Reference #: RAV-1017-917314
Submit Date: 04/04/2002 04:21:43-0500

Singlet oxygen-mediated oxidation of cellular DNA

ABSTRACT:
We have recently synthesized (Martinez G., et al. (2000). J. Am. Chem. Soc., 122, 10212-10213) the [¹⁸O]-labeled endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide (DHPN¹⁸O₂). Such an endoperoxide was prepared by methylene blue-mediated photosensitization of the naphthalene derivative (DHPN) in the presence of [¹⁸O]-labeled molecular oxygen. Thermal decomposition of the endoperoxide could be used as a clean source of labeled singlet oxygen. In order to determine whether singlet oxygen, when released within cells, is able to oxidize cellular DNA, cells were incubated with the labeled endoperoxide DHPN¹⁸O₂. First, incubation is performed at 4°C to allow the penetration of DHPN¹⁸O₂ into cells. Then, samples are maintained at 37°C to induce the thermal decomposition of the endoperoxide together with the intracellular release of singlet oxygne. Thereafter, DNA was extracted and the amount of 8-oxodGuo was determined using HPLC coupled to tandem mass spectrometry (HPLC-MS/MS). Using this approach, we were able to show that a significant formation of [M+2] ¹⁸O-labeled 8-oxodGuo could be detected in cells treated with DHPN¹⁸O₂ (Ravanat, J.-L. et al. (2000) J. Biol. Chem., 275, 40601-40604). This demonstrates that singlet oxygen, when released within cells, is able to induce the formation of 8-oxodGuo .

Keywords: singlet oxygen, DNA damage, 8-oxo-7,8-dihydro-2'-deoxyguanosine, tandem mass spectrometry

Reference #: HE-1017-350077
Submit Date: 03/28/2002 14:23:36-0500

UVA-induced efflux of reduced glutathione (GSH) in human keratinocytes

ABSTRACT:
Glutathione is the principal low-molecular-weight thiol and critical antioxidant in cellular defense against oxidative stress. The GSH/GSSG ratio is considered to be a reflection of cellular redox status. Transport of both GSH and GSSG plays an important role in maintaining cellular redox environment in response to oxidative stress, and this process is energy-dependent. We have studied the effect of UVA on glutathione efflux and GSH/GSSG ratio in normal and immortalized HaCaT keratinocytes. After UVA irradiation (30 J/cm²) total intracellular glutathione content (i.e. GSH+GSSG), decreased 2-fold in the presence of glucose, while the extracellular total glutathione increased 2-fold, indicating an increased efflux of glutathione. However, UVA irradiation in the absence of glucose resulted in a 40-fold decrease in the GSH/GSSG ratio, while in the presence of glucose the decrease was 1.5-fold. Interestingly the extracellular GSH levels were much higher in UVA-irradiated keratinocytes in the presence of glucose than in the absence of glucose, while GSSG levels remained the same. These findings indicate that, under UVA irradiation, GSH rather than GSSG was exported in an energy-dependent manner thereby producing a higher extracellular GSH/GSSG ratio. The addition of GSH (1 mM) reduced the UVA-induced efflux of intracellular GSH, while depletion of intracellular GSH by buthionine sulfoximine (BSO) decreased UVA-induced GSH efflux. These findings suggest that GSH transport is dependent on both intracellular and extracellular GSH levels. Glutathione efflux was also observed in the presence of H₂O₂ (1 mM) and glucose. However, in contrast to UVA both extracellular GSH and GSSG levels were higher than in the absence of glucose, although the total extracellular glutathione was much lower than that induced by UVA. This implies that UVA induced GSH efflux is not mediated by H₂O₂. Verapamil and cyclosporin A decreased UVA-induced GSH efflux. Taken together the results indicate that GSH efflux is both energy-dependent and carrier mediated. A likely candidate transporter is a multidrug resistance-associated protein (MRP), which may increase activity after exposure to UVA.

Keywords: UVA, glutathione, keratinocytes, multidrug resistance-associated protein (MRP)

Reference #: CAS-1016-653562
Submit Date: 03/20/2002 12:49:39-0500

The effects of UDCA on photodynamic induced apoptosis

ABSTRACT:
In previous studies, we reported that UDCA markedly enhanced the efficacy of photodynamic therapy(PDT)in cell culture, using photosensitizing agents known to target the anti-apoptotic protein bcl-2 for photodamage. These studies have now been extended with a view toward establishing an explanation for this effect. Mouse leukemia L1210 were employed in these studies, using photosensitizing agents that target either bcl-2 (the porphycene CPO), lysosomes (N-aspartyl chlorin e6) or both (SnET2) for photodamage. Bcl-2 photodamage is sufficiently selective so that the pro-apoptotic protein bax is not affected, and we previously established that overexpression of bcl-2 leads to the appearance of elevated bax levels and an enhanced apoptotic response following bcl-2 photodamage. In contrast, lysosomal photodamage led to bid cleavage and a subsequent pro-apoptotic interaction between t-bid and mitochondria. We found that UDCA alone did not affect growth of L1210 cells at levels as high as 100uM, but markedly promoted the apoptotic reponse to bcl-2 photodamage. when lysosomes were the sole target for PDT, no effect of UDCA could be detected. PDT induced apoptosis and loss of viability were promoted by either CPO or SnET2. Furthermore, Immunohistochemical staining experiments indicated that UDCA promoted the targeting of the pro-apoptotic protein bax to the mitochondria after bcl-2 photodamage. These results suggest an explanation for the mode of action of UDCA: the promotion of bax:mitochondrial but not t-bid:mitochondrial interactions.

Keywords: photodynamic therapy, UDCA

Reference #: WAN-1017-070861
Submit Date: 03/25/2002 09:14:14-0500

Heat Shock Protein 27 Plays A Critical Role in the Cellular Response to Photodynamic Therapy Mediated Oxidative Damage

ABSTRACT:
Photofrin-resistant cell line (HT29-P14) was used in the present study to investigate the mechanism(s) involved in Photofrin-mediated photodynamic therapy (PDT). We compared gene expression profiles between the resistant cell line and its parental cell line (HT29) using DNA microarray analysis. A significant upregulation of small heat shock protein 27 (Hsp27) was found in HT29-P14 cells. The elevated Hsp27 may play an important role in the resistance of HT29-P14 to Photofrin-PDT. To test this hypothesis we stably transfected HT29 cells with human Hsp27 cDNA. The potential role of Hsp27 in the resistance to PDT was examined in Hsp27 overexpression cells. Stable trasnfected cells (H13) showed an increased survival following Photofrin-PDT, suggesting that upregulation of Hsp27 is causally related to the induced resistance to Photofrin-PDT. Phosphorylation of Hsp27 has been suggested to play an important role in the cytoprotection. We have examined the phosphorylation activity of Hsp27 among the parental and resistant cells, as well as the overexpression cells. Elevated level of Hsp27 resulted in an increased ability of phosphorylation in both resistant and overexpression cells following PDT. These data suggest that Hsp27 may play an important role in mediating the adaptive response to Photofrin PDT-induced oxidative stress, and that the pathways leading to Hsp27 phosphoryaltion may contribute to the resistance of cells to photooxidative damage.

Keywords: photodynamic therapy, Hsp27, phosphoryaltion, microarray

Reference #: THI-1017-331263
Submit Date: 03/28/2002 09:26:11-0500

Indirect Detection of Photosensitizer ex vivo

ABSTRACT:
Photodynamic therapy induces the production of reactive oxygen species (ROS) within tissues exposed to laser light after administration of a sensitizer. In the context of continuing clinical and commercial development of chemicals with sensitizing properties, a minimally invasive assay is needed to determine the tissue kinetics of fluorescent or non-fluorescent photoreactive drugs. The level of ROS was determined ex vivo from 1 mm3 biopsy samples using 2'-7' dichlorofluorescin diacetate (DCFH-DA), a fluorescent probe which was converted into highly fluorescent dichlorofluorescein (DCF) in the presence of ROS. This assay was tested on meta(tetrahydroxyphenyl)chlorin (m-THPC, FOSCAN®), a powerful and fluorescent sensitizer, and bacteriochlorophyl derivative WST09 (TOOKAD®), a near-infrared absorbing sensitizer that is only slightly fluorescent. In conjunction with the ROS assay, the tissue accumulation of m-THPC was determined on biopsy samples using an optic fibre spectrofluorometer (OFS). DCF fluorescence was proportional to the level of oxidation induced by horseradish peroxidase used as a control and to the concentration (range: 0 to 5 g ml-1) of both selected photosensitizers irradiated in a tube together with DCFH. Regardless of the organ studied, an excellent correlation was found between fluorescence measurement by OFS and ROS determination for m-THPC. m-THPC (2 mg kg-1 iv) accumulation in tumour tissues was best after 48 h, and the best signal was obtained in liver. With non-fluorescent WST09 (2 mg kg-1), ROS determination showed the best tumour uptake 48 h after injection, with a tumour/muscle ratio of 5.4. The ROS assay appears to be feasible for determining sensitizer concentration in regular grip biopsy tissue samples.

Keywords: Photodynamic therapy, Reactive Oxygen species, 2'-7' dichlorofluorescin diacetate, biopsy

Reference #: RED-1018-036054
Submit Date: 04/05/2002 13:23:16-0500

Does lipid hydroperoxide formation mediate cellular DNA damage ?

ABSTRACT:
The formation and accumulation of lipid hydroperoxides (LOOH) are key detrimental events involved in the mechanism of some membrane-targeted photosensitizers. Singlet oxygen reacts with polyunsaturated fatty acids in cell membranes leading to formation of LOOH and further lipid peroxidation reactions. In this study, extranuclear singlet oxygen formation was ultimately found to cause extensive DNA modification, as evidenced by single cell gel electrophoresis (comet) assay. Singlet oxygen reactions caused accumulation of LOOH in the plasma membrane of WTK-1 lymphocytes treated with rose bengal and light. LOOH formation was shown to play a key role in the formation of single strand DNA breaks. Levels of LOOH and DNA strand breaks were both reduced by the addition of a-tocopherol, a potent antioxidant involved in protection against lipid peroxidation. Reducing intracellular GSH concentration using buthionine sulfoxamine (BSO) led to an increase in both LOOH accumulation and DNA strand breaks, whereas incubation with N-acetylcysteine (NAC) to enhance GSH levels decreased LOOH accumulation and DNA strand breaks. Trolox, a water soluble a-tocopherol analogue that resides in the cytoplasm of exposed cells, was shown to reduce DNA strand breaks, without affecting LOOH accumulation. These results suggested that singlet oxygen mediated formation of LOOH in the plasma membrane ultimately led to nuclear DNA strand breaks. Secondary species derived from LOOH breakdown rather than LOOH are more likely to mediate the DNA damage. In accordance with this hypothesis the addition of iron caused an increase in DNA damage. At least a partial role for free radical- mediated DNA strand breaks is suggested, since trolox was able to reduce but not remove the damage seen. Non-radical species, such as malondiahdehyde and 4-hydroxy-2-nonenal, produced as end products of lipid peroxidation, may also represent a non-radical route to the observed DNA damage.

Keywords: DNA Strand Breaks, Lipid Hydroperoxide, Singlet Oxygen

Reference #: NIZ-1017-707664
Submit Date: 04/01/2002 17:12:49-0500

Antioxidant and cytoprotective effects of nitric oxide on ALA/light-treated tumor cells.

ABSTRACT:
Nitric oxide (^.NO) can be converted to toxic prooxidant and nitrating species in cells. However, under appropriate conditions, ^.NO itself can function as an antioxidant, e.g. by intercepting lipid-derived free radicals. The purpose of this study was to test the hypothesis that low flux ^.NO acting in the latter fashion can render tumor cells more resistant to photodynamic killing, specifically that sensitized by 5-aminolevulinic acid (ALA)-derived protoporphyrin IX (PpIX). A breast tumor subline (COH-BR1) deficient in lipid hydroperoxide (LOOH)-detoxifying capacity was studied. In a typical approach, cells in serum-free medium were incubated with 1 mM ALA for 15 min, then without ALA for 3.75 h, allowing biogenerated PpIX to diffuse to extramitochondrial sites, including plasma membrane. Immediately after exposing to fresh medium either lacking or containing the NO donor PAPA/NO (0.4 mM; lifetime ~22 min), the cells were irradiated with broad-band visible light, then switched to serum containing medium. Using a Hoechst stain to assess viability 24 h after irradiation, we observed a progressive increase in necrotic cell death with increasing light exposure up to 30 min (2 J/cm² fluence), which was inhibited nearly 75% by PAPA/NO. Decomposed PAPA/NO had no effect, confirming that ^.NO was the responsible agent. Adding lipophilic iron to ALA-treated cells before irradiation exacerbated photokilling and ^.NO's protective effect was even greater. ALA/light-treatment of [14C]cholesterol-labeled cells produced oxidation products (ChOX) arising from free radical turnover of photogenerated LOOHs. ChOX levels were significantly reduced by PAPA/NO, consistent with ^.NO being antinecrotic by interfering with chain lipid peroxidation at the plasma membrane level. These findings provide new insights into the possible role of ^.NO in tumor resistance to ALA-PDT and other photodynamic treatments. (Supported by NIH: CA70823)

Keywords: Nitric oxide, Antioxidant, Lipid peroxidation, Photodynamic therapy

Reference #: SIM-1017-674858
Submit Date: 04/01/2002 09:24:20-0500

The Effect of Aggregation on the Photogeneration of Reactive Oxygen Species by Eumelanin

ABSTRACT:
Melanins protect tissue by absorption and rapid non-radiative, non-reactive dissipation of ultraviolet (UV) light. However, melanins also produce reactive oxygen species (ROS) upon UV illumination. A chemical understanding of this dichotomy of photoprotection and phototoxicity has not been established. Herein this issue is examined by studying the UV-B induced oxidation and reduction of cytochrome c by ROS generated by different aggregation states of eumelanin. The quantum yield for superoxide anion by unaggregated oligomers is 7.4*10^-3, an order of magnitude greater than that characteristic of the bulk pigment. The quantum efficiency of hydrogen peroxide production by oligomers is 5.7*10^-3, and its production is attributed to reaction between superoxide anion and hydroquinone groups on eumelanin oligomers. Aggregation of oligomers results in a reduction of these quantum yields, having a significantly greater effect on the efficiency of hydrogen peroxide production. This effect is attributed to the decrease in surface concentration of hydroquinone sites upon aggregation. The effect of aggregation on the photogeneration of ROS serves to provide a foundation for the understanding of the dichotomy of photoprotective and phototoxic properties of melanin.

Keywords: melanin, oxygen activation, aggregation