In vivo protective effect of beta-carotene against psoralen phototoxicity

The protective effect of β-carotene against PUVA- and UVB-induced erythema was determined in groups of albino female Osborne Mendel rats fed standard, placebo or β-carotene-fortified diets. After 13 weeks, the backs of selected animals were clipped and the animals received 8MOP orally, 20 mg/kg in corn oil, followed by 5 J/cm² UVA irradiation 2 h after dose. A significant β-carotene protective effect was observed against phototoxicity induced by 8MOP, as measured by erythema production. A correlation was noted between the observed protective effect and β-carotene serum and skin levels, determined by HPLC. No protective effect against erythema induced by UVB (0.7 J/cm²) was observed in animals fed the β-carotene-fortified diet. This report represents the first in vivo demonstration of a protective effect of dietary p-carotene against psoraten-induced phototoxicity. Possible mechanisms for the observed β-carotene-protective effect are discussed.     

The Endogenous Tryptophan‐derived Photoproduct 6‐formylindolo[3,2‐b]carbazole (FICZ) is a Nanomolar Photosensitizer that Can be Harnessed for the Photodynamic Elimination of Skin Cancer Cells in Vitro and in Vivo

UV‐chromophores contained in human skin may act as endogenous sensitizers of photooxidative stress and can be employed therapeutically for the photodynamic elimination of malignant cells. Here, we report that 6‐formylindolo[3,2‐b]carbazole (FICZ), a tryptophan‐derived photoproduct and endogenous aryl hydrocarbon receptor agonist, displays activity as a nanomolar sensitizer of photooxidative stress, causing the photodynamic elimination of human melanoma and nonmelanoma skin cancer cells in vitro and in vivo. FICZ is an efficient UVA/Visible photosensitizer having absorbance maximum at 390 nm (ε = 9180 L mol^?1 cm^?1), and fluorescence and singlet oxygen quantum yields of 0.15 and 0.5, respectively, in methanol. In a panel of cultured human squamous cell carcinoma and melanoma skin cancer cells (SCC‐25, HaCaT‐ras II‐4, A375, G361, LOX), photodynamic induction of cell death was elicited by the combined action of solar simulated UVA (6.6 J cm^?2) and FICZ (≥10 nm ), preceded by the induction of oxidative stress as substantiated by MitoSOX Red fluorescence microscopy, comet detection of Fpg‐sensitive oxidative genomic lesions and upregulated stress response gene expression (HMOX1, HSPA1A, HSPA6). In SKH1 “high‐risk” mouse skin, an experimental FICZ/UVA photodynamic treatment regimen blocked the progression of UV‐induced tumorigenesis suggesting feasibility of harnessing FICZ for the photooxidative elimination of malignant cells in vivo.     

INVOLVEMENT OF SINGLET OXYGEN IN THE PHOTOTOXICITY MECHANISM FOR A METABOLITE OF PIROXICAM

It has been previously shown that a metabolite of piroxicam but not piroxicam itself causes phototoxicity to cells in vitro after exposure to UVA (320–400 nm) radiation. The phototoxicity mechanism for this metabolite, 2-methyl-4-oxo-2H-l,2-benzothiazine-l,l-dioxide (Compound I), was investigated. In vitro phototoxicity to human mononuclear cells was assayed using 0.5 m M Compound I and UVA radiation. The UVA fluence required for phototoxicity of Compound I was lower by a factor of 2-3 in D₂O buffer compared to H₂O buffer. Superoxide dismutase and mannitol, which remove O₂^- and OH", respectively, do not decrease the phototoxicity. The photodecomposition of Compound I was inhibited by sodium azide, enhanced by human serum albumin and unaffected by mannitol. Stable photoproducts of Compound I were not toxic to the cells. The quantum yield of singlet oxygen based on its emission at 1270 nm was 0.19 and 0.35 for Compound I and s2 ± 10^-3 and 10^-2 for piroxicam in D₂O and C₆H₆, respectively. While the extremely low quantum yield for singlet oxygen from piroxicam appears to account for its lack of phototoxicity, the phototoxicity mechanism for its metabolite, Compound I, most likely does involve singlet oxygen.     

Long-wavelength Ultraviolet A1 and Visible Light Photoprotection: A Multimodality Assessment of Dose and Response

Human skin is exposed to visible light (VL; 400–700 nm) and long-wavelength ultraviolet A1 (UVA1) radiation (370–400 nm) after the application of organic broad-spectrum sunscreens. The biologic effects of these wavelengths have been demonstrated; however, a dose–response has not been investigated. Ten subjects with Fitzpatrick skin phototype IV-VI were enrolled. Subjects were irradiated with 2 light sources (80–480 J cm⁻²): one comprising VL with less than 0.5% UVA1 (VL+UVA1) and the other pure VL. Skin responses were evaluated for 2 weeks using clinical and spectroscopic assessments. 4-mm punch biopsies were obtained from nonirradiated skin and sites irradiated with 480 J cm⁻² of VL+UVA1 and pure VL 24 h after irradiation. Clinical and spectroscopic assessments demonstrated a robust response at VL+UVA1 sites compared with pure VL. Histology findings demonstrated a statistically significant increase in the marker of inflammation (P < 0.05) and proliferation (P < 0.05) at the irradiated sites compared with nonirradiated control. Threshold doses of VL+UVA1 resulting in biologic responses were calculated. Results indicate that approximately 2 h of sun exposure, which equates to VL+UVA1 dose (~400 J cm⁻²), is capable of inducing inflammation, immediate erythema and delayed tanning. These findings reinforce the need of photoprotection beyond the UV range.     

An Endoplasmic Reticulum Targeting Type I Photosensitizer for Effective Photodynamic Therapy against Hypoxic Tumor Cells

Organelle-targeted type I photodynamic therapy (PDT) shows great potential to overcome the hypoxic microenvironment in solid tumors. The endoplasmic reticulum (ER) is an indispensable organelle in cells with important biological functions. When the ER is damaged due to the production of reactive oxygen species (ROS), the accumulation of misfolded proteins will interfere with ER homeostasis, resulting in ER stress. Here, an ER-targeted benzophenothiazine-based photosensitizer NBS-ER was presented. ER targeting modification significantly reduced the dark toxicity and improved phototoxicity index (PI). NBS-ER could effectively produce O₂⁻⋅ with near-infrared irradiation, making its phototoxicity under hypoxia close to that under normoxia. Meanwhile, the photoinduced ROS triggered ER stress and induced apoptosis. In addition, NBS-ER possessed excellent photodynamic therapeutic effect in 4T1-tumor-bearing mice.     

ULTRAVIOLET RADIATION-INDUCED PHOSPHOLIPASE A2 ACTIVATION OCCURS IN MAMMALIAN CELL MEMBRANE PREPARATIONS

Ultraviolet erythema in human skin is mediated in part by membrane derivatives of arachidonic acid (AA). UVA (320–400nm) and UVB (290–320nm) have been shown to induce release of AA from intact mammalian cells in culture. In order to investigate the mechanism of this release we examined the effect of UVA and UVB on release of [³H] AA from membrane preparations of murine fibroblasts. C3H 10T1/2 cells were prelabelled for 24 h with [³H] AA. The membrane fractions of the cells were separated after lysis by differential centrifugation. The membranes were irradiated in suspension and the [³H] AA released from the membranes was determined by scintillation spectroscopy of supernatants3–4 h after irradiation. Both UVA and UVB induced release of AA from the membrane preparations. The response to UVB was small but significant, reaching levels approximately 150% of control release at doses of 1,200-4,000 J/m². The response to UVA was larger; doses of 2.5-5.0 J/cm² induced release equal to twice control (200%) levels, while doses of10–20 J/cm² induced maximal release at levels approximately 400% of control. Time course studies with UVB and UVA showed maximal release at 4 h after irradiation. When the membrane preparations were incubated with a polyclonal anti-phospholipase A₂ antibody the UV induced release of [³H] AA was completely inhibited in both UVB (1200 J/m²) and UVA (10 J/cm²) treated cells. These data suggest that activation of phospholipase A₂ is responsible for the UV induced release of AA in mammalian cells and that the mechanism of this activation is due, in part at least, to direct photon-membrane interaction.     

Intracellular uptake and photodynamic activity of water-soluble [60]- and [70]fullerenes incorporated in liposomes

Water-soluble fullerenes have attracted attention as promising compounds that have been used to forge new paths in the field of photo-biochemistry. To prepare water-soluble fullerenes, we employed lipid-membrane-incorporated fullerenes (LMICx; x=60 or 70) by using the fullerene exchange method from a gamma-cyclodextrin (gamma-CD) cavity to vesicles. LMIC60 have low toxicity in the dark and engender cell death by photoirradiation (lambda>350 nm). Furthermore, the photodynamic activity of LMIC70 is 4.7-fold that of LMIC60 for the same photon flux (lambda>400 nm). One of the reasons for the higher phototoxicity of LMIC70 is the higher generation of singlet oxygen (1O2) in LMIC70 than in LMIC60. The difference between LMIC60 and LMIC70 is considered to be simply derived from the amount of light absorption in the 400-700 nm region that is suitable for photodynamic therapy (PDT). To the best of our knowledge, this is the first case in which biological activity of C70 and its derivatives toward HeLa cells has been assayed.     

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells

Hypericin, the major component of St. John's Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.     

Antitumor Effect of Sinoporphyrin Sodium‐Mediated Photodynamic Therapy on Human Esophageal Cancer Eca‐109 Cells

The aim of this study was to evaluate the photodynamic effect of Sinoporphyrin sodium (DVDMS). In this study, Eca‐109 cells were treated with DVDMS (5 μg mL^?1) and subjected to photodynamic therapy (PDT). The uptake and subcellular localization of DVDMS were monitored by flow cytometry and confocal microscopy. The phototoxicity of DVDMS was studied by MTT assay. The morphological changes were observed by scanning electron microscopy (SEM). DNA damage, reactive oxygen species (ROS) generation and mitochondria membrane potential (MMP) changes were analyzed by flow cytometry. Studies demonstrated maximal uptake of DVDMS occurred within 3 h, with a mitochondrial subcellular localization. MTT assays displayed that DVDMS could be effectively activated by light and the phototoxicity was much higher than photofrin under the same conditions. In addition, SEM observation indicated that cells were seriously damaged after PDT treatment. Furthermore, activation of DVDMS resulted in significant increases in ROS production. The generated ROS played an important role in the phototoxicity of DVDMS. DVDMS‐mediated PDT (DVDMS‐PDT) also induced DNA damage and MMP loss. It is demonstrated that DVDMS‐mediated PDT is an effective approach on cell proliferation inhibition of Eca‐109 cells.     

Photogenotoxicity of fluoroquinolones in Chinese hamster V79 cells: dependency on active topoisomerase II

The Chinese hamster V79 lung cell in vitro micronucleus assay was adapted to detect and quantify phototoxicity and photogenotoxicity of fluoroquinolones. Using this assay, the quinolones were ranked in terms of decreasing phototoxicity: clinafloxacin > lomefloxacin, sparfloxacin > trovafloxacin, nalidixic acid, ofloxacin, ciprofloxacin > enoxacin, norfloxacin. This rank order agrees well with published studies utilizing various other phototoxicity models and establishes this approach as a fast and sensitive way to characterize the phototoxic potential of quinolones. Nearly complete inhibition of phototoxicity was observed if the cells were pretreated for as little as 1 min with 10-20 mM sodium azide prior to the addition of quinolone. An identical azide effect was seen in unirradiated quinolone- and etoposide-treated cells. These findings are consistent with a model in which sodium azide renders DNA topoisomerase II catalytically inactive. In this state, topoisomerase II cannot initiate DNA strand cleavage and the DNA/topoisomerase complex becomes insensitive to quinolones and other topoisomerase II inhibitors. The fact that azide reduces both UV-dependent and UV-independent toxicity and clastogenicity strongly suggests a common mechanism of toxicity dependent on the formation of topoisomerase-induced DNA double-strand breaks.     

UVA and UVB‐Induced 8‐Methoxypsoralen Photoadducts and a Novel Method for their Detection by Surface‐Enhanced Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (SELDI‐TOF MS)

UVA‐activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320–400 nm) is more commonly used clinically, studies have shown that UVB (280–320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB‐induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm^?1 M^?1 at 300 nm) compared with the UVA region (2016 cm^?1 M^?1 at 365 nm), a greater extent of adduct formation is expected. SELDI‐TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A–T oligonucleotide. 8‐Methoxypsoralen (8‐MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI‐TOF. For UVB‐activated 8‐MOP, the extent of adducts was three times greater than for UVA. HPLC ESI‐MS analysis showed that UVB irradiation yielded high levels of 3,4‐monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4′,5′‐monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.     

RESEARCH NOTE PHOTOTOXICITY OF CITRAL TO Trichoplusia ni (LEPIDOPTERA: NOCTUIDAE) AND ITS AMELIORATION BY VITAMIN A

Abstract Citral, a monoterpene aldehyde synthesized by several plant families, was recently shown to exhibit ultraviolet A light (320–400 nm) (UVA)-enhanced oxygen-dependent toxicity against bacteria and fungi ( Escherichia coli and Fusarium species). In this study, we report for the first time that citral is phototoxic to insects; at concentrations of 300 ppm in artificial diet, mortality of first instar Trichoplusia ni (cabbage loopers) approached 30% after 120 h of UVA exposure, approximately double the mortality level in the absence of UVA. At 300 ppm, the antioxidant vitamin A reduces citral phototoxicity by about 50% in this species, suggesting that citral phototoxicity against insects is oxygen dependent.     

The Effects of the Broadband UVA Radiation on Myeloid Leukemia Cells: The Possible Role of Protein Kinase C in Mediation of UVA-lnduced Effects

Abstract— We examined the effects of broadband UVA radiation (320–400 nm) on a rat myeloid leukemia cell line–chlo-roma (ChL). A Phillips face tanner model HB 171/A was used as a light source. Chloroma were irradiated through a 5 mm thick glass Alter that cut off all of the UVB contamination. The irradiances were measured, from 250 to 400 nm, with a well-characterized and calibrated double-grating spectroradiometer Optronic 742. The overall uncertainty of dose evaluation was estimated to be <15% (2s?). The cells were irradiated with UVA doses of 4 and 8 J/cm² and cultured thereafter for 24 h. After this period of time, a marked decline up to 50% was observed in cell proliferation in UVA-irradiated ChL cultures. The cell proliferation decline was found to be caused by simultaneously occurring G2/M phase cell cycle arrest and apoptosis in part of the UVA-irradiated ChL population. Concomitantly, with the decline in cell proliferation, an increase was observed in the expression of the major histocompatibility (MHC) class I and II antigens. Because protein kinase C (PKC) is known to regulate cell proliferation, apoptosis and expression of MHC antigens, and because UVA was shown to regulate PKC activity/expression, we therefore examined whether UVA irradiation has any effect on the expression of isozymes of PKC. Western blots revealed that ChL express α, βI, δ, α, γ, and π isozymes of PKC and that expression of all isozymes declined 24 h after UVA irradiation (8 J/cm²). Finally, PKC activation in ChL by exposure to phorbol ester caused cell cycle arrest in G1 phase but did not induce apoptosis. This suggests that the previously shown UVA-induced PKC activation in ChL might be responsible for the induction of MHC antigens but the simultaneously observed ChL apoptosis is likely to be mediated by PKC down-regulation. All together, our results suggest that UVA, at irradiance levels that resemble the outdoor exposure, may have profound effects on the immune-related properties of leukocytes. Thus, we speculate that in vivo the immune functions of leukocytes passing through dermal capillaries might be altered by exposure to solar UVA radiation.     

DETERMINATION OF RESIDUAL 4-AMINOMETHYL-4,5',8-TRIMETHYLPSORALENAND MUTAGENICITY TESTING FOLLOWING PSORALEN PLUS UVA TREATMENT OF PLATELET SUSPENSIONS

Abstract— Psoralens and UVA light have been used in the laboratory to study the inactivation of viruses that may be infrequently present in platelet concentrates that are prepared for transfusion. In order to evaluate safety aspects of the treatment of platelet suspensions with 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), we have investigated the residual levels and mutagenic potential of AMT after UVA phototreatment. 4'-aminomethyl-4,5',8-trimethylpso-ralen, at a final concentration of 40 μg/mL, was added to platelet suspensions which contained 16% plasma and a synthetic medium. Platelet suspensions containing AMT were irradiated with up to 7.2 J/cm² UVA light under normal oxygen levels. Residual levels of AMT were determined by HPLC and a bioassay based on bacteriophage 0.6 inactivation. The photodestruction of AMT or its activity by UVA was characterized by a D₃₇ value of 0.6 and 0.3 J/cm² with HPLC or bioassay, respectively. At 2.4 J/cm² UVA, which results in approximately 5 log₁₀ inactivation of vesicular stomatitis virus (VSV) and retention of platelet in vitro properties, 12% (HPLC) to 9% (bioassay) AMT remained. Like other psoralens, AMT was found to bind to serum proteins as shown by ultrafiltration. Results are consistent with approximately 36% of the initial drug load binding primarily to serum albumin. It was determined using ³H-AMT that 9 to 18% of radioactivity was bound to platelets in the absence of irradiation. Similar fractions (13 to 18%) of AMT were bound to platelets after 3.6 J/cm² UVA irradiation, and 8 to 10% of total AMT was associated with saline-washed irradiated platelets and is presumably tightly bound. Mutagenicity testing (Ames test, in the absence of UVA) was also carried out on the UVA irradiated platelet samples. With Salmonella tester strains which detect primarily base substitution mutations (TA100, TA1535 and TA102), no increase from background mutagenesis levels was observed with any of the samples. However, tester strains which detect frameshift mutations (TA98, TA1537, and TA1538) displayed significant increases in histidine revertants over background levels for irradiated and non-irradiated AMT-containing samples tested in the presence of S9 microsomal enzymes. In the absence of S9 activation, a mutagenic response was observed only with tester strain TA1537. All frameshift tester strains exhibited decreased numbers of induced revertants with lower residual AMT concentrations (which correlated with higher UVA dose). Significant mutagenesis was still observed for platelet suspensions irradiated with virucidal levels of UVA which maintain platelet in vitro function (2.4 J/cm²). These results suggest that residual available AMT is mutagenic in the Ames test and that the observed frameshift mutations may be caused by binding of AMT or its metabolites to nucleic acids in the absence of UVA light.     

THE LETHAL EFFECT OF LONGWAVE ULTRAVIOLET LIGHT AND PUVA. AN ANALYSIS BASED UPON HUMAN MESENCHYMAL CELLS IN VITRO

Abstract— The lethal effect of UVA and PUVA radiation was studied in cultures of fresh and mature monocytes. UVA radiation alone was shown to possess a lethal effect at doses which are attained in the dermis in vivo. The synergistic action of 8-methoxypsoralen and UVA radiation predominated in PUVA radiation, but again a residual effect of UVA alone was demonstrated mathematically. Mature cells were less sensitive than fresh monocytes.
Our results indicate that a monolayer culture of non-dividing, mesenchymal cells offers considerable advantages over in vivo systems as a model for the study of phototoxicity.     

THE PHOTOACTIVE ANTIMICROBIAL PROPERTIES OF EUDISTOMINS FROM THE CARIBBEAN TUNICATE Eudistoma olivaceum

Abstract— Five eudistomins, β-carboline derivatives isolated from a Caribbean tunicate, were tested for phototoxicity against several viruses, bacteria, yeast, and mammalian cells. The five compounds showed varying degrees of UVA dependant phototoxicity (i.e. long wavelength UV dependant) against murine cytomegalovirus (MCMV), Sindbis virus (SV) and mouse 3T3 cells, although the relative order of potency was the same for these three organisms. Eudistomin N was the most active (approximately the same as the β-carboline, harmine), while eudistomins M and O were moderately phototoxic, and H and I had little activity. To some degree the relative phototoxicity was correlated with fewer side chain substituents. A similar relative order of phototoxic potency was seen against phage T4, but in this case the magnitude of the effect was considerably reduced, in contrast to harmine. The antibacterial and antifungal activities were not correlated with antiviral effects, and some UVA-independent activities were seen. Thus the eudistomins may possess different mechanisms of action against different organisms, depending upon the presence or absence of UVA.     

Sunscreen Protection Against Ultraviolet Radiation-induced Pyrimidine Dimers in Mouse Epidermal DNA

Pyrimidine dimers were measured in epidermal DNA of SKH:HRI mice following exposure to solar-simulated UV radiation (SSUV, 290–400 nm) or to UVA (320–400 nm). Mice were exposed to SSUV or UVA after topical application (2 mg/cm²) of vehicle, a UVB absorber (5% 2-ethylhexyl p-methoxycinnamate [2-EHMC]), or a broad-spectrum UVA absorber (5% Mexoryl®SX). The rates of induction of pyrimidine dimers in untreated animals were 5.4 ± 0.57 times 10^-4 (mean ± SEM) and 7.6 ± 0.95 times 10^-6 dimers per 10⁸ Da of epidermal DNA per J/m² of SSUV and UVA, respectively. Topical application of Mexoryl®SX reduced the rate of induction of pyrimidine dimers in SSUV-exposed animals to 4.7 ± 0.44 times 10^-5 dimers per 10⁸ Da per J/m² for a dimer induction protection factor (PF) of 11.5 (5.4 times 10 ⁴/4.7 times 10^-5). The rate of dimer induction in Mexoryl®SX-treated, UVA-ex-posed mice was 0.95 ± 0.2 times 10^-6 dimers per 10⁸ Da per J/m² (PF = 8.0). The 2-EHMC at a concentration of 5% (wt/wt) was significantly less effective than Mexoryl®SX in preventing the induction of pyrimidine dimers in animals exposed to either SSUV or UVA. The rates of dimer induction in 2-EHMC-treated mice were 8.2 ± 1.1 times 10^-5 and 3.8 ± 0.33 times 10^-6 dimers per Da per J/m² of SSUV (PF = 6.6) and UVA (PF = 2.0), respectively. Upon normalizing to the efficacy for edema induction, UVA induced approximately one-fourth the number of pyrimidine dimers per equivalent edematous response when compared to SSUV.     

Photosensitized oxidation of membrane lipids: reaction pathways, cytotoxic effects, and cytoprotective mechanisms.

Unsaturated lipids in cell membranes, including phospholipids and cholesterol, are well-known targets of oxidative modification, which can be induced by a variety of stresses, including ultraviolet A (UVA)- and visible light-induced photodynamic stress. Photodynamic lipid peroxidation has been associated with pathological conditions such as skin phototoxicity and carcinogenesis, as well as therapeutic treatments such as antitumor photodynamic therapy (PDT). Lipid hydroperoxides (LOOHs), including cholesterol hydroperoxides (ChOOHs), are important non-radical intermediates of the peroxidative process which can (i) serve as in situ reporters of type I vs. type II chemistry; (ii) undergo one-electron or two-electron reductive turnover which determines whether peroxidative injury is respectively intensified or suppressed; and (iii) mediate signaling cascades which either fortify antioxidant defenses of cells or evoke apoptotic death if oxidative pressure is too great. The purpose of this article is to review current understanding of photodynamic (UVA- or visible light-induced) lipid peroxidation with a special focus on LOOH generation and reactivity. Future goals in this area, many of which depend on continued development of state-of-the-art analytical techniques, will also be discussed.     

PSORALEN-MEDIATED VIRUS PHOTOINACTIVATION IN PLATELET CONCENTRATES: ENHANCED SPECIFICITY OF VIRUS KILL IN THE ABSENCE OF SHORTER UVA WAVELENGTHS

Abstract— Treatments with psoralens and long-wavelength ultraviolet radiation (UVA, 320–400 nm; PUVA) have shown efficacy for virus sterilization of platelet concentrates (PC). Our laboratory has employed the psoralen derivative 4'-aminomethyl-4, 5', 8-trimethylpsoralen (AMT), and we have found that platelet integrity is best preserved when rutin, a flavonoid that quenches multiple reactive oxygen species, is present during AMT/UVA treatment of PC. In this report, we examine the effects of different UVA spectra under our standard PC treatment conditions (i.e. 50 μg/mL AMT, 0.35 mM rutin and 38 J/cm² UVA). Added vesicular stomatitis virus (VSV; ≥5.5 log₁₀) was completely inactivated with the simultaneous maintenance of the platelet aggregation response (>90% of control) when a UVA light source with transmission mainly between 360 and 370 nm (narrow UVA1) was used. In contrast, with a broad-band UVA (320–400 nm; broad UVA) light source, the aggregation response was greatly compromised (<50% of control) with only a minor increase in the rate of VSV kill. With this lamp, platelet function could be improved to about 75% of the control by adding a long-pass filter, which reduced the transmission of shorter (≤345 nm) UVA wavelengths (340–400 nm; UVA1). At equivalent levels of virus kill, aggregation function was always best preserved when narrow UVA1 was used for PUVA treatment. Even in the absence of AMT, and with or without rutin present, narrow UVA 1 irradiation was better tolerated by platelets than was broad UVA. Our results suggest that for PUVA treatment of PC, the UVA dose range in which complete virus kill is obtained with the simultaneous maintenance of in vitro platelet function is smallest with broad UVA irradiation and largest with narrow UVA1. Thus, the virus specificity of PC treatment with AMT, UVA and quenchers can be further enhanced by the exclusion of the shorter UVA wavelength range.     

PHOTOCYTOTOXICITY OF CURCUMIN

Curcumin, bis (4-hydroxy-3-methoxyphenyl)-l,6-diene-3,5-dione, is a yellow-orange dye derived from the rhizome of the plant Curcuma longa. Curcumin has demonstrated phototoxicity to several species of bacteria under aerobic conditions (Dahl, T. A., et al. , 1989, Arch. Microbiol. 151 183), denoting photodynamic inactivation. We have now found that curcumin is also phototoxic to mammalian cells, using a rat basophilic leukemia cell model, and that this phototoxicity again requires the presence of oxygen. The spectral and photochemical properties of curcumin vary with environment, resulting in the potential for multiple or alternate pathways for the exertion of photodynamic effects. For example, curcumin photogenerates singlet oxygen and reduced forms of molecular oxygen under several conditions relevant to cellular environments. In addition, we detected carbon-centered radicals, which may lead to oxidation products (see accompanying paper). Such products may be important reactants in curcumin's phototoxicity since singlet oxygen and reduced oxygen species alone could not explain the biological results, such as the relatively long lifetime (t¹²= 27 s) of the toxicant responsible for decreased cell viability.