N-t-butyl hydroxylamine, a hydrolysis product of α-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts

alpha-Phenyl-N-t-butyl nitrone (PBN), a spin trap, scavenges hydroxyl radicals, protects tissues from oxidative injury, and delays senescence of both normal human lung fibroblasts (IMR90) and senescence-accelerated mice. N-t-butyl hydroxylamine and benzaldehyde are the breakdown products of PEN. N-t-Butyl hydroxylamine delays senescence of IMR90 cells at concentrations as low as 10 mu M compared with 200 mu M PEN to produce a similar effect, suggesting that N-t-butyl hydroxylamine is the active form of PEN. N-Benzyl hydroxylamine and N-methyl hydroxylamine compounds unrelated to PEN were also effective in delaying senescence, suggesting the active functional group is the N-hydroxylamine. All the N-hydroxylamines tested significantly decreased the endogenous production of oxidants, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin and the increase in the GSH/GSSG ratio. The acceleration of senescence induced by hydrogen peroxide is reversed by the N-hydroxylamines. DNA damage, as determined by the level of apurinic/apyrimidinic sites, also decreased significantly following treatment with N-hydroxylamines. The N-hydroxylamines appear to be effective through mitochondria; they delay age-dependent changes in mitochondria as measured by accumulation of rhodamine-123, they prevent reduction of cytochrome C-FeIII by superoxide radical, and they reverse an age dependent decay of mitochondrial aconitase, suggesting they react with the superoxide radical.