Nitroxide radicals protect DNA from damage when illuminated in vitro in the presence of dibenzoylmethane and a common sunscreen ingredient

Indolinonic nitroxide radicals efficiently scavenge oxygen- and carbon-centered radicals. They protect lipid and protein systems against oxidative stress, but little is known about their capacity to protect DNA against radical-mediated damage. We compare indolinonic nitroxides and the piperidines TEMPO and TEMPOL for their ability to inhibit strand breaks inflicted on DNA when it is illuminated in vitro in the presence of dibenzoylmethane (DBM) and a relative, Parsol 1789, used as a UVA-absorbing sunscreen. We used spin-trapping EPR to examine the formation of radicals and plasmid nicking assays to evaluate DNA strand breakage. The results have a two-fold interest. First, they show that all the nitroxides tested efficiently prevent DNA damage in a dose-dependent fashion. Vitamin E had no effect under the conditions used. Second, they show that carbon-centered radicals are produced on illumination of DBM and its relative and that their formation is probably responsible for the direct strand breaks found when naked DNA is illuminated in vitro in their presence. Additional work on the ability of sunscreens to enter human cells and their response to the light that penetrates sunscreen-protected skin would be necessary before any conclusion could be drawn as to whether the results reported here are relevant to human use of sunscreens.     

The effects of manganese doping on UVA absorption and free radical generation of micronised titanium dioxide and its consequences for the photostability of UVA absorbing organic sunscreen components.

The effect of manganese doping on the free radical generation rate, free radical scavenging and UVA absorption properties of micronised sunscreen grade titania has been studied with respect to enhancement of the UVA photostability of test sunscreen formulations containing the organic UVA absorber Parsol 1789. Manganese doping has been shown to increase the UVA:UVB absorption ratio of titania, reduce free radical generation rates by over 90%, and provide free radical scavenging behaviour. Adding manganese-doped titania to a test formulation incorporating Parsol 1789 shows that manganese doping increases UVA attenuation stability by up to 3 times the amount achieved by comparable commercial undoped titania materials. HPLC data shows this to be related to an improved stabilisation of the organic sunscreen components. Manganese doped titania shows improved efficacy over undoped titania in sunscreen formulations containing organic UV absorbers.     

High-performance liquid chromatographic assay for common sunscreening agents in cosmetic products,bovine serum albumin solution and human plasma

This paper reports the development of a reversed-phase high-performance liquid chromatographic assay for quantifying five of the most common sunscreen agents, namely 2-ethylhexyl-p-dimethyl aminobenzoate (Escalol 507), 2-ethylhexyl-p-methoxycinnamate (Parsol MCX); 4-tert.-butyl-4′-methoxydibenzoylmethane (Parsol 1789), 2-hydroxy-4-methoxybenzophenone-3 (oxybenzone) and 2-ethylhexyl-salicylate (octylsalicylate). The assay permits analysis of the sunscreen agents in formulations and in biological fluids, including bovine serum albumin (BSA) solution, a common additive to in vitro skin diffusion cell receptor fluids, as well as human plasma. Separation was achieved using an ODS C₁₈ column with a methanol-water (88:12) mobile phase. The analytes were detected by ultraviolet light absorption at a wavelength of 315 nm. The assay was linear with minimum detectable limits, calculated as greater than 3-times the baseline noise level: for oxybenzone and Escalol 507, 0.05 μg/ml; for Parsol 1789 and Parsol MCX, 0.1 μg/ml; for octylsalicylate, 1 μg/ml. Recoveries from both plasma and 2% BSA were within the range 89–107%. The inter- and intra-day coefficients of variation for the five agents were not more than 4% at the upper end of the linear range and not more than 10% at the lower end. Preliminary stability studies of the sunscreen agents in a commercial product and in two diffusion cell receptor fluids were also conducted.     

The effects of derivatives of the nitroxide tempol on UVA-mediated in vitro lipid and protein oxidation

Derivatives of tetramethylpiperidines are extensively employed in polymers to prevent photooxidation, and their stabilizing effect is attributed to the activity of the nitroxide radical derived from the parent amine. In this study, we examined the photoprotective effect of a commercial polymer photostabilizer, HALS-1, its corresponding nitroxide, bis(2,2,6,6-tetramethyl-piperidine-1-oxyl-4-yl)sebacate (TINO), and two derivatives of the piperidine nitroxide TEMPOL, 2,2,6,6-tetramethyl-piperidin-4-acetyloxy-1-oxyl (TEMP2) and 2,2,6,6-tetramethyl-piperidin-4-octanoyloxy-1-oxyl (TEMP8) synthesized by us, in liposomes exposed to ultraviolet A (UVA) radiation. For comparison, the UVA-absorber, 4-tert-butyl-4'-methoxydibenzoylmethane (Parsol 1789) used in many suncream formulations, was also included. The nitroxide TINO resulted extremely efficient at inhibiting aldehydic breakdown products deriving from 30 min exposure of liposomes to UVA and the protection was dose-dependent (10-100 microM). The corresponding amine HALS-1 was the least efficient while protection increased in the order: TEMP2 < Parsol 1789 < TEMP 8. HALS-1, TINO, and the two TEMPOL derivatives were also tested in a simple protein system consisting of bovine serum albumin (BSA) exposed to UVA. In this case, these compounds did not inhibit nor enhance UVA-mediated protein carbonyl formation in BSA. The differences in protection between the compounds are discussed in relation to their chemical reactivity, UVA-absorbing capacities, and their molecular structure. Overall, the results obtained envisage the potential use of nitroxide compounds as topical antioxidants.     

Synthesis and application of a novel sunscreen-antioxidant

Background information on the inefficacy of sunscreens to provide free radical protection in skin, despite their usefulness in preventing sunburn/erythema, prompted us to synthesize a compound which would display in the same molecule both UV-absorbing and antioxidant capacities. For this purpose, the UVB absorber, 2-ethylhexyl-4-methoxycinnamate (OMC) was combined with the piperidine nitroxide TEMPOL, which has antioxidant properties. The spectral properties of the new nitroxide-based sunscreen (MC-NO) as well as its efficacy to prevent photo-oxidative damage to lipids induced by UVA, natural sunlight and 4-tert-butyl-4-methoxydibenzoylmethane (BMDBM), a photo-unstable sunscreen which generates free radicals upon UV radiation, was studied. The results obtained demonstrate that MC-NO: (a) absorbs in the UVB region even after UVA irradiation; (b) acts as free radical scavenger as demonstrated by EPR experiments; (c) strongly reduces both UVA-, sunlight- and BMDBM-induced lipid peroxidation in liposomes, measured as reduced TBARS levels; and (d) has comparable antioxidant activity to that of commonly used vitamin E and BHT in skin care formulations. These results suggest that the use of the novel sunscreen-antioxidant or of other nitroxide-based sunscreens in formulations aimed at reducing photoinduced skin damage may be envisaged.     

Measuring sunscreen protection against solar-simulated radiation-induced structural radical damage to skin using ESR/spin trapping: development of an ex vivo test method

The in vitro star system used for sunscreen UVA-testing is not an absolute measure of skin protection being a ratio of the total integrated UVA/UVB absorption. The in vivo persistent-pigment-darkening method requires human volunteers. We investigated the use of the ESR-detectable DMPO protein radical-adduct in solar-simulator-irradiated skin substitutes for sunscreen testing. Sunscreens SPF rated 20+ with UVA protection, reduced this adduct by 40-65% when applied at 2 mg/cm(2). SPF 15 Organic UVA-UVB (BMDBM-OMC) and TiO(2)-UVB filters and a novel UVA-TiO(2) filter reduced it by 21, 31 and 70% respectively. Conventional broad-spectrum sunscreens do not fully protect against protein radical-damage in skin due to possible visible-light contributions to damage or UVA-filter degradation. Anisotropic spectra of DMPO-trapped oxygen-centred radicals, proposed intermediates of lipid-oxidation, were detected in irradiated sunscreen and DMPO. Sunscreen protection might be improved by the consideration of visible-light protection and the design of filters to minimise radical leakage and lipid-oxidation.     

Mexoryl: a review of an ultraviolet a filter

It is widely known that ultraviolet light causes skin damage and melanoma. Different wavelengths of ultraviolet light penetrate the skin at different depths, causing varying levels of damage. Higher wavelengths tend to penetrate deeper and, consequently, are thought to induce a myriad of skin conditions, thereby playing a significant role in the photoaging process. Sunscreens containing the ultraviolet A blocker Mexoryl are important in impeding ultraviolet A light, potentially reducing many of the characteristics of skin aging and preventing biochemical changes that can lead to nonmelanoma carcinoma. Until now, sunscreen products sold in the United States focused on blocking ultraviolet B light. Those that did provide ultraviolet A filtering contained physical blocks (zinc oxide or titanium dioxide) or the chemical block Parsol 1789 (avobenzone). These broad-spectrum sunscreens have limitations, such as degradation under ultraviolet exposure, that resulted in decreased effectiveness. Mexoryl, a novel ultraviolet A filter, provides efficient ultraviolet A coverage, better photostability, and enhanced water resistance. Sunscreens containing Mexoryl are widely used in Europe and Canada. It was not until July 24, 2006, that the U.S. Food and Drug Association approved the compound.     

Illumination of human keratinocytes in the presence of the sunscreen ingredient Padimate-O and through an SPF-15 sunscreen reduces direct photodamage to DNA but increases strand breaks.

On illumination with simulated sunlight, the UVB-absorbing sunscreen chemical 2-ethylhexyl-4-dimethylaminobenzoate (Padimate-O) generates excited species which inflict non-ligatable strand breaks on DNA in vitro and it also becomes mutagenic to yeast in vivo. Padimate-O is known to penetrate human skin but its effects on human cells are not clear. Here, we first simulate the sunlight which penetrates human skin and use it to illuminate human keratinocytes. The DNA damage observed in terms of UV-endonuclease-sensitive sites (ESS) and direct strand breaks per kilobase (kb) of DNA per joule per square metre agrees well with that predicted from action spectra based on monochromatic light. Using plasmid DNA in vitro, we find a very similar pattern of results. Next, we simulate the spectrum that results when the incident light is first attenuated by a film of sunscreen (SPF-15; 2 mg/cm(2)) containing benzophenone-3 (a UVA absorber), octyl methoxycinnamate (a UVB absorber), and Padimate-O. If the sunscreen is not in contact with keratinocytes it reduces direct DNA damage from sunlight (ESS). However, any Padimate-O in contact with the cells substantially increases indirect damage (strand breaks) even though the film of sunscreen reduces direct photodamage. We estimate that applying an SPF-15 sunscreen which contains Padimate-O to human skin followed by exposure to only 5 minimum erythemal doses (MED) of sunlight could, while suppressing the formation of ESS, increase strand breaks in cells under the epidermis by at least 75-fold compared to exposure to 1 MED in the absence of sunscreen.     

Interactions between different solar UVB/UVA filters contained in commercial suncreams and consequent loss of UV protection.

A systematic investigation of two well-known and popular commercial suncreams reveals significant degradation when exposed to simulated UV sunlight at an irradiance corresponding to natural sunlight. We have examined the photochemistry of two widely used sunscreen active agents in pure solvents separately and together (in solution), and in neat form, as well as their photochemistry when present in the actual suncream emulsion (as thin films on a glass substrate) since their combination typically produces suncreams with high sun protection factors (SPF): (1a) octyl methoxycinnamate (OMC; octinoxate) and (2a) 4-tert-butyl-4'-methoxydibenzoylmethane (also known as avobenzone and Parsol 1789), present in the two suncream formulations in combination with others (one also contained TiO2). Intermediates and/or photoproducts were identified by UV/visible spectroscopy, HPLC and liquid chromatographic/mass spectral methods, and by both 1H and 13C-NMR techniques. Structural assignments of the substrates produced were aided by examining model systems {viz. ethyl cinnamate (1b) and dibenzoylmethane (2b)} of the two sunscreen active agents. Irradiation of the cinnamates and the diketones together led to a [2 + 2] photocycloaddition process yielding cinnamate dimers and cyclobutylketone photoadducts that subsequently fragmented into substituted oxopentanoates and oxobutanoates. Similar findings were observed when the two active agents were simultaneously present in the same suncream emulsion.     

Inorganic and organic UV filters: Their role and efficacy in sunscreens and suncare products

Minerals such as titanium dioxide, TiO₂, and zinc oxide, ZnO, are well known active semiconductor photocatalysts used extensively in heterogeneous photocatalysis to destroy environmental pollutants that are organic in nature. They are also extensively used in sunscreen lotions as active broadband sunscreens that screen both UVB (290-320 nm) and UVA (320-400 nm) sunlight radiation and as high SPF makers. When so photoactivated by UV light, however, these two particular metal oxides are known to generate highly oxidizing radicals (OH and ) and other reactive oxygen species (ROS) such as H₂O₂ and singlet oxygen, ¹O₂, which are known to be cytotoxic and/or genotoxic. Hydroxyl (OH) radicals photogenerated from photoactive TiO₂ specimens extracted from commercial sunscreen lotions [R. Dunford, A. Salinaro, L. Cai, N. Serpone, S. Horikoshi, H. Hidaka, J. Knowland, FEBS Lett. 418 (1997) 87] induce damage to DNA plasmids in vitro and to whole human skin cells in cultures. Accordingly, the titanium dioxide particle surface was modified to produce TiO₂ specimens of considerably reduced photoactivity. Deactivation of TiO₂ diminishes considerably, in some cases completely suppresses damage caused to DNA plasmids, to human cells, and to yeast cells compared to non-modified specimens exposed to UVB/UVA simulated solar radiation. The photostabilities of sunscreen organic active agents in neat polar and apolar solvents and in actual commercial formulations have been examined [N. Serpone, A. Salinaro, A.V. Emeline, S. Horikoshi, H. Hidaka, J. Zhao, Photochem. Photobiol. Sci. 1 (2002) 970]. With rare exceptions, the active ingredients undergo photochemical changes (in some cases form free radicals) and the sunscreen lotions lose considerable Sun protection efficacy only after a relatively short time when exposed to simulated sunlight UVB/UVA radiation, confirming the recent findings by Sayre et al. [R.M. Sayre, J.C. Dowdy, A.J. Gerwig, W.J. Shields, R.V. Lloyd, Photochem. Photobiol. 81 (2005) 452].     

Free radical-mediated degradation of proteins: the protective and deleterious effects of membranes

Lipid membranes have been shown to scavenge free radicals generated by various means. However, under oxidative conditions, unsaturated lipids within membranes can produce damaging free radicals. We have determined the relative importance of these two conflicting properties of lipid membranes with the use of liposomal membrane studies. (1) Liposome membranes can protect extra-liposomal albumin from free radicals derived from sources other than peroxidizing lipid. When albumin or copper (an essential component of the free radical generating systems used) were encapsulated, protein damage was further reduced. (2) Using sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) we demonstrate that the exposure of albumin to peroxidizing liposome membranes results in both cross-linking and degradation. Our results indicate that protein damage is substantially less than in the case of other biologically relevant free radical generating systems. We discuss our findings with respect to membrane function and the in vivo exposure of cells to free radicals.     

In vivo generation of free radicals in the skin of live mice under ultraviolet light, measured by L-band EPR spectroscopy

Although free radicals may be involved in various types of UV-induced injuries, only a few in vivo studies of the generation of free radicals, including oxygen radicals, during exposure to ultraviolet light (UV) have been reported. In this study, the nitroxyl probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-N-oxyl was intravenously injected into hairless mice, and its decay was monitored in the skin with an in vivo EPR spectrometer equipped with a surface-coil-type resonator. The rate of decay of the EPR signal increased during UV (UVA+B) irradiation. This increase in signal decay was suppressed by preadministration of a spin trap, N-tert-butyl-alpha-phenylnitrone (PBN). PBN did not change the rate of signal decay in nonirradiated mice. The correlation between signal decay rate and physiological parameters such as blood velocity, blood mass, or skin temperature was low. The decay rate responded rapidly and reversibly to starting and stopping the UV illumination. Hydroxyl and peroxyl radicals caused reduction of the probe signal in vitro, and PBN inhibited only the peroxyl radical-induced signal reduction. These observations suggest that peroxyl radicals are generated in the skin of live mice during UVA+B irradiation.     

Human erythema and matrix metalloproteinase-1 mRNA induction, in vivo, share an action spectrum which suggests common chromophores

Matrix metalloproteinase 1 (MMP-1) is widely regarded as a biomarker of photoageing. We tested the hypothesis that MMP-1 mRNA expression and erythema share a common action spectrum by comparing the effects of erythemally equivalent doses of UVB, UVA1 and solar simulated radiation (SSR) on acute MMP-1 mRNA expression in whole human skin in vivo. Our results show comparable MMP-1 expression with all three spectra, which supports our hypothesis. The sharing of an action spectrum implies common chromophores, one of which is likely to be DNA. We have previously shown that all spectra that we used readily induce cyclobutane thymine dimers (T<>T) in human epidermis in vivo but we lack quantitative data on damage to dermal DNA. This is important because we do not know if dermal MMP-1 induction occurs via direct damage to the dermis, or indirectly via damage to the epidermis. Our results show that UVB induces about 3 times more T<>T compared with erythemally equivalent doses of UVA1, which is similar to our published epidermal data. This supports previously published work that also implicates an unknown UVA1 chromophore for erythema and MMP-1 induction. However, the distribution of the dermal DNA damage varies considerably with spectrum. In the case of UVB it is primarily in the upper dermis, but with UVA1 it is evenly distributed. Thus, irrespective of chromophores, MMP-1 induction by direct dermal damage by both spectra is possible. The practical conclusions of our data are that the small (<5%) UVB content of solar UVR is likely to be the main cause of photoageing, at least in terms of MMP-1 expression. Furthermore, prevention of erythema by sunscreen use is likely to result in reduced MMP-1 expression.     

3-hydroxypyridine chromophores are endogenous sensitizers of photooxidative stress in human skin cells

Photocarcinogenesis and photoaging are established consequences of chronic exposure of human skin to solar irradiation. Accumulating evidence supports a causative involvement of UVA irradiation in skin photo-damage. UVA photodamage has been attributed to photosensitization by endogenous skin chromophores leading to the formation of reactive oxygen species and organic free radicals as key mediators of cellular photooxidative stress. In this study, 3-hydroxypyridine derivatives contained in human skin have been identified as a novel class of potential endogenous photosensitizers. A structure-activity relationship study of skin cell photosensitization by endogenous pyridinium derivatives (pyridinoline, desmosine, pyridoxine, pyridoxamine, pyridoxal, pyridoxal-5'-phosphate) and various synthetic hydroxypyridine isomers identified 3-hydroxypyridine and N-alkyl-3-hydroxypyridinium cation as minimum phototoxic chromophores sufficient to effect skin cell sensitization toward UVB and UVA, respectively. Photosensitization of cultured human skin keratinocytes (HaCaT) and fibroblasts (CF3) by endogenous and synthetic 3-hydroxypyridine derivatives led to a dose-dependent inhibition of proliferation, cell cycle arrest in G2/M, and induction of apoptosis, all of which were reversible by thiol antioxidant intervention. Enhancement of UVA-induced intracellular peroxide formation and p38 mitogen-activated protein kinase-dependent stress signaling suggest a photooxidative mechanism of skin cell photosensitization by 3-hydroxypyridine derivatives. 3-hydroxypyridine derivatives were potent photosensitizers of macromolecular damage, effecting protein (RNase A) photocross-linking and peptide (melittin) photooxidation with incorporation of molecular oxygen. Based on these results, we conclude that 3-hydroxypyridine derivatives comprising a wide range of skin biomolecules, such as enzymatic collagen cross-links, B6 vitamers, and probably advanced glycation end products in chronologically aged skin constitute a novel class of UVA photosensitizers, capable of skin photooxidative damage.     

Enhancement of DNA damage and involvement of reactive oxygen species after exposure to bitumen with UVA irradiation.

This study was carried out to evaluate whether bitumen cytotoxicity is enhanced when bitumen treatment is combined with UVA exposure. We also evaluated the oxidative processes in bitumen-induced DNA damage, and attempted to identify the DNA damage caused by bitumen and UVA exposures, either alone or in combination. The effects of bitumen and UVA on cell proliferation were examined using HL 60 cells. DNA-protein crosslinks (DPCs) were assessed using a K-SDS assay, and reactive oxygen species formation was detected by 8-OH-dG formation. We evaluated the formations of double-strand breaks (DSB) using lambdaDNA/HindIII and single-strand breaks (SSB) using PM2 DNA. The cytotoxicity assay showed enhanced suppression of cell proliferation when bitumen exposure and UVA exposure were combined. Combined exposure caused significant increases in DPCs over either exposure alone. Incubation of deoxyguanosine (dG) with bitumen or UVA showed an increase in 8-hydroxy-2'-deoxyguanosine (8-OH-dG) levels when compared with controls, and combined exposure enhanced this effect. An evaluation of agarose gel bands showed that DSB and SSB were not formed following exposure to bitumen and UVA. This fact indicates that bitumen and UVA may be involved in genotoxic processes by producing oxygen free radicals and that combined exposure enhances these effects.     

Protein, lipid, and DNA radicals to measure skin UVA damage and modulation by melanin

Afro-Caribbeans have a lower incidence of skin cancer than Caucasians, but the effectiveness of melanin as a photoprotective pigment is debated. We investigated the UVA and solar irradiation of ex vivo human skin and DMPO using electron spin resonance spectroscopy, to determine whether pigmented skin is protected by melanin against free radical damage. Initial ascorbate radicals in Caucasian skin were superseded by lipid and/or protein radical adducts with isotropic (a(H)=1.8 mT) and anisotropic spectra comparable to spectra in irradiated pig fat (a(H)=1.9 mT) and BSA. DNA carbon-centered radical adducts (a(H)=2.3 mT) and a broad singlet were detected in genomic DNA/melanin but were not distinguishable in irradiated Caucasian skin. Protein and lipid radicals (n=6 in Caucasian skin) were minimal in Afro-Caribbean skin (n=4) and intermediate skin pigmentations were variable (n=3). In irradiated Afro-Caribbean skin a shoulder to the melanin radical (also in UVA-irradiated pigmented melanoma cells and genomic DNA/melanin and intrinsic to pheomelanin) was detected. In this sample group, protein (but not lipid) radical adducts decreased directly with pigmentation. ESR/spin trapping methodology has potential for screening skin susceptibility to aging and cancer-related radical damage and for measuring protection afforded by melanin, sunscreens, and antiaging creams.     

UVA filters in sun-protection products: regulatory and biological aspects

This review of published in vitro and in vivo studies concerning the biological effects of ultraviolet A (UVA; 320-400 nm) radiation illustrates the evidence for combining UVA and UVB filters in sun-protection products. These data have led to the development of new sunscreens as well as methods to evaluate their efficacy. After listing the UVA filters available and briefly noting the requirements for a high SPF, broad-spectrum sunscreen, the methods for evaluating the level of UVA protection will be described. This article also summarizes several studies looking at the prevention of erythema, pigmentation, DNA damage, photoimmunosuppression, photoaging and photodermatoses. These data demonstrate in vitro and in vivo that only well-balanced UVA-UVB sunscreens, absorbing over the entire UV spectrum are able to prevent or significantly reduce the associated biological damage.     

Cytotoxicity and genotoxicity induced by the photochemical alkoxyl radical source N-tert-butoxypyridine-2-thione in L5178Y mouse lymphoma cells under UVA irradiation

The cell-damaging effects of N-tert-butoxypyridine-2-thione (tBuOPT), which generates alkoxyl and thiyl radicals on photolysis, have been investigated in L5178Y mouse lymphoma cells. The UVA irradiation of 2.5 microM tBuOPT inhibits strongly cell growth and cell viability, causes pronounced membrane damage, and induces micronuclei. Without irradiation, tBuOPT does not cause any cell damage at 2.5 microM concentration. The phototoxicity of tBuOPT is effectively inhibited by the radical scavenger glutathione, while the photogenotoxicity (micronuclei induction) is not affected by this strong hydrogen-atom donor. Thus, for the cytotoxicity and genotoxicity different reactive species seems to be responsible. The cytotoxicity is presumably caused by oxyl radicals, which are derived from tert-butoxyl radicals generated by photocleavage of tBuOPT, while in the genotoxicity the less reactive pyridyl-2-thiyl radicals appear to play a role. These results demonstrate that N-alkoxypyridinethiones are useful photochemical sources of oxyl and thiyl radicals to elucidate biological effects caused by these free radicals.     

Light-dependent degradation of the D1 protein in photosystem II is accelerated after inhibition of the water splitting reaction

Strong illumination of oxygen-evolving organisms inhibits the electron transport through photosystem II (photoinhibition). In addition the illumination leads to a rapid turnover of the D1 protein in the reaction center of photosystem II. In this study the light-dependent degradation of the D1 reaction center protein and the light-dependent inhibition of electron-transport reactions have been studied in thylakoid membranes in which the oxygen evolution has been reversibly inhibited by Cl- depletion. The results show that Cl(-)-depleted thylakoid membranes are very vulnerable to damage induced by illumination. Both the D1 protein and the inhibition of the oxygen evolution are 15-20 times more sensitive to illumination than in control thylakoid membranes. The presence, during the illumination, of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) prevented both the light-dependent degradation of the D1 protein and the inhibition of the electron transport. The protection exerted by DCMU is seen only in Cl(-)-depleted thylakoid membranes. These observations lead to the proposal that continuous illumination of Cl(-)-depleted thylakoid membranes generates anomalously long-lived, highly oxidizing radicals on the oxidizing side of photosystem II, which are responsible for the light-induced protein damage and inhibition. The presence of DCMU during the illumination prevents the formation of these radicals, which explains the protective effects of the herbicide. It is also observed that in Cl(-)-depleted thylakoid membranes, oxygen evolution (measured after the readdition of Cl-) is inhibited before electron transfer from diphenylcarbazide to dichlorophenolindophenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Major differences in oxysterol formation in human low density lipoproteins (LDLs) oxidized by *OH/O2*- free radicals or by copper.

The aim of our study was to determine the oxysterol formation in low density lipoproteins (LDLs) oxidized by defined oxygen free radicals (*OH/O2*-). This was compared to the oxysterol produced upon the classical copper oxidation procedure. The results showed a markedly lower formation of oxysterols induced by *OH/O2*- free radicals than by copper and thus suggested a poor ability of these radicals to initiate cholesterol oxidation in LDLs. Moreover, the molecular species of cholesteryl ester hydroperoxides produced by LDL copper oxidation seemed more labile than those formed upon *OH/O2*(-)-induced oxidation, probably due to their degradation by reaction with copper ions.