Effects of UVB radiation on 4-hydroxy-2-trans-nonenal metabolism and toxicity in human keratinocytes.
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 microM) 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 UVB-induced 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.
Aldini
Giancarlo
G
Istituto di Chimica Farmaceutica e Tossicologica Pietro Pratesi, Faculty of Pharmacy, University of Milan, Viale Abruzzi 42, I-20131 Milan, Italy. giancarlo.aldini@unimi.it
Granata
Paola
P
Marinello
Cristina
C
Beretta
Giangiacomo
G
Carini
Marina
M
Facino
Roberto Maffei
RM
D000447:Aldehydes / Q000378:metabolism* / Q000633:toxicity*; D002336:Carnosine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism* / Q000633:toxicity*; D004307:Dose-Response Relationship, Radiation; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism / Q000528:radiation effects*; D018384:Oxidative Stress / Q000502:physiology / Q000528:radiation effects; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet; D014466:Ultraviolet Rays
D000447:Aldehydes; D015853:Cysteine Proteinase Inhibitors; D002336:Carnosine; D005978:Glutathione; C027576:4-hydroxy-2-nonenal