The FASEB Journal express article10.1096/fj.03-0359fje. Published online October 2, 2003.
The presence of nitrite during UVA irradiation protects from apoptosis Christoph V. Suschek,* Peter Schroeder,† Olivier Aust,† Helmut Sies,† Csaba Mahotka,‡ Markus Horstjann,¶ Heiko Ganser,¶ Manfred Mürtz,¶ Peter Hering,¶ Oliver Schnorr,* Klaus-Dietrich Kröncke,* and Victoria Kolb-Bachofen* *
Department of Immunobiology, †Institute of Biochemistry and Molecular Biology I, ‡Institute of Pathology, ¶Institute of Laser Medicine, Heinrich-Heine-University of Düsseldorf, 40225 Düsseldorf, Germany *Corresponding author: Christoph V. Suschek, Department of Immunobiology, Bldg: 23.12.02, Heinrich-Heine-University of Düsseldorf, P.O. Box 10 10 07 D-40001 Düsseldorf, Germany. E-mail:
[email protected] ABSTRACT Nitrite occurs ubiquitously in biological fluids such as blood and sweat, representing an oxidation product of nitric oxide. Nitrite has been associated with a variety of adverse effects such as mutagenicity, carcinogenesis, and toxicity. In contrast, here we demonstrate that the presence of nitrite, but not nitrate, during irradiation of endothelial cells in culture exerts a potent and concentration-dependent protection against UVA-induced apoptotic cell death. Protection is half-maximal at a concentration of 3 mM, and complete rescue is observed at 10 mM. Nitritemediated protection is mediated via inhibition of lipid peroxidation in a similar manner as seen with butylated hydroxytoluene, a known inhibitor of lipid peroxidation. Interestingly, nitritemediated protection is completely abolished by coincubation with the NO scavenger cPTIO. Using electron paramagnetic resonance (EPR) spectroscopy or Faraday modulation spectroscopy, we directly prove UVA-induced NO formation in solutions containing nitrite. In conclusion, evidence is presented that nitrite represents a protective agent against UVA-induced apoptosis due to photodecomposition of nitrite and subsequent formation of NO. Key words: apoptosis • DNA fragmentation • endothelial cells • lipid peroxidation • nitrite
N
itrite salts are chemical agents with widespread use in manufacturing processes but are also used as a preservative for meat and fish. Moreover, nitrite is used in medicine as a broncho- and vasodilating agent and as an antidote against cyanide poisoning. Nitrite has been associated with several toxic consequences such as methemoglobinemia, mutagenicity, and carcinogenesis. Recently, the National Toxicology Program of the U.S. Food and Drug Administration (1) began investigating these questions, and the outcome gave surprisingly little evidence for toxic consequences of long-term intake of daily doses up to 345 mg nitrite/kg by rats and mice. In addition, the 2-year intake study showed a significant protection from mononuclear cell leukemia with a nonsignificant tendency toward increases in forestomach hyperplasia and papilloma with highest doses (1). Nitrite is present in blood as a stable oxidation
product of NO synthesis or as a consequence of intake of nitrite-containing food, and it is also a constituent of sweat, assumed to be formed on the skin surface by commensural bacteria (2). It has been known for some time that daylight irradiation of vessels induces dilation, a phenomenon called photorelaxation (3). This effect is markedly potentiated by solutions containing nitrite (4, 5), indicating that under certain circumstances, nitrite may exhibit relaxing activities comparable to NO. Indeed, studies in environmental chemistry revealed that both the nitrite anion and nitrous acid (HNO2) in aqueous solutions undergo photodecomposition when irradiated with UV light at 200–400 nm, resulting in the formation of NO (6–9). The purpose of the present study was to examine whether nitrite may exhibit a similar protective activity against UVA-induced cell death as we had previously shown for endogenously produced iNOS-derived NO (10, 11) and thus might represent a NO source when irradiated with UVA light. Here we demonstrate that indeed during UVA exposure nitrite leads to formation of NO and to protection from apoptosis. Thus, the presence of nitrite on or in human skin as a constituent of sweat, or applied exogenously, might be considered as a nontoxic and effective NO donor during UVA exposure, conferring protection from UV-induced cell death. Implications extend to regulation of epidermal proliferation and differentiation, suppression of immunocompetent cell function, or pigmentation and tanning (12, 13). MATERIALS AND METHODS Materials Nitrite, azide, and nitrate, as sodium salts, were from Merck (Darmstadt, Germany). Catalase, butylated hydroxytoluene (BHT), endothelial cell growth supplement (ECGS), the Hoechst dye H33342, Neutral Red solution (3%), type I collagen, collagenase (from Cl. histolyticum), rabbit anti-human von Willebrand Factor (vWF) antiserum were from Sigma (Deisenhofen, Germany). The rat endothelium-specific monoclonal antibody Ox43 was from Serotec (Camon, Wiesbaden, Germany). The monoclonal anti-eNOS antibodies were from Transduction Laboratories (Lexington, KY). Peroxidase-conjugated porcine anti-rabbit IgG was from DAKO (Hamburg, Germany). Peroxidase-conjugated goat anti-mouse IgG was from Zymed Laboratories (San Francisco, CA). Trypsin and EDTA were from Roche (Mannheim, Germany). The caspase inhibitor ZVAD was from Enzyme Systems (Livermore, CA). 1H-imidazol-1-yl-oxy-2-(4carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-3-oxide (cPTIO) and N-(dithiocarbamoyl)-N-methyl-Dglucamine (MGD) were from Alexis Biochemicals (Grünberg, Germany). RPMI 1640 (endotoxin free) and fetal calf serum (FCS, endotoxin free) were from PAA Systems (Linz, Austria). The RPMI medium was custom made by substituting Mg nitrate for Mg sulfate. S-Nitrosocysteine (SNOC) and (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAMA/NO), respectively, were synthesized as previously described (14, 15). The UVA source (340–400 nm with a maximum peak at 366 nm, 4000 W lamp) and the dosimeter were purchased from Sellas Medizinische Geräte (Gevelsberg, Germany). All buffer and culture media were free from nitrate and nitrite.
Animals Male Wistar rats (~30 days old and 150 g body weight) were obtained from the university breeding facility. All animals received a standard diet and tap water ad libitum. Cell culture and cellular characterization Rat aorta endothelial cells (EC) were isolated by outgrowth from rat aortic rings as previously described (16). In briefly, aortic segments were placed on top of a collagen gel (1.8 mg collagen/ml) in 24-well tissue culture plates and incubated in RPMI 1640 with 20% FCS and 100 µg/ml ECGS for 4–6 days depending on the degree of cellular outgrowth. Aortic explants were then removed and cells were detached with 0.25% collagenase in HBSS and replated onto plastic culture dishes in RPMI 1640+20% FCS. Cells were subcultured for up to 10 passages, and removal from culture dishes for each passage was performed by treatment with 0.05% trypsin/0.02% EDTA in isotonic NaCl for 3 min. EC were characterized by using a crossreacting rabbit-anti-human-vWF antiserum, the rat vascular endothelium-specific monoclonal antibody Ox43, the monoclonal mouse anti-eNOS-antibody, and the respective secondary peroxidase-conjugated porcine anti-rabbit IgG or peroxidase-conjugated goat anti-mouse IgG antisera exactly as previously described (16). EC showed the antigen phenotype vWFhigh Ox43high eNOShigh exactly as previously described (17). The labeling experiments showed that the cell cultures were free from contaminating cells, because the respective staining patterns were found in virtually all cells (data not shown). Experimental design All measurements were performed with cells from passages 2–8. EC (2×105) were cultured in 12-well tissue culture plates in a humidified incubator at 37°C in RPMI 1640/10% FCS. Apoptosis of endothelial cells was induced by irradiation with UVA (3–38 J/cm2). During UVA challenge, cells were maintained in nitrate- and nitrite-free RPMI 1640 medium without phenol red and only supplemented with 0.25 mM HEPES. In addition, during UVA irradiation, resident EC were incubated with the respective additives at concentrations indicated, and 24 h later, the relative number of living EC was determined. Inhibition of caspases was obtained by incubation with the pan-caspase inhibitor ZVAD (40 µM). Depletion of H2O2 or inhibition of lipid peroxidation was achieved by incubation with 2000 U/ml catalase or with 10 µM BHT present for 4 h before and during and for 2 h after UVA challenge. We used the NO scavenger cPTIO to examine NO-mediated effects during UVA exposure. The colored NO scavenger oxyhemoglobin could not be used in our experiments, whereas cPTIO at the concentrations used does not adsorb UVA radiation. In accordance to the current literature, cPTIO is the most frequently used NO scavenger and is used as a “specific” NO scavenger in systems where NO and reactive oxygen species such as superoxide, hydroxyl radical, and hydrogen peroxide are produced in parallel (18–20). Determination of growth rates and viability of cell cultures Cell growth was determined by Neutral Red staining (21). EC were incubated for 90 min with Neutral Red (1:100 dilution of a 3% solution), washed twice with PBS, dried completely, and
lysed with isopropanol containing 0.5% of 1N HCl. Extinction of the supernatants, which show a linear correlation to the number of living cells, was then measured at 530 nm. In addition, viability of EC was routinely controlled at the beginning and the end of every experiment using the Trypan blue exclusion assay or propidium iodide staining. Viable cells were defined as cells excluding Trypan blue or propidium iodide and positive for respiratory activity as determined by Neutral Red. Detection of nuclear chromatin condensation and nuclear fragmentation At various time points after UVA challenge, EC (2×105) grown in 6-well culture plates were washed with PBS and stained with Hoechst H33342 (8 µg/ml) for 5 min. Nuclei were visualized using a Zeiss fluorescence microscope using the Zeiss filter set 02 (excitation: 320–380 nm, emission: LP 420 nm). In each sample, a minimum of 400 cells were counted, and condensed or fragmented nuclei were expressed as percent of total nuclei. Detection of DNA strand breaks DNA strand breaks of EC (1×104) grown on 8-well chamber-tec slides were visualized by the in situ nick translation method (22) at various time points after UVA irradiation. Endogenous peroxidase activity was blocked in acetone-fixed cells with methanol containing 0.3% H2O2 for 30 min. The nick translation mixture consisted of 3 µM biotin-dUTP; 5 U/100 µl Kornberg polymerase; and 3 µM each dGTP, dATP, dCTP, 5 mM MgCl2, 0.1 mM dithiothreitol, and 50 mM Tris-HCl, pH 7.5, and the reaction was performed at room temperature for 20 min. Slides were washed in PBS and processed for immunocytochemical detection of biotin-labeled UTP by peroxidase-labeled avidin, followed by staining with diaminobenzidine. In each sample, a minimum of 500 cells were counted, and labeled nuclei were expressed as percent of total nuclei. Determination of lipid peroxidation Lipid peroxidation was determined in resting EC (2×107) or cells irradiated with UVA (33 J/cm2) in the absence or presence of the respective additives. After a period of 18 h after UVA challenge, lipid peroxidation in EC cultures was stopped by addition of BHT (10 µM). Cells were then lysed by repeated freezing and thawing. Lipid peroxidation was measured by determination of thiobarbituric acid-reactive substances (TBARS) with HPLC and expressed as malondialdehyde (MDA) equivalents. In brief, 500 µl cell lysate were deproteinized with 350 µl trichloroacetic acid (TCA, 20%). Thiobarbituric acid (TBA; 500 µl, 2%, stabilized with 100 µM BHT) was added to the reaction mixture. After heating for 15 min at 95°C, the reaction mixture was centrifuged and 1 ml of the supernatant was chromatographed on a 5 µm endcapped C-18 reversed-phase column (LiChrospher, 250×5 mm, RP-18, Merck) equipped with LaChrom L7100 (Merck-Hitachi) solvent delivery system and an autosampler (655 A 40, Merck-Hitachi). The flow rate was held at 0.75 ml/min. Detection of MDA was carried out using a fluorescence detector (LaChrom L-7480, Merck-Hitachi) and a D-2500 Chromato-integrator (Merck-Hitachi). Diluted malonaldehyde (bis methyl acetal) solutions served as calibration standards. Peak heights were determined.
Detection of NO release from UVA-irradiated nitrite solution using electron paramagnetic resonance spectroscopy and Faraday modulation spectroscopy The NO concentration in solution was estimated using electron paramagnetic resonance (EPR) spectroscopy. To trap NO, we used the water-soluble MGD2-Fe3+ complex (23). This iron-MGD complex was prepared immediately before use by mixing appropriate amounts of FeCl3 and MGD in 100 mM Tris-HCl (pH 7.2). To quantitate in situ generated NO, we added 55 µl 10 mM NaNO2 solution irradiated with 33 J/cm2 UVA light to 5 µl concentrated MDG2-Fe3+, resulting in a final concentration of 75 mM MGD/15 mM Fe3+ (24). As controls, nonirradiated NaNO2 solution or irradiated buffer without NaNO2 were treated the same way. For comparison, 75 mM MGD/15 mM Fe3+ were treated with various concentrations of the NO donors SNOC (10 min at room temperature) or MAMA/NO (30 min at 37°C). A Miniscope MS 100 EPR spectrometer (Magnettech GmbH, Berlin, Germany) was used to record the EPR spectra at room temperature. The spectrometer settings were center field, 335 mT; sweep width, 18.1 mT; modulation frequency, 100 kHz; modulation amplitude, 0.1 G; scan time, 30 s; and microwave power, 100 mW. Faraday modulation spectroscopy (FAMOS) is a sensitive method to measure paramagnetic molecules (e.g., radicals and ions) in the gas phase. It exploits the fact that the interaction of paramagnetic molecules with laser light changes in the presence of a magnetic field. This method is similar to laser magnetic resonance (LMR) (25) except that a frequency-tunable laser is used. Probing a NO absorption line at 5.211 µm, where NO is the only stable paramagnetic molecule showing absorption, ensures that NO is detected without cross-interference. Here, a quantum cascade laser (QCL) (26) sends linearly polarized mid-infrared light (λ=5.2 µm) into a sample cell containing the gas sample. In the absence of a magnetic field, the light is completely blocked by a crossed polarizer placed behind the sample cell. If NO is present and a magnetic field is applied, the polarization axis of the light is slightly tilted (Faraday effect). This tilted fraction passes the polarizer and is detected. The signal strength is proportional to the NO concentration in the gas sample. With our experimental setup, the detection accuracy is 50 ppb NO with a time resolution of 300 ms. Note that no cross-interference by nonparamagnetic molecules such as water disturb the measurements. Because of this and the high sensitivity, no further processing of the gas sample is needed. Statistical analysis Values were reported as means ± SD. For statistical analysis, we used ANOVA followed by an appropriate post hoc multiple comparison test (Tukey method). P < 0.05 was considered significant. RESULTS When present during irradiation, nitrite but not nitrate protects from UVA-induced apoptosis Irradiation of endothelial cells with UVA leads to apoptosis in a dose-dependent manner as described previously (10, 11). Using irradiation with a dose of 33 J/cm2 UVA, the appearance of apoptotic markers such as nuclear chromatin condensation and nuclear fragmentation as well as
DNA strand breaks are highly increased after a delay of 4–8 h as quantified by Hoechst staining or in situ nick translation (Fig. 1A, Table 1). UVA-induced cell death is completely inhibited by the pan-caspase inhibitor ZVAD (40 µM), by catalase (2000 U/ml), or by the inhibitor of lipid peroxidation BHT (10 µM), indicating the involvement of H2O2 formation as well as lipid peroxidation as main events (Fig. 1B, Table 1). Importantly, the presence of nitrite (1–10 mM) in culture supernatants during UVA challenge protects from apoptosis in a concentration-dependent manner (Fig. 2, Table 1). Protection from onset of apoptosis is significant at ≥3 mM nitrite. At 10 mM, nitrite-mediated protection is optimal. As controls, nitrate at concentrations of up to 10 mM during UVA exposure does not show detectable effects nor is any protection achieved when adding nitrite (10 mM) after irradiation is completed (data not shown). Nitrite but not nitrate blocks the UVA-induced lipid peroxidation We had shown previously that the cascade of UVA-induced damage involves formation of different reactive oxygen species (ROS), leading to lipid peroxidation that represents the pivotal event initiating endothelial apoptosis (11), Thus, we next examined the effect of nitrite on UVAinduced lipid peroxidation. Resting EC were irradiated with UVA (33 J/cm2) in the absence or presence of additives indicated, and after 18 h, lipid peroxidation was assayed by quantitating malondialdehyde formation. As shown in Figure 2D, UVA irradiation leads to the known, substantial increase in lipid peroxidation. Control compounds such as catalase (2000 U/ml) that abolish UVA-induced H2O2 formation or BHT (10 µM), an effective inhibitor of lipid peroxidation, gave the expected protection from UVA-induced lipid peroxidation. Nitrite (1–10 mM) present during UVA challenge lowered MDA formation to the same extent as seen with the antioxidants. In contrast, nitrate (10 mM) did not lower MDA formation. Nitrite-mediated protection is abolished in the presence of the NO scavenger cPTIO Because UVA-irradiation and thus photodecomposition of nitrite may lead to NO formation, we next investigated whether NO might be the effector molecule of nitrite-mediated protection from UVA-induced cell death. We added the NO scavenger cPTIO (10–40 µM) to the nitritecontaining (10 mM) culture during UVA exposure and observed a concentration-dependent abrogation of the protective actions, as reflected by the level of cell viability (Fig. 3A) and lipid peroxidation (Fig. 3B). These data are in agreement with controls, showing that in the presence of the NO donor, SNOC (0.5 mM) cells are completely protected from UVA-induced lipid peroxidation and the onset of apoptosis (Fig. 4). Without UVA treatment, cPTIO or SNOC did not result in MDA formation (data not shown). UVA light converts nitrite to NO We next directly measured NO formation resulting from UVA-induced photodecomposition of nitrite solutions using FAMOS as well as electron paramagnetic resonance spectroscopy. For this, 10 mM NaNO2 dissolved in 0.9% NaCl/10 mM HEPES was irradiated with 33 J/cm2 UVA light and incubated with the specific NO trap MGD-iron complex as described in Materials and Methods. As shown in Figure 5A–C, EPR spectroscopy revealed the characteristic three-line spectrum of the MGD2(NO)Fe2+ complex. Measurements performed every 2.5 min during 15
min irradiation revealed no significant change of the signal size (data not shown), suggesting a steady-state NO formation. Neither nonirradiated NaNO2 nor irradiated buffer without NaNO2 nor irradiation of a NaNO3 solution (10 mM) gave any positive signal (data not shown). To quantify the NO-induced signal size, we compared NO generated from various concentrations of the NO donors SNOC or MAMA/NO (Fig. 5D–F). Irradiation of 10 mM NaNO2 generated a signal size comparable to 50 µM SNOC or 25 µM MAMA/NO, which generates 2 NO per molecule (data not shown). In addition, we determined gas-phase NO concentration and production rate during UVA exposure of nitrite solutions as a further parameter for photodecomposition of nitrite in solution using FAMOS (Fig. 5G). Irradiation of a 1 mM nitrite solution led to a steady-state NO concentration of 372 ppb in the gas phase and a NO production rate of 55 pmol/s, with 3 mM nitrite 990 ppb and 152 pmol/s, and with 10 mM nitrite, 4800 ppb and 768 pmol/s. The presence of antioxidants such as the singlet oxygen quenchers azide (40 mM), histidine (100 mM), and imidazole (100 mM) or the ROS-degrading enzymes superoxide dismutase (1000 U/ml) and catalase (1000 U/ml) during or immediately after UVA exposure did not lead to any significant alterations in the efficiency of UVA-induced nitrite-derived NO formation (data not shown). Thus, ROS species are not involved in UVA-induced photodecomposition of nitrite. DISCUSSION The cytotoxic action of UVA radiation on mammalian cells is known to be oxygen-dependent, and ROS are involved in the action of UVA light on cells (27). Formation of singlet oxygen, superoxide, hydrogen peroxide, and lipid hydroperoxides is involved in the onset of endothelial cell apoptosis following irradiation with UVA light (11). Lipid peroxidation represents a late step in UVA-induced apoptosis as a prerequisite for mitochondrial cytochrome c leakage, the initiation step for the mitochondrial pathway of apoptosis (11). Lipid peroxidation results from the net abstraction of an allylic hydrogen atom of an unsaturated fatty acid by an initiating radical species. The lipid radical generated then reacts with O2, resulting in an alkylperoxy radical (LOO•), which can then react with a neighboring lipid to form another lipid radical that can also react with O2 and so on (28). Thus, a single initiating event can lead to the destruction/modification of numerous lipid molecules, resulting in loss of membrane integrity. Lipid peroxidation can be avoided or limited by inhibiting the generation or quenching of the initiating radical species. However, using the same experimental system in a previous investigation, data did not corroborate this role for NO but gave evidence for a radical-chain terminating activity (11). Indeed, the reaction of ·NO• with LOO• species predominates over the slower initiation of secondary peroxidation propagation reactions by LOO• with vicinal unsaturated lipids (29, 30). Irradiation of aqueous solutions of nitrite under various conditions with doses in the range of 200–400 nm has been previously reported to result in NO formation (6–9). In accordance with the prevailing opinion, in aqueous solutions the N-O bond of the nitrite ion will be cracked by the energy of light at a wavelength of 340–360 nm. ROS appear not to be involved in this process. Using the chemiluminescence reaction with ozone as a detection system for NO, we examined this possibility and found that neither the presence of singlet oxygen quenchers nor ROS-degrading enzymes during UV -irradiation significantly alters the NO formation (data not
shown). Thus, ROS apparently are not involved in the UVA-induced N-O bond splitting of the nitrite ion. We have examined the extent of NO generated during irradiation of nitrite in solution using UVA light (340–400 nm) at doses corresponding to doses used in skin photoprovocation studies. To quantify the amount of NO generated in situ in cell cultures, we performed EPR spectroscopy. We found that irradiation of a 10 mM nitrite solution yields a signal similar to that obtained with 50 µM SNOC or 25 µM MAMA/NO, compounds that both spontaneously generate 1 or 2 molecules of NO, respectively, per molecule. The signal size was constant when NO formation was determined during an irradiation time of 15 min. This suggests that irradiation of a neutral nitrite solution with UVA light generates a steady-state flux of NO, as was confirmed by FAMOS. Photodecomposition-mediated effects of nitrite are efficiently abolished in the presence of a NO scavenger, thus they are apparently a result of the action of NO. Indeed, we had previously shown that NO applied exogenously via NO-generating compounds or synthesized endogenously fully protects endothelial cells from ROS-mediated apoptosis or necrosis via inhibition of lipid peroxidation as well as enhanced expression of the antiapoptotic protein Bcl-2 (10, 11). Detailed analysis of the mechanism of light-induced nitrite decomposition (Fig. 6) revealed the formation of very reactive and potentially cytotoxic radical species such as O•–, OH•, or NO2• (7, 9). However, adverse effects on endothelial cell viability due to radical formation other than NO were not observed. The question arises whether photodecomposition of nitrite during sun exposure may serve a biological function. In skin, NO can be produced by all dermal cell types either via constitutively expressed NO synthases or via iNOS expression following cytokine challenge or exposure to UV light (31), and at least some of this NO remains in the skin in the form of its oxidation products, nitrate and nitrite. The nitrate or nitrite content of sweat appears to reflect the rates of cutaneous NO formation, and concentrations of up to 40 or 10 µM have been reported (2). However, under normal sun- and heat-exposed conditions, the sweat surface layer will undergo rapid concentration by fluid evaporation, and thereby local concentrations of nitrate/nitrite may be many times higher. Because NO has been found to play important roles in regulating skin pigmentation, cell growth, and cell differentiation (for review see refs 12, 13), it appears likely that photodecomposition of these sources and thus intradermal NO formation might exert an important physiological role for human skin. Regarding the question of the biological function and practicability of our findings, one might speculate as to what extent a supplementation of skin fluids with nitrite plus UVA might support physiological parameters of the skin or therapy of infectious diseases such as fungal infections, leishmaniosis or psoriasis, respectively, diseases for which evidence for a fundamental role of NO is suggested (12, 13, 32, 33). As already mentioned, in several animal studies, orally applied nitrite at daily concentrations of up to 74 mM displayed neither acute toxicity nor carcinogenic or teratogenic activity (1, 34–36). Thus, topical application of nitrite, representing less of a burden as compared with the mentioned animal studies, might be an easy and safe enhancing tool for clinical therapy involving UV irradiation but may also be used for UV protection, mimicking a naturally occurring principle.
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Table 1 Effect of antioxidants or UV-decomposed nitrite on UVA-induced apoptosis of endothelial cella Apoptotic Cells (%) +cPTIO (40 µM) Resident
1±1b
1±1b
UVA (33 J/cm2)
63±7
61±7
+ Nitrite (5 mM)
22±5b
59±10c
+ Nitrite (10 mM)
7±5b
58±13c
+ Nitrate (10 mM)
57±9
64±5
+ SNOC (500 µM)
3±1b
8±5b
+ Cysteine (500 µM)
58±9
57±8
4±2b
10±4b
8±8b
9±3b
9±3b
12±4b
+ Catalase (2000 U/ml) + BHT (10 µM) + ZVAD (40 µM)
a
Resting endothelial cells grown in the presence or absence of additives indicated were exposed to UVA radiation. The number of apoptotic cells, as determined by the appearance of cells with pyknotic nuclei, nuclear chromatin condensation, and nuclear fragmentation in Hoechst 33343-stained cells, was determined 16 h after the light stimulus. Values are the mean ± SD of five individual experiments. b P
