APP-mediated photosensitization because the HCT-116 cell line expressing the dominant negative mutant of inhibitor-kappa B alpha was more sensitive to apoptosis as measured by DNA fragmentation and caspase activation. These data unambiguously show that a membrane-located photosensitizer can lead to effective apoptosis, reinforcing the idea that PDT can be an effective means to eradicate colon cancer cells.

Lysosomes are sites of fluoroquinolone photosensitization in human skin fibroblasts: a microspectrofluorometric approach.

The fluoroquinolone antibiotics are widely used despite their strong phototoxicity under solar UV irradiation. Although they are known as good photodynamic photosensitizers, other factors than production of activated oxygen species may play a role in the effectiveness of the phototoxic effect. Subcellular localization is one of the important parameters that may determine this strength. Using microspectrofluorometry, it is shown that norfloxacin, ofloxacin, lomefloxacin, ciproflaxin and BAYy3118 are readily incorporated into lysosomes of HS68 human skin fibroblasts although weak staining of the whole cytoplasm also occurs especially with norfloxacin. Consistent with their photoinstability in solutions, the fluoroquinolones under study are readily photobleached by UVA in the HS68 fibroblasts. The BAYy3118 derivative that has the fastest bleaching rate also shows the strongest phototoxicity toward HS68 fibroblasts. Photosensitization with these fluoroquinolones induces lysosomal membrane damage as shown by the increased rate of leakage of the lysosomal probe lucifer yellow as compared to that observed with untreated cells.

Pyropheophorbide-a methyl ester-mediated photosensitization activates transcription factor NF-kappaB through the interleukin-1 receptor-dependent signaling pathway.

Pyropheophorbide-a methyl ester (PPME) is a second generation of photosensitizers used in photodynamic therapy. We demonstrated that PPME photosensitization activated NF-kappaB transcription factor in colon cancer cells. Unexpectedly, this activation occurred in two separate waves, i.e. a rapid and transient one and a second slower but sustained phase. The former was due to photosensitization by PPME localized in the cytoplasmic membrane which triggered interleukin-1 receptor internalization and the transduction pathways controlled by the interleukin-1 type I receptor. Indeed, TRAF6 dominant negative mutant abolished NF-kappaB activation by PPME photosensitization, and TRAF2 dominant negative mutant was without any effect, and overexpression of IkappaB kinases increased gene transcription controlled by NF-kappaB. Oxidative stress was not likely involved in the activation. On the other hand, the slower and sustained wave could be the product of the release of ceramide through activation of the acidic sphingomyelinase. PPME localization within the lysosomal membrane could explain why ceramide acted as second messenger in NF-kappaB activation by PPME photosensitization. These data will allow a better understanding of the molecular basis of tumor eradication by photodynamic therapy, in particular the importance of the host cell response in the treatment.

Pro-oxidant role of superoxide dismutase in ultraviolet-A-induced lipid peroxidation in cultured normal human skin fibroblasts.

Exposure of cultured normal human skin fibroblasts to ultraviolet A triggers lipid peroxidation. In sharp contrast with the tert-butylhydroperoxide-induced lipid peroxidation, the ultraviolet-A-induced lipid peroxidation is inhibited by treating cells with diethyldithiocarbamate. Diethyldithiocarbamate decreases superoxide dismutase activity and, to a lesser extent, the total glutathione level. Catalase and glutathione peroxidase, however, are unaffected. The decrease in the superoxide dismutase activity parallels an inhibition of H2O2 formation in both irradiated and unirradiated cells. The protection against lipid peroxidation may thus be associated with superoxide dismutase inhibition. Membrane damage revealed by neutral red uptake is not prevented by diethyldithiocarbamate.

Ultraviolet-A-dependent inhibition of cytoplasmic aconitase activity of iron regulatory protein-1 in NCTC 2544 keratinocytes.

The aconitase activity of the cytoplasmic iron regulatory protein-1 of NCTC 2544 keratinocytes is effectively inhibited by physiological doses of UVA. The time course of the photoinactivation is biphasic. A fast step is first observed corresponding to about 50% inactivation after exposure to 5 J/cm2 of UVA followed by a much slower photoinactivation at higher doses. The water-soluble antioxidant N-acetylcysteine only partially inhibits the photoinduced inactivation of the cytoplasmic aconitase function, whereas the lipophilic vitamin E, the iron chelator, desferrioxamine and the superoxide dismutase inhibitor, diethyldithiocarbamate do not protect at all. As a consequence, reactive oxygen species such as O2-., H2O2 and lipid peroxides and hydroperoxides seem to play a rather minor role in the inactivation induced by the UVA photooxidative stress although an oxidative stress produced by O2-. and H2O2 is known to inhibit reversibly and effectively cytoplasmic aconitase activity in mammalian cells.

Tolyporphin: a natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo.

Tolyporphin (TP), a porphyrin extracted from cyanobacteria, was found to be a very potent photosensitizer of EMT-6 tumor cells grown both in vitro as suspensions or monolayers and in vivo in tumors implanted on the backs of C.B17/Icr severe combined immunodeficient mice. Thus, during photodynamic treatment (PDT) of EMT-6 tumor cells in vitro, the photokilling effectiveness of TP measured as the product of the reciprocal of D50 (the light dose necessary to kill 50% of cells) and the concentration of TP is approximately 5000 times higher than that of Photofrin II (PII), the only PDT photosensitizer thus far approved for clinical trials. TP almost exclusively localizes in the perinuclear region and specifically in the endoplasmic reticulum (ER), as shown by microspectrofluorometry on single living EMT-6 cells costained with the ER and/or Golgi fluorescent vital probes, 3,3'-dihexyloxacarbocyanine iodide and N-[4,4-difluoro-(5,7-dimethyl-BODIPY)-1-pentanoyl]-D-erythro-sphin gosine (Molecular Probes, Eugene, OR). As a result, the singlet oxygen-mediated photodynamic activity of TP induces an effective inactivation of the acyl CoA:cholesterol-O-acyltransferase, a sensitive marker of ER membrane integrity and alterations of the nuclear membrane. In vivo, with the EMT-6 mouse tumor model, an exceptional effectiveness is also observed as compared to that of PII and other second generation photosensitizers of the pheophorbide class, which are themselves much more potent than PII. The outstanding PDT activity of TP observed in vivo may be due to its unique biodistribution properties, in particular much less extraction by the liver, resulting in a higher delivery to other tissues, including tumor.