Antioxidant status in J774A.1 macrophage cell line during chronic exposure to glycated serum

Advanced glycation end-products (AGEs) are linked to aging and correlated diseases. The aim of present study was to evaluate oxidative stress related parameters in J774A.1 murine macrophage cells during chronic exposure to a subtoxic concentration of AGE (5% ribose-glycated serum (GS)) and subsequently for 48 h to a higher dose (10% GS). No effects on cell viability were evident in either experimental condition. During chronic treatment, glycative markers (free and bound pentosidine) increased significantly in intra- and extracellular environments, but the production and release of thiobarbituric acid reactive substances (TBARs), as an index of lipid peroxidation, underwent a time-dependent decrease. Exposure to 10% GS evidenced that glycative markers rose further, while TBARs elicited a cellular defence against oxidative stress. Nonadapted cultures showed an accumulation of AGEs, a marked oxidative stress, and a loss of viability. During 10% GS exposure, reduced glutathione levels in adapted cultures remained constant, as did the oxidized glutathione to reduced glutathione ratio, while nonadapted cells showed a markedly increased redox ratio. A constant increase of heat shock protein 70 (HSP70) mRNA was observed in all experimental conditions. On the contrary, HSP70 expression became undetectable for a longer exposure time; this could be due to the direct involvement of HSP70 in the refolding of damaged proteins. Our findings suggest an adaptive response of macrophages to subtoxic doses of AGE, which could constitute an important factor in the spread of damage to other cellular types during aging.Key words: in vitro cytotoxicity, AGE, pentosidine, glycoxidation, oxidative stress, TBARs.

Increased expression of transglutaminase-1 and PPARgamma after vitamin E treatment in human keratinocytes

In skin, vitamin E acts as the predominant lipophilic antioxidant with a protective function against irradiation and oxidative stress. In addition to that, vitamin E can also modulate signal transduction and gene expression. To study whether the four natural tocopherol analogues (α-, β-, γ-, δ-tocopherol) can influence transcriptional activity by modulating the activity of nuclear receptors, a human keratinocytes cell line (NCTC 2544) was transfected with plasmids containing the luciferase reporter gene under control by direct repeat elements (DR1–DR4), representing binding sites for four different classes of nuclear receptors. In this model, the tocopherols positively modulated only the reporter construct containing a consensus element for peroxisome proliferator-activated receptors (PPARs). The induction was strongest with γ-tocopherol and was most likely the direct consequence of stimulation of PPARγ protein expression in keratinocytes. Vitamin E treatment also led to increased expression of a known PPARγ target gene involved in terminal keratinocytes differentiation, the transglutaminase-1.