Effects of UVB Radiation on 4-Hydroxy-2-trans-nonenal Metabolism

and Toxicity in Human Keratinocytes. Giancarlo Aldini,* Paola Granata, Cristina Marinello, Giangiacomo Beretta,. Marina Carini, and Roberto Maffei Fac...
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Chem. Res. Toxicol. 2007, 20, 416-423

Effects of UVB Radiation on 4-Hydroxy-2-trans-nonenal Metabolism and Toxicity in Human Keratinocytes Giancarlo Aldini,* Paola Granata, Cristina Marinello, Giangiacomo Beretta, Marina Carini, and Roberto Maffei Facino Istituto di Chimica Farmaceutica e Tossicologica “Pietro Pratesi”, Faculty of Pharmacy, UniVersity of Milan, Viale Abruzzi 42, I-20131 Milan, Italy ReceiVed July 21, 2006

Endogenous lipid peroxidation (LPO)-derived aldehydes accumulate in human skin after photoexposure and contribute to the development of skin cytotoxicity and cancer. This study employed LC-ESI-MS and HPLC-UV-DAD techniques to investigate the effect of UVB radiation on the biotransformation and detoxification of the prototype aldehyde 4-hydroxy-2-trans-nonenal (HNE) using the human keratinocyte cell line (NCTC 2544). In parallel we followed the keratinocytes’ cytotoxic response to HNE through morphological analysis and cell viability assay. In UVB-unstressed keratinocytes, even a supraphysiological dose of the aldehyde (200 µM) was rapidly and completely cleared in metabolized form (free and GSH-conjugated metabolites) from the cell, with no signs of cytotoxicity. By contrast, UVB preexposure already at 1 MED (50 mJ/cm2, the minimal erythemal dose in humans) markedly impaired HNE metabolism. After 2 h of incubation, the relative amount of GSH-conjugated adducts dose-dependently dropped from 44% (unirradiated cells) to 22% at 3 MED as a consequence of UVBinduced GSH depletion (no impairment of GST A4.4 nor of G6PD activities was observed). The levels of free metabolites, 1,4-dihydroxy-trans-nonene (DHN) and 4-hydroxy-trans-2-nonenoic acid (HNA), were modified (+30% DHN, -22% HNA) only at 3 MED, in parallel to the AR and ALDH enzyme activity modulation. In addition, a dose-dependent increase of unmodified HNE was found in the extracellular medium, paralleled by a significant fraction of the HNE-incubated dose not recovered at the intra- or extracellular level. The impairment of HNE metabolism paralleled a dramatic cytotoxic response. These results provide a reasonable explanation for the massive accumulation of carbonyl toxins in human skin in vivo after photoexposure and shed light on the detrimental effects of UVB radiation in the presence of unmetabolized LPO metabolites. Introduction Several human keratinocyte-based culture models have been established for investigating skin toxicology (cytotoxicity), since keratinocytes are the most important cell population of the outermost layer of the skin involved in the metabolism of (1) endogenous lipid peroxidation (LPO)1 products, (2) exogenous agents topically applied, and (3) environmental and occupational toxins. It is therefore important to characterize the metabolic profile of different substances and the enzyme systems involved in their detoxification to understand the various manifestations of cutaneous toxicity (e.g., skin sensitization or irritation) in susceptible humans in response to endogenous or exogenous agents. The oxidative fragmentation of arachidonate and linoleate phospholipids present in the skin generates a set of highly reactive γ-hydroxyalkenals, the prototype being 4-hydroxy* To whom correspondence should be addressed. E-mail: [email protected]. Phone: +39-02-50317545. Fax: +39-0250317565. 1 Abbreviations: ALDH, aldehyde dehydrogenase; AR, aldose reductase; BSO, buthionine sulfoximine; DHN, 1,4-dihydroxy-trans-nonene; G6PD, glucose-6-phosphate dehydrogenase; 6PGD, 6-phosphogluconate dehydrogenase; HNA, 4-hydroxy-2-trans-nonenoic acid; HNE, 4-hydroxy-2-transnonenal; HNEm, unrecovered HNE fraction; HNEu, unmodified HNE fraction; LC-DAD, liquid chromatography with diode array detection; LCESI-MS/MS, liquid chromatography-electrospray ionization tandem mass spectrometry; LPO, lipid peroxidation; MED, minimal erythemal dose; OPA, o-phthaldialdehyde; UVB, 280-320 nm wavelengths.

trans-2-nonenal (HNE). HNE reactivity is because of the conjugation of the double bond with the aldehyde function, which, by tautomeric equilibrium, makes the C-3 carbon a strong electrophilic center, able to react with cellular nucleophiles and in particular with the nucleophilic sites of protein (through a Michael addition mechanism), such as the sulfhydryl groups of cysteines, the imidazole moiety of histidines, and the -amino group of lysine residues (1). HNE covalently modifies several proteins, as demonstrated by immunological and mass spectrometric methods, as recently reviewed by Carini et al. (2), among them glucose-6-phosphate dehydrogenase (3) and actin (4). Lipid peroxidation-derived carbonyls are responsible for intrinsic and photoinduced premature skin aging and ultimately for skin melanoma and squamous and basal cell carcinoma (5). Electrophilic R,β-unsaturated aldehydes can react with the stratum corneum epidermis proteins, inducing cytotoxicity and atopic dermatitis (6), with loss of skin hydration and barrier function. These aldehydes, when absorbed through the skin, can react with amino acid residues of nuclear proteins to initiate malignancy. In addition it has been found in vivo, using immunohistochemical techniques, that the content of carbonyl derivatives rises in parallel with physiological aging but peaks after photoexposure (7); in addition, investigations using the monoclonal antibodies anti-HNE, anti-acrolein, and anti-elastin detected HNE and acrolein in the elastotic material from sundamaged skin (actinic elastosis) (8).

10.1021/tx0601657 CCC: $37.00 © 2007 American Chemical Society Published on Web 02/17/2007

HNE Metabolism Impairment by UVB in Keratinocytes

In a previous study, working with a keratinocyte culture (NCTC 2544 cell line), we used LC-ESI-MS/MS and LCDAD to characterize the metabolic products of HNE and clarify its detoxification mechanism (9). Since solar radiation plays a primary role in skin photooxidative damage (10, 11) and markedly affects the cutaneous detoxification pathway, the aim of the present study was to investigate the effects of UVB radiation on the qualitative and quantitative profile of HNE metabolism in human keratinocytes. This is in view to explaining whether the accumulation of reactive carbonyl species (RCS) covalent adducts with macromolecules observed in vivo after sun exposure (i.e., carbonylated proteins in actinic elastosis) (8) is in any way related to an impairment of RCS metabolism. In parallel, we evaluated the cell cytotoxic response to HNE in native and UVB-prestressed keratinocytes. The results indicate that preexposure of keratinocytes to even minimal erythemal UVB radiation (1 MED, minimal erythemal dose) drastically impairs HNE metabolism, leading HNE to covalently form adducts with macromolecules and thus to induce carbonylation damage.

Materials and Methods Chemicals. All chemicals and reagents were of analytical grade, purchased from Sigma-Fluka-Aldrich Chemical Co. (Milan, Italy). HPLC-grade and analytical-grade organic solvents were from Merck (Bracco, Milan, Italy). HPLC-grade water was prepared with a Milli-Q water purification system. 4-Hydroxynon-2-enal diethyl acetal (HNE-DEA), 1,4-dihydroxy-trans-nonene (DHN), and 4-hydroxy-trans-2-nonenoic acid (HNA) were synthesized according to the literature (12, 13). HNE was prepared from HNE-DEA by 1 mM HCl hydrolysis (1 h at room temperature) and quantified by UV spectroscopy (λmax ) 224 nm,  ) 13750 M-1 cm-1). Calcein acetoxymethyl ester (calcein AM) was purchased from Molecular Probes (Space Import-Export, Milan, Italy). Carnosine was kindly supplied by Flamma SpA (Chignolo d’Isola, Bergamo, Italy). Cell Cultures. Human skin epithelial cells NCTC 2544 (Flow Laboratories, Irvine, U.K.) were maintained in a phenol red-free culture medium (Dulbecco’s modified Eagle’s medium, DMEM, Sigma), supplemented with a solution containing 10% (v/v) fetal calf serum (Celbio), L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 µg/mL) (Sigma). Cultures were maintained at 37 °C in a humidified 5% CO2 incubator. The cells were grown to confluence (80-90%) in 25 cm2 flasks. For the UVB/HNE experiments, cells were seeded at a density of 1.0 × 106/mL in a 24-well plate (Iwaki, Bibby srl, Milan, Italy). Apparatus. Spectrophotometric and fluorimetric studies were performed with a computer-aided Perkin-Elmer Lambda 16 spectrophotometer and a computer-aided Perkin-Elmer LS50B luminescence spectrometer (Perkin-Elmer, Monza, Italy). For the calcein assay, the hydrolyzed fluorescent product was measured by a Victor2 Wallac multiwell fluorescent plate reader (Perkin-Elmer, Monza, Italy). HPLC analyses were done on an HPLC Surveyor LC system (Thermoquest, Milan, Italy) equipped with a quaternary pump, a Surveyor UV/vis diode array programmable detector (6000 LP) at high sensitivity (operating at fixed wavelengths, 223 and 202 nm, and in scan mode), a vacuum degasser, a thermostated column compartment, and a Surveyor autosampler (200-vial capacity). Data processing was performed by the ChromQuest 4.0 version software (Thermo Finnigan, Milan, Italy). The LC-MS experiments were carried out on a Surveyor LC system (ThermoQuest), equipped with a quaternary pump, a UV/vis diode array programmable detector (6000 LP), a Surveyor AS autosampler, a vacuum degasser, and Xcalibur software and connected to a Finnigan LCQ Advantage ion trap mass spectrometer (ITMS) (ThermoQuest). Morphological analyses of living cells were carried out in contrast phase by using normal-phase microscopy and a 20× lens (Eclipse TS 100, Nikon). Treatment of Keratinocytes. In the first set of experiments (Figure 1A) keratinocytes were washed three times with prewarmed

Chem. Res. Toxicol., Vol. 20, No. 3, 2007 417

Figure 1. Experimental protocol: (A) keratinocytes treated only with HNE (200 µM), (B) keratinocytes preexposed to different UVB doses (50, 100, and 150 mJ/cm2 corresponding to 1, 2, and 3 MED, respectively) and then treated with HNE (200 µM).

Dulbecco’s phosphate-buffered saline (PBS) to remove the culture medium and then exposed to HNE dissolved in PBS at a final concentration of 200 µM. The plates were incubated at 37 °C in 5% CO2 for 2 h, after which we checked the formation of HNE metabolites, GSH content, and cell viability (Figure 1, observation time 1). The last parameter was checked again 10 h after the beginning of the experiment (Figure 1, observation time 2). In the second set of experiments (Figure 1B) keratinocytes treated as above were preexposed to UVB radiation (1, 2, and 3 MED corresponding to 50, 100, and 150 mJ/cm2) in the presence of PBS. For UVB radiation two Philips TL20W:12 fluorescence tubes (Sara srl, Castellanza, VA, Italy) were used, emitting a continuous spectrum between 280 and 320 nm, with peak emission at 312 nm (the delivered UVB doses were measured with a Vilber Lourmat VLX-3W radiometer UVB probe, 312 nm). After irradiation, PBS was replaced with culture medium, the cells were incubated for an additional 4 h, and phase I (AR and ALDH) and phase II (GSTA44, G6PD) enzyme activities were determined (observation time 3). The prestressed keratinocytes were then washed free of the culture medium and incubated with 200 µM HNE in PBS for 2 h, after which we evaluated the (1) HNE metabolism (in extracellular and intracellular media), (2) intracellular GSH content, and (3) cell viability and morphology (observation time 1). Keratinocytes were washed again, as above, and incubated with culture medium for 4 h, and the cell viability and morphology were checked as in the first experiment (10 h from the beginning of the experiment) (Figure 1, observation time 2). Native cells were keratinocytes subjected to the various washing procedures but not exposed to chemical or physical stresses. When carnosine was used as the HNE quencher, keratinocytes were preexposed to UVB radiation (1 MED); after 4 h the cells were incubated with HNE (200 µM) together with carnosine (20 mM) for an additional 2 h and then processed as above. GSH Analysis. Intracellular GSH was determined by the method of Hissin and Russell (14), suitably modified. Briefly, the culture medium was discarded, and the cells were washed three times, scraped off, and suspended in PBS-EDTA (0.1 M, pH 7.4, 0.5 mL/well). Aliquots of the keratinocyte suspension were centrifuged at 2000 rpm, and the supernatant was discarded. The pellets were homogenized in 1 mL of a 6.25% solution of metaphosphoric acid and centrifuged for 5 min at 18000 rpm. A 0.7 mL sample of the supernatants was added to 3.3 mL of phosphate-EDTA buffer, the resulting mixture was vortexed, and 500 µL aliquots were transferred to the reaction mixture consisting of 4.4 mL of phosphate-EDTA buffer and 100 µL of OPA (1 mg/mL in methanol). Fluorescence was measured after incubation at 25 °C for 30 min at 350 nm (λexc, bandwidth 5 nm) and 420 nm (λem, bandwidth 5 nm). The GSH content was calculated as nmol/mg of protein. Protein was determined by a modified Lowry’s method (15) using bovine serum albumin as the standard. Cell Viability. Cell viability was checked by the calcein assay (16) and by morphological analysis (phase contrast microscopy).

418 Chem. Res. Toxicol., Vol. 20, No. 3, 2007 The cytotoxicity of treated keratinocytes (UVB, HNE, UVB/HNE) was calculated in relation to that of native keratinocytes. Enzyme Activities. Glutathione transferase (GST A4-4), glucose6-phosphate dehydrogenase (G6PD), aldehyde dehydrogenase (ALDH), and aldose reductase (AR) were determined in native, BSO-treated, and UVB-irradiated keratinocytes (1, 2, and 3 MED). At the end of the incubation period (4 h after UVB irradiation, 27 h after BSO treatment), cell homogenates obtained by scraping the monolayers followed by cell sonication at 50 W for 5 s were centrifuged at 18000 rpm for 10 min, and different aliquots of the supernatant were used for the enzyme assays. GST activity using HNE as the substrate was measured by suitably modifying the original spectrophotometric method (17), since it was not sensitive enough in keratinocytes. The standard assay mixture, containing 50 µM HNE, 0.25 mM GSH, 0.05 mM DTPA, and 600 µL of the supernatant from the cell lysate (1 mL final volume in PBS 0.1 M at pH 6.5), was incubated for 5 min at 30 °C. To estimate the amount of HNE undergoing nonenzymatic conjugation with GSH, a blank was prepared by replacing the cell supernatant with 600 µL of PBS. A 200 µL aliquot of each incubate was deproteinized with 200 µL of 0.7 M perchloric acid and centrifuged at 18000 rpm for 10 min, and the supernatant (20 µL) was injected into the HPLC Surveyor system. Separations were performed on a Phenomenex Synergi Fusion RP column (150 mm × 2 mm i.d., 4 µm) protected by a guard column, Synergi Fusion RP (CPS Analitica, Milan, Italy), and kept at 37 °C under the following conditions: isocratic elution with 60% solvent A (H2O/CH3CN/H3PO4, 90:10: 0.1, v/v/v) and 40% solvent B (CH3CN/H2O, 90:10, v/v), flow rate 0.35 mL/min, detection 224 nm. The results were calculated as (nmol of HNE consumed/min)/mg of protein by subtracting the amount of HNE determined in blanks from that found in the assay mixture and expressed as mU/mg of protein, where mU ) 1 (nmol of conjugated HNE/min)/mL. Determinations were run in quadruplicate. The G6PD activity was determined as previously described (18) by spectrophotometrically measuring at 340 nm the rate of production of NADPH over a 6 min observation period. Since 6PGD, the second enzyme of the pentose phosphate pathway (PPP), also produces NADPH, the total dehydrogenase activity (G6PD + 6PGD) and the 6PGD activity were determined separately. The G6PD activity was calculated by subtracting the activity of 6PGD from the total. Briefly, 50 µL of cell lysates (native, UVB-stressed, and BSO-treated keratinocytes) was added to 950 µL of assay buffer (50 mM Tris, 1 mM MgCl2, pH 8.1) containing NADP (100 µM) and the two substrates glucose-6-phosphate (G6P; 200 µM) and 6-phosphogluconate (6PG, 200 µM) to obtain the total dehydrogenase activity and only 6PG to determine the 6PGD activity. The results were expressed as mU/mg of protein, where mU ) 1 (nmol of NADPH formed/min)/mL. The ALDH activity was determined according to the method reported by Boesch et al. (19) with slight modifications. Briefly, 0.5 mL of the supernatant from the cell lysate was added to the assay mixture containing 2 mM NAD and 10 mM propionaldehyde in 50 mM PBS (pH 7.4, final volume 1 mL) preincubated for 3 min at 30 °C. The increase in absorbance due to NADH production was monitored at 340 nm using an extinction coefficient () of 6300 M-1 cm-1. The enzyme activity was expressed as mU/mg of protein, where mU is defined as 1 (nmol of NADH formed/min)/mL. The AR activity was determined as described by Shrivastava et al. (20) using 0.5 mL of the keratinocyte supernatant. The enzyme activity was expressed as mU/mg of protein, where mU is defined as 1 (nmol of NADPH oxidized/ min)/mL. Qualitative and Quantitative Determination of HNE Metabolites. GSH-dependent biotransformation products of HNE were characterized by LC-ESI-MS directly on the extracellular medium and on the cell homogenate supernatants obtained by scraping off the monolayer, homogenizing, precipitating the protein with trichloroacetic acid (TCA; 10%, v/v), and centrifuging at 18000 rpm for 20 min (9). Aliquots (20 µL) of each sample were injected into the LC-MS apparatus using the following conditions: isocratic elution with trifluoroacetic acid (TFA)/CH3CN/H2O (0.05:4:6, v/v/v) at a flow rate of 0.2 mL/min, capillary temperature 250 °C, spray voltage

Aldini et al. 5 kV, capillary voltage 5 V, sheath gas flow rate 2 L/min, auxiliary gas flow rate 0.5 L/min. The spectra were acquired in negative and positive ion modes, with a scan range from m/z 100 to m/z 500 (scan rate 0.5 scans/s). In unirradiated cells, the amount of each GSH-conjugated metabolite (1b, 2b, 3b, 4b) was calculated as the mass chromatogram peak area (arbitrary units, au) (9) and the overall GSHdependent HNE metabolism by subtracting the amounts of native HNE and of oxidized and reduced HNE metabolites (HNA, DHN) formed in the unirradiated cells from the nominal dose of incubated HNE (200 µM). In irradiated keratinocytes the formation of HNEGSH adducts was calculated with respect to the amount determined in unirradiated cells thus to evaluate the amount of unrecovered HNE fraction (HNEm). Unmodified HNE (HNEu) and the HNA and DHN metabolites were determined directly (without sample manipulation/extraction) on (a) an extracellular medium and (b) supernatants from protein precipitation of keratinocytes. Dichloromethane-ethanol (2:1) extraction (in the presence of trans-2-hexenoic acid as an internal standard) was used only to monitor HNE and metabolites in UVBstressed keratinocytes (to increase sensitivity). The extracts were dried under nitrogen and dissolved in the mobile phase. All the samples were then injected into the HPLC system. HNE and the metabolites HNA and DHN were characterized on the basis of their retention times (RTs) and UV-DAD spectra and by comparison with authentic standards and separated and quantified as previously described (9). The extraction recoveries for HNE, HNA, and DHN were calculated by spiking cell cultures with known amounts of the analytes (from 1 to 200 µM) before extraction. After five replicates, the mean recovery was 101.2% for HNE, 98.5% for HNA, and 97.8% for DHN. In UVB-prestressed keratinocytes, the percentage of HNE material (HNEm) that was not recovered as HNEu and/or metabolites even after exhaustive extraction with organic solvents of increasing polarity, was determined indirectly as the difference between the nominal dose of HNE (200 µM) and the total amount of HNE metabolites (HNEΣ) plus HNEu, according to the following equation: HNEm ) 100[200 - (HNEΣ + HNEu)]/200.

GSH Depletion. To induce GSH depletion, a specific inhibitor of GSH synthesis (BSO, 1 mM) was added to the culture medium, and the cells were incubated for 27 h. After washing, keratinocytes were treated with HNE (200 µM) and processed according to protocol A (Figure 1) to study the HNE metabolism and cytotoxicity or processed as described for determination of GST, G6PD, ALDH, and AR enzyme activities. Carnosine and Carnosine-HNE Adduct Identification by LC-ESI-MS Analysis. Carnosine and carnosine-HNE adduct were identified in the cell supernatant by the LC-ESI-MS method previously reported (21, 22). Separations were done by gradient elution from 100% H2O/CH3CN/heptafluorobutyric acid (A) (9:1: 0.01, v/v/v) to 80% CH3CN (B) in 12 min at a flow rate of 0.2 mL/min (injection volume 10 µL); the composition of the eluent was then restored to 100% A within 1 min, and the system was reequilibrated for 6 min. The sample rack was maintained at 4 °C. ESI interface parameters (positive ion mode) were set as follows: middle position, capillary temperature 270 °C, spray voltage 4.0 kV, sheath gas flow rate 2 L/min, auxiliary gas flow rate 0.5 L/min. Statistical Analysis. All data were expressed as the mean ( SD of four independent experiments. Prism software (GraphPad Inc., San Diego, CA) was used for one-way ANOVA (Tukey’s posttest). Differences were considered significant when p < 0.05.

Results and Discussion HNE Metabolism in Cultured Keratinocytes. As previously observed with lower HNE doses (9), a supraphysiological dose of the aldehyde (200 µM) was rapidly and completely cleared from the cell. Six different nonreactive, highly polar metabolites were found in the extracellular medium, all of phase I and phase

HNE Metabolism Impairment by UVB in Keratinocytes

Chem. Res. Toxicol., Vol. 20, No. 3, 2007 419

Figure 3. Morphological analysis (phase-contrast microscopy, observation time 2) of keratinocytes incubated for 2 h with PBS (native cells, A) or with 200 µM HNE in PBS (B).

Figure 2. Metabolic fate of HNE in a keratinocyte cell culture (200 µM, 2 h incubation): 4-hydroxy-2-nonenoic acid (HNA), 1,4-dihydroxy-2-nonene (DHN), S-(4-hydroxynonanal-3-yl)glutathione (1b), S-(1,4-dihydroxynonan-3-yl)glutathione (2b), S-(4-oxononan-1-ol-3-yl)glutathione (3b), and S-(4-oxononanal-3-yl)glutathione (4b). Table 1. Effects of UVB Radiation and GSH Depletion (BSO Treatment) on the HNE Metabolic Profile in Keratinocytesa DHN controls 1 MED 2 MED 3 MED BSO

13.1 ( 0.7 12.8 ( 0.5 14.1 ( 0.8 17.2 ( 0.4b 33.2 ( 2.1b

HNA

HNEu

HNEm

42.8 ( 1.0 0 0 41.3 ( 1.0 6.42 ( 0.08b 6.53 ( 0.15b b 40.8 ( 1.2 8.15 ( 0.17 9.11 ( 0.18b 33.5 ( 0.8c 11.42 ( 0.11b 15.37 ( 0.15b 65.1 ( 2.9b

GSH adducts 44.1 ( 0.5 33.0 ( 0.3b 27.9 ( 0.3b 22.5 ( 0.2b

a Data (relative formation of different metabolites) are expressed as a percentage of the incubated dose (200 µM). Each value is the mean ( SD of quadruplicate measurements. HNEu ) unmodified free HNE, and HNEm ) HNE fraction (% of the incubated dose) unrecoverable as free HNE plus metabolites, corresponding to HNE/metabolites covalently adducted to proteins/nucleic acids. b Significantly different from the value of control keratinocytes: p < 0.001. c Significantly different from the value of control keratinocytes: p < 0.01.

II enzyme products. These were HNA, DHN, S-(4-hydroxynonanal-3-yl)glutathione (1b), S-(1,4-dihydroxynonan-3-yl)glutathione (2b), S-(4-oxononan-1-ol-3-yl)glutathione (3b), and S-(4-oxononanal-3-yl)glutathione (4b), which were characterized by HPLC/UV-DAD and LC-MS as previously described (Figure 2) (9). Quantitative analysis indicated that the bulk of free metabolites (∼56% of dose) consisted of HNA (42.8 ( 1.0%) with a smaller amount of DHN (13.1 ( 0.7%) (Table 1). The remainder (∼44%) was made up of the conjugated GSH adducts: 1b (the precursor “Michael adduct”) is the primary metabolite, reaching 38.1 ( 0.9% of the total GSH adducts, followed by 4b (34.2 ( 0.9%), by 2b (20.5 ( 0.7%), and in a smaller amount by 3b (7.2 ( 0.4%). The amount of total conjugated adducts was further confirmed by calculating at the end of the incubation period the GSH consumption by HNE (by considering the basal and final levels of GSH and the amount of incubated HNE). Morphological analysis of the keratinocyte layer (at the last observation time, 10 h) showed no loss of cell adhesion, no membrane alterations, and no decrease in cell viability (Figure 3). Thus, the keratinocyte has a highly efficient detoxifying machinery against HNE, since it is able to eliminate the aldehyde through the cooperative interaction of phase I and II enzyme systems. This efficient metabolic pathway permits a rapid HNE detoxification even if the aldehyde is present at supraphysi-

ological concentrations (60 times those commonly found in vivo in nonirradiated skin) (1, 23). HNE Metabolism and Cytotoxicity in UVB Preexposed Keratinocytes. The metabolic profile of HNE in UVBprestressed keratinocytes was qualitatively identical to that in unstressed cells, but there was an UVB-dependent, significant alteration in the amount of metabolites formed (Table 1). In particular, the overall formation of oxidized and reduced metabolites was slightly affected by UVB irradiation (56% in unirradiated cells, 54% at 1 MED, 55% at 2 MED, 51% at 3 MED). Only at the highest UVB dose was a significant decrease of the oxidative pathway (HNA formation - 22%) in favor of the reductive one (DHN + 30%) observed. By contrast, the formation of GSH adducts dropped more significantly already at the lowest UVB dose: by 25% at 1 MED, 37% at 2 MED, and ∼50% at 3 MED (p < 0.001) (Table 1). Unlike unirradiated keratinocytes, at 1 MED we already found a fraction of unmodified HNE (6.42% of the nominal dose) in the extracellular medium and a fraction of incubated HNE (approximately 8%) which we were unable to characterize as HNE-derived metabolites by LC-MS or HPLC (Table 1). Different structurally related metabolites have been postulated: 4-oxo-2-trans-nonenal, S-(4-oxononanoic-3-yl)glutathione, S-(4hydroxyonanoic-3-yl)glutathione, and thioether HNE-cysteine (24); a detailed LC-MS investigation was made by searching in SIM (single ion monitoring) mode the [M + H]+ ions corresponding to the above proposed metabolites, but no chemical entities were found. In light of these results, we believe that the unrecovered HNE fraction must be HNE covalently adducted to keratinocyte proteins and nucleic acids, since it is not extractable from protein pellets even after exhaustive washings with organic solvents of increasing polarity. Very likely it is the fraction of HNE/ metabolites covalently bound to DNA through the formation of bulky exocyclic DNA adducts (25) or to cell proteins (26). Following exposure to 2 MED, the changes were much more evident: unmodified HNE in the extracellular medium rose to ∼8% of the dose, while the unextractable HNE material was around 9%; at 3 MED there was still an increase in both fractions (to 11% and ∼15%, respectively) (Table 1). Cell viability and morphology checked after 2 h of HNE incubation (observation time 1 in Figure 1) were found not significantly changed in all the experimental groups (over 90%, p > 0.05). At observation time 2, cell viability (Figure 4) and morphological analyses (Figure 5) showed that UVB radiation alone caused limited morphological damage and a slight, not significant, decrease in cell viability from 93.5 ( 2.1% to 91.6 ( 3.7% at the highest dose (p > 0.05). This finding is consistent with our previous study carried out in NCTC 2544 keratinocytes (27) using trypan blue exclusion and LDH leakage as viability assays and further confirmed in human keratinocytes (primary culture), where cell survival was assayed by measuring mito-

420 Chem. Res. Toxicol., Vol. 20, No. 3, 2007

Figure 4. Cell viability (observation time 2) measured by the Calcein assay. Keratinocytes were incubated in the absence (empty bars) and in the presence (filled bars) of 200 µM HNE following UVB exposure to 0, 1, 2, and 3 MED. Values are means ( SD of four independent experiments. Key: ***, p < 0.001; n.s., not significant with respect to the value of unirradiated cells.

Aldini et al.

Figure 6. Effect of UVB on GSH levels in keratinocytes. Values are means ( SD of four independent experiments. Key: ***, p < 0.001. Table 2. Effects of UVB Radiation and GSH Depletion (BSO Treatment) on the Enzyme Activities (Phase I and Phase II) Involved in HNE Detoxification in Keratinocytesa

controls UVB, 1 MED UVB, 2 MED UVB, 3 MED BSO

GST

G6PD

ALDH

AR

7.16 ( 0.80 6.44 ( 0.12 6.29 ( 0.35 6.55 ( 0.67 6.98 ( 0.72

442 ( 13 433 ( 15 453 ( 22 423 ( 16 445 ( 18

23.1 ( 0.7 18.2 ( 0.3c 20.2 ( 0.6d 10.3 ( 0.9b 27.6 ( 0.6b

11.8 ( 0.8 12.0 ( 0.9 13.1 ( 1.8 15.2 ( 0.2c 17.8 ( 0.6b

a Data are expressed as mU/mg of protein (mU ) (nmol/mL)/min). Each value is the mean ( SD of quadruplicate measurements. b Significantly different from the value of the controls (MED 0): p < 0.001. c Significantly different from the value of the controls (MED 0): p < 0.01. d Significantly different from the value of the controls (MED 0): p < 0.05.

Figure 5. Morphological analysis (phase-contrast microscopy, observation time 2) of unirradiated keratinocytes incubated for 2 h with PBS (A) and HNE (200 µM, B) and of UVB (1 MED)-irradiated keratinocytes treated with PBS (C) and HNE (200 µM, D).

chondrial activity (XTT test) (26). Contradictory results have been obtained in HaCaT keratinocytes, which seem to be more prone to cytotoxicity induced by UVB: at doses above 20 mJ/ cm2 cell survival, measured as LDH leakage, is not affected within 8 h postirradiation, but significantly decreases at 24 h (28). In contrast, Handa et al. (29) reported that UVB doses up to 30 mJ/cm2 have no effect on cellular viability quantified by MTT-based assay. All discrepancies may be the result of differences in the sunlamps employed in each study (amount of UVA/UVC emitted). In UVB- and HNE-stressed keratinocytes there was a striking cytotoxic response. At 1 MED morphological analysis already showed marked alterations to the keratinocyte structure (see phase-contrast microscopic analyses, Figure 5, panel d) and the appearance of several birefringent cells, indicative of massive cell necrosis. In parallel, cell viability (Figure 4) dropped from 93.5 ( 2.1% in unirradiated cells to 33.6 ( 3.1% at 1 MED (p < 0.001), 18.7 ( 1.6% at 2 MED, and 6.4 ( 2.9% at 3 MED (p < 0.001), indicating a synergistic cytotoxic effect between UVB stress and HNE. Effects of UVB on Glutathione Levels and Enzyme Activities in Keratinocytes. Four hours after UVB exposure to 1 MED, there was already a significant loss of GSH (∼24%) in keratinocytes compared to native cells (82.8 ( 3.6 vs 109.1 ( 4.2 nmol/mg of protein), which dose-dependently increased to 38% at 3 MED (66.8 ( 2.6 nmol/mg of protein) (Figure 6). These findings, and the previous ones on the metabolic pattern, indicate that the conjugative pathway (GSH adduct formation) is primarily affected by UVB radiation (already at 1 MED, Table

1) and that a high UVB dose alters the reductive/oxidative pathways. We therefore investigated whether UVB affectss besides the endogenous content of GSHsalso the key enzyme systems involved in the GSH-dependent detoxification pathway (GST and G6PD) and those involved in HNA and DHN formation (ALDH and AR, respectively) (Table 2). After 4 h of UVB exposure to 1 MED, no significant changes in all the enzyme activities (p > 0.05) were observed, except for that of ALDH, which decreased by ∼20% (p < 0.01). This effect was maximal (55% decrease) following exposure to 3 MED, a UVB dose that induced an opposite effect on AR activity (∼30% increase), in agreement with the reduced formation of HNA and the increased levels of DHN, respectively. The reduction in ALDH activity at 1 and 2 MED is not paralleled by a concomitant reduced production of HNA (Tables 1 and 2). This can be explained by considering a significant increase of the HNE substrate (due to the concomitant reduction in the GSHdependent pathway) that counteracts the ALDH activity impairment. Although no studies are reported in the literature on the effects of UVB on these enzymes in keratinocytes, a decrease in ALDH expression and induction of its aggregation was observed in cornea from mice exposed to 0.2 J/cm2 (30). Conversely, AR and G6PD were found to be decreased in rat lens (31), but under drastic conditions (UVB irradiation for 24 h). In addition, the results in Table 2 indicate that UVB radiation, even at high doses, does not affect the function of GST, nor that of G6PD, the latter involved in the formation of reducing equivalents responsible for regeneration of GSH from oxidized glutathione (GSSG). Opposite results have been reported for GST upon UVB exposure, but they are not comparable due to differences in experimental protocols (in vitro or in vivo studies, UVB dose, single or multiple doses, observation time after irradiation, etc.) For example, marked expression of GST A4-4 (not detectable in unirradiated rat skin cytosol) was observed only following very high UVB doses (4000-24000 mJ/cm2) (32). In cultured human keratinocytes and the epidermis of hairless mice, a single or multiple UVB radiation (50 mJ/cm2) significantly decreases GST activity 24 h postirradiation, without

HNE Metabolism Impairment by UVB in Keratinocytes

any changes in mRNA expression or protein amount, indicating a direct inhibitory effect on the enzyme activity (33). Mechanistic Explanation. Because the UVB-induced decrease of GSH content (up to 38% compared to that of native cells) seems to be a key factor in inducing impairment of HNE metabolism, leading to cell damage, we made a deeper investigation on the role of GSH in HNE metabolism, a study never done before in this cell culture model. Keratinocytes were preincubated with the specific inhibitor of GSH biosynthesis, BSO (1 mM for 27 h), and the HNE metabolic profile was evaluated in GSH-depleted keratinocytes (21.0 ( 1.8 nmol/mg of protein) by measuring the levels of HNE metabolites, the enzyme activities involved in their formation, and the cell toxicity response. As reasonably expected (Table 1), the levels of GSH adducts fell near the LC-MS quantization limit, but surprisingly, the cell cultures retained a highly satisfactory detoxifying capacity toward HNE. There was in fact a complete shift of the conjugative pathway toward the redox pathway, with the massive formation of HNA (130.2 ( 9.6 µM) and DHN (66.5 ( 3.6 µM), whose levels were close to 100% of the incubated dose (200 µM). In addition, no HNEu was found, and there were no signs of cytotoxicity: no significant difference in cell viability with respect to that of native keratinocytes (93.5 ( 5.9% vs 92.8 ( 4.8%, p > 0.05). From a mechanistic point of view, the different metabolic profile indicates that the selective depletion of GSH in the keratinocyte induces the activation of a cooperative mechanism between reducing and oxidizing enzymes, which act together to eliminate the cytotoxic aldehyde completely from the cell, maintaining survival and normal function. This assumption is confirmed by the up-regulation of ALDH and AR enzymes found in BSO-treated keratinocytes, whose activities were increased by 20% and 50%, respectively (Table 2). BSO did not significantly modify GST and G6PD activities. The role of ALDH in protecting cells from UV- and HNE-induced toxicity has already been demonstrated by transfecting a human corneal epithelial cell line lacking endogenous enzyme with human ALDH3A1 (34). In addition, the enhanced levels of the NAD(P)H pool generated by the catalytic function of the enzyme may help to maintain a reducing potential for pyridine nucleotide-dependent redox enzymes. Presently, we do not know whether other pyridine coenzyme-linked oxidoreductases such as alcohol dehydrogenase, catalyzing HNE reduction in the liver (35), are expressed in keratinocytes and/or up-regulated by GSH depletion, thus justifying in BSO-treated cells the significant shift toward the reductive pathway (DHN levels more than doubled with respect to those of native cells). Unlike that observed in the BSO model, in UVB-stressed keratinocytes the loss of GSH adducts was not paralleled by an increase in redox metabolites, overall accounting for 50-55% (as in controls) at all the UVB doses. Probably, some signaling molecule/mediator, which in the BSO model regulates the crosstalk between enzyme systems and is needed for up-regulation of enzyme defense mechanisms, is destroyed by aerobic UVB radiation, and the cell becomes at least partially unable to metabolize HNE and to survive the burden of electrophilic species. This finding was clearly UVB dose-dependent, since the increase of the radiation energy led to a parallel increase of both unmetabolized and unrecoverable HNE material from the keratinocyte (Table 1). The aldehyde, while inside the cell in unmodified form, can interact with nuclear macromolecules (25), giving stable nucleic adducts such as the bulky exocyclic DNA adducts already identified by 32P-postlabeling in different tissues

Chem. Res. Toxicol., Vol. 20, No. 3, 2007 421

Figure 7. LC-MS analysis of extracellular medium from keratinocytes exposed to UVB/HNE in the presence of carnosine (20 mM): (A) total ion current (TIC) (mass range m/z 50-600); (B) selected ion chromatogram (SIC) at m/z 227, relative to [M + H]+ of carnosine; (C) SIC at m/z 383 relative to [M + H]+ of the carnosine/HNE Michael adduct.

of humans and rats and consequent massive necrotic damage (1, 36, 37). Alternatively, HNE can modify cell proteins, as demonstrated by immunoistochemical detection in human keratinocytes irradiated with UVA (10 J/cm2) plus UVB (0.05 J/cm2) (26) or in epidermal cells of hairless mice after chronic UVB exposure to 2 and 10 MED (38). Studies are in progress to identify in keratinocytes, through a combined immunohistochemical and mass spectrometric approach recently applied to other biological matrixes (39, 40), the target protein(s) for HNE adduction in our model. When carnosine, a specific HNE quencher (21), was coincubated with HNE (200 µM) after UVB exposure, keratinocytes were totally protected; there were no signs of cytotoxicity (data not shown). This maintenance of cell viability by carnosine was paralleled by a significant sparing of intracellular GSH (99.9 ( 0.6 nmol/mg of protein), by a reduced extrusion into the incubation medium of the GSH/HNE adducts ((1.97 ( 0.45) × 106 au vs (9.88 ( 0.10) × 106 au), and by the presence in the extracellular medium of the HNE/carnosine Michael adduct, detected by LC-MS analysis (Figure 7). These results unequivocally show that the necrotic damage in UVB/HNEexposed keratinocytes was due to a specific, direct interaction of HNE with DNA/proteins and not to an HNE-mediated GSH depletion, leading to intranuclear oxidative stress. Thus, in our model, the UVB-induced drop in GSH content and the parallel decrease in the formation of GSH adducts extruded from the cell after 1 MED irradiation must be considered onesthough not the onlysfactor in the loss of HNE metabolism; the seconds and most importantsfactor is the loss of the cooperative interaction between HNE-detoxifying enzymes. This leads to an accumulation of unmodified HNE inside and outside the cell, with subsequent diffusion to nucleic acids/proteins and ultimately necrotic cell damage.

Conclusions The present findings clearly indicate that keratinocytes in cell culture are strongly resistant to HNE toxicity, since phase I and II enzymes actively cooperate in its removal, ensuring cell survival and a high degree of protection. This cooperation is lost in UVB-prestressed keratinocytes, in connection with a loss

422 Chem. Res. Toxicol., Vol. 20, No. 3, 2007

of endogenous GSH and impairment of the interplay between phase II proteins more than their inactivation. In addition, GSH depletion by itself (as demonstrated in BSO-treated cells) did not affect the cells’ resistance to HNE toxicity, since HNE can still be effectively metabolized and removed through compensatory redox pathways due to induction of aldehyde dehydrogenase and aldose reductase, an effect never previously reported. These compensatory redox pathways are clearly impaired following UVB exposure, and further studies are required to gain a deeper insight into the underlying molecular mechanisms. Whatever the mechanismsand several other factors could be involvedswhat is clear from this study is that UVB impairs the HNE metabolism, and this leads to the synergistic cytotoxic effect of HNE and ultimately to cell death. If we extrapolate these in vitro findings to an in vivo situation, we can easily explain the increased accumulation of LPO-derived carbonyl species in the skin after photostress, as well as the presence in the elastotic skin of R,β-unsaturated aldehydes such as HNE and acrolein (5, 8). Another interesting finding regards the toxicological risk to the skin when exposed to UVB radiations in the presence of preformed, still unmetabolized HNE. In these conditions, the skin unable to detoxify the toxin is exposed to its detrimental effect since this reactive chemical, when absorbed, can react through Michael addition with nucleophilic residues, causing skin cytotoxicity and ultimately even carcinogenicity. Finally, sequestering agents of R,β-unsaturated aldehydes such as carnosine can be considered as useful tools for preventing UVinduced skin damage.

Aldini et al.

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Acknowledgment. This study was supported by COFIN2004 (Cofinanziamento Programma Nazionale 2004, Ministero dell’Istruzione, dell’Universita` e della Ricerca) and FIRST 2005 (Fondo Interno Ricerca Scientifica e Tecnologica, University of Milan).

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References

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(1) Esterbauer, H., Schaur, R. J., and Zollner, H. (1991) Chemistry and biochemistry of 4-hydroxynonenal, malondialdehyde and related aldehydes. Free Radical Biol. Med. 11, 81-128. (2) Carini, M., Aldini, G., and Maffei Facino, R. (2004) Mass spectrometry for detection of 4-hydroxy-trans-2-nonenal (HNE) adducts with peptides and proteins. Mass Spectrom. ReV. 23, 281-305. (3) Szweda, L. I., Uchida, K., Tsai, L., and Stadtman, E. R. (1993) Inactivation of glucose-6-phosphate dehydrogenase by 4-hydroxy-2nonenal. Selective modification of an active-site lysine. J. Biol. Chem. 268, 3342-3347. (4) Aldini, G., Dalle-Donne, I., Vistoli, G., Maffei Facino, R., and Carini, M. (2005) Covalent modification of actin by 4-hydroxy-trans-2-nonenal (HNE): LC-ESI-MS/MS evidence for Cys374 Michael adduction. J. Mass Spectrom. 40, 946-954. (5) Sander, C. S., Hamm, F., Elsner, P., and Thiele, J. J. (2003) Oxidative stress in malignant melanoma and non-melanoma skin cancer. Br. J. Dermatol. 148, 913-922. (6) Niwa, Y., Sumi, H., Kawahira, K., Terashima, T., Nakamura, T., and Akamatsu, H. (2003) Protein oxidative damage in the stratum corneum: Evidence for a link between environmental oxidants and the changing prevalence and nature of atopic dermatitis in Japan. Br. J. Dermatol. 149, 248-54. (7) Sander, C. S., Chang, H., Salzmann, S., Muller, C. S., EkanayakeMudiyanselage, S., Elsner, P., and Thiele, J. J. (2002) Photoaging is associated with protein oxidation in human skin in vivo. J. InVest. Dermatol. 118, 618-625. (8) Tanaka, N., Tajima, S., Ishibashi, A., Uchida, K., and Shigematsu, T. (2001) Immunohistochemical detection of lipid peroxidation products, protein-bound acrolein, and 4-hydroxynonenal protein adducts, in actinic elastosis of photodamaged skin. Arch. Dermatol. Res. 293, 363-367. (9) Aldini, G., Granata, P., Orioli, M., Santaniello, E., and Carini, M. (2003) Detoxification of 4-hydroxynonenal (HNE) in keratinocytes: characterization of conjugated metabolites by liquid chromatography/

(23) (24) (25)

(26) (27) (28) (29) (30)

(31) (32)

electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 38, 1160-1168. Thiele, J. J., Hsieh, S. N., Briviba, K., and Sies, H. (1999) Protein oxidation in human stratum corneum: susceptibility of keratins to oxidation in vitro and presence of a keratin oxidation gradient in vivo. J. InVest. Dermatol. 113, 335-339. Afaq, F., and Mukhart, H. (2001) Effects of solar radiation on cutaneous detoxification pathways. J. Photochem. Photobiol., B 63, 61-69. Alary, J., Bravais, F., Cravedi, J. P., Debrauwer, L., Rao, D., and Bories, G. (1995) Mercapturic acid conjugates as urinary end metabolites of the lipid peroxidation product 4-hydroxy-2-nonenal in the rat. Chem. Res. Toxicol. 8, 34-39. Rees, M. S., van Kuijk, F. J. G. M., Siakotos, A. N., and Mundy, B. P. (1995) Improved synthesis of various isotope-labelled 4-hydroxyalkenals and peroxidation intermediates. Synth. Commun. 25, 32252236. Hissin, P. J., and Russell, H. (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214-26. Markwell, M. A., Haas, S. M., Tolbert, N. E., and Bieber, L. L. (1981) Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol. 72, 296-303. Oral, H. B., George, A. J., and Haskard, D. O. (1998) A sensitive fluorimetric assay for determining hydrogen peroxide-mediated sublethal and lethal endothelial cell injury. Endothelium 6, 143-151. Ålin, P., Danielson, H., and Mannervick, B. (1985) 4-Hydroxyalk-2enals are substrates for glutathione transferase. FEBS Lett. 179, 267270. Tian, W. N., Pignatare, J. N., and Stanton, R. C. S. (1994) Signal transduction proteins that associate with the platelet-derived growth factor (PDGF) receptor mediate the PDGF-induced release of glucose6-phosphate dehydrogenase from permeabilized cells. J. Biol. Chem. 269, 14798-14805. Boesch, J. S., Lee, C., and Lindhal, R. G. (1996) Constituitive expression of Class 3 aldehyde dehydrogenase in cultured rat corneal epithelium. J. Biol. Chem. 271, 5150-5157. Srivastava, S., Chandra, A., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998). Identification of cardiac oxidoreductase(s) involved in the metabolism of the lipid peroxidation-derived aldehyde4-hydroxynonenal. Biochem. J. 329 (Pt 3), 469-475. Aldini, G., Granata, P., and Carini, M. (2002) Detoxification of cytotoxic alpha,beta-unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by liquid chromatography/mass spectrometry in rat skeletal muscle. J. Mass Spectrom. 37, 1219-1228. Aldini, G., Orioli, M., Carini, M., and Maffei Facino, R. (2004) Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry. J. Mass Spectrom. 39, 1417-1428. Uchida, K. (2003) 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res. 42, 318-343. Alary, J., Fernandez, Y., Debrauwer, L., Perdu, E., and Gueraud, F. (2003) Identification of intermediate pathways of 4-hydroxynonenal metabolism in the rat. Chem. Res. Toxicol. 16, 320-327. Feng, Z., Hu, W., and Tang, M. (2004) Trans-4-hydroxy-2-nonenal inhibits nucleotide excision repair in human cells: a possible mechanism for lipid peroxidation-induced carcinogenesis. Proc. Natl. Acad. Sci. U.S.A. 101, 8598-8602. Bulteau, A. L., Moreau, M., Nizard, C., and Friguet, B. (2002) Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes. Free Radical Biol. Med. 32, 1157-1170. Carini, M., Aldini, G., Piccone, M., and Maffei Facino, R. (2000) Fluorescent probes as markers of oxidative stress in keratinocyte cell lines following UVB exposure. Farmaco 55, 526-34. Cimino, F., Ambra, R., Canali, R., Saija, A., and Virgili, F. (2006) Effect of cyanidin-3-O-glucoside on UVB-induced response in human keratinocytes. J. Agric. Food Chem. 54, 4041-7. Handa, O., Kokura, S., Adachi, S., Takagi, T., Naito, Y., Tanigawa, T., Yoshida, N., and Yoshikawa, T. (2006) Methylparaben potentiates UV-induced damage of skin keratinocytes. Toxicology 227, 62-72. Manzer, R., Pappa, A., Estey, T., Sladek, N., Carpenter, J. F., and Vasilion, V. (2003) Ultraviolet radiation decreases expression and induces aggregation of corneal ALDH3A1. Chem.-Biol. Interact. 143144, 45-53. Reddy, G. B., and Bhat, K. S. (1999) Protection against UVB inactivation (in vitro) of rat lens enzymes by natural antioxidants. Mol. Cell Biochem. 194, 41-45. Hiratsuka, A., Saito, H., Hirose, K., and Watabe, T. (1999) Marked expression of glutathione S-transferase A4-4. detoxifying 4-hydroxy2(E)-nonenal in the skin of rats irradiated by ultraviolet B-band light (UVB). Biochem. Biophys. Res. Commun. 260, 740-746.

HNE Metabolism Impairment by UVB in Keratinocytes (33) Seo, K. I., Cho, K. H., Park, K. C., Youn, J. I., Eun, H. C., Kim, K. T., and Park, S. C. (1996) Change of glutathione S-transferases in the skin by ultraviolet B irradiation. J. Dermatol. Sci. 13, 153-60. (34) Pappa, A., Chen, C., Koutalos, Y., Townsend, A. J., and Vasiliou, V. (2003) ALDH3A1 protects human corneal epithelial cells from ultraviolet- and 4-hydroxy-2-nonenal-induced oxidative damage. Free Radical Biol. Med. 34, 1178-89. (35) Sellin, S., Holmquist, B., Mannervick, B., and Vallee, B. L. (1991) Oxidation and reduction of 4-hydroxyalkenals catalyzed by isozymes of human alcohol dehydrogenase. Biochemistry 30, 2514-2518. (36) Chung, F. L., Nath, R. G., Ocando, J., Nishikawa, A., and Zhang, L. (2000) Deoxyguanosine adducts of t-4-hydroxy-2 nonenal are endogenous DNA lesions in rodents and human: detection of potential sources. Cancer Res. 60, 1507-1511. (37) Wacker, M., Schuler, D., Wanek, P., and Eder, E. (2000) Development of a 32P-postlabelling method for the detection of 1,N-propanodeoxy-

Chem. Res. Toxicol., Vol. 20, No. 3, 2007 423 guanosine adducts of trans-4-hydroxy-2-nonenal in vivo. Chem. Res. Toxicol. 13, 1165-1173. (38) Hattori, Y., Nishigori. C., Tanaka, T., Uccida, K., Nikaido, O., Osawa, T., Hiai, H., Imamura, S., and Toyokuni, S. (1996) 8-hydroxy-2′deoxyguanosine is increased in epidermal cells of hairless mice after chronic ultraviolet B exposure. J. InVest. Dermatol. 107, 733-737. (39) Aldini, G., Carini, M., Vistoli, G., Shibata, T., Kusano, Y., Gamberoni, L., Dalle-Donne, I, Milzani, A., and Uchida, K. (2007) Identification of actin as a 15-deoxy-∆12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational and functional approaches to investigate the effect on cytoskeletal derangement. Biochemistry, in press. (40) Aldini G., Orioli M., and Carini M. (2007). R,β-Unsaturated aldehydes adducts to actin and albumin as potential biomarkers of carbonylation damage. Redox Rep. 12, 20-25.

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