L-arginine increases UVA cytotoxicity in irradiated human keratinocyte cell line: potential role of nitric oxide

Human fibroblasts and keratinocytes possess nitric oxide synthases (NOS), w hich metabolize L-arginine (L-Arg) for producing nitric oxide (NO.), This r eport delineates the relations between NO. and UVA in the human keratinocyt e cell line HaCaT. NOS activity was stimulated by exposure of cells to L-Ar g just after irradiation. L-Arg (5 mM) supply led to an increase in UVA (25 .3 J/cm(2)) cytotoxicity (% of viability 18 +/- 3%) whereas neither L-Arg i tself nor UVA irradiation induced cell death at the doses used in this stud y. Cells were also treated either with L-thiocitrulline (L-Thio), an irreve rsible inhibitor of NOS, or with exogenous superoxide dismutase (SOD) and c atalase, L-Thio and SOD prevented L-Arg-mediated deleterious effects in irr adiated cells, whereas catalase was ineffective. Intracellular antioxidant enzyme activities were also determined. UVA/L-Arg stress altered catalase ( 66% decrease) and glutathione peroxidase (83% decrease). DNA damage was eva luated using the 'comet assay' and quantified using the 'tail moment'. UVA alone was genotoxic (mean tail moment: 25.43 +/- 1.23, P < 0.001 compared c ontrol cells), The addition of L-Arg potentiated DNA damage (mean tail mome nt: 41.05+/-3.9) whereas L-Thio prevented them (mean tail moment 9.86 +/- 0 .98). We attempted to assess the effect of poly(ADP-ribose) polymerase (PAR P) inhibition on cell death. Using the PARP inhibitor 3-aminobenzamide, we established that PARP determines both cell lysis and DNA damage induced by UVA and/or L-Arg, Our findings demonstrated that L-Arg was able to increase UVA-mediated deleterious effects in keratinocytes (both DNA damage age and cytotoxicity) and that the ratio NO./O-2(.-) plays a key role in these pro cesses.