Deleterious in vitro effects of ammonia and accumulated urea cycle amino acids on leukocyte DNA: assessment of the antioxidant effects of N-acetylcysteine and L-carnitine.

The urea cycle is essential for ammonia detoxification. Deficiencies in this pathway cause urea cycle disorders (UCDs), characterized by hyperammonemia and severe systemic and neurological manifestations. While ammonia toxicity is well established, the contribution of urea cycle altered amino acids that accumulate in specific UCDs to cellular stress and DNA damage-associated responses remains poorly understood. This study investigated the DNA damage effects of ornithine (1 mM), homocitrulline (0.5 mM), citrulline (1 mM), and arginine (1 mM), alone and combined with ammonia (1 mM), in human peripheral leukocytes, as well as the protective effects of N-acetylcysteine (1 mM) and L-carnitine (60 µM). DNA damage was evaluated using the comet assay and statistically analyzed. All treatments induced significant DNA damage compared with controls. When tested individually at pathophysiologically relevant concentrations, urea cycle-related metabolites produced measurable increases in DNA damage, with citrulline and arginine inducing damage comparable to ammonia. Homocitrulline also caused DNA damage, to a lesser extent, while ornithine induced the lowest levels among isolated treatments. Combined exposure to ammonia and amino acids markedly increased DNA damage levels compared with isolated conditions, emphasizing the impact of metabolic imbalance in UCDs. The highest DNA damage indices were observed with ornithine and homocitrulline in the presence of ammonia, indicating potential synergistic interactions. L-carnitine significantly reduced DNA damage in all experimental conditions. N-acetylcysteine also reduced DNA damage effects in most combinations, except for ornithine with ammonia. Overall, these findings demonstrate that both isolated and combined UCD-related metabolites are associated with increased DNA damage under hyperammonemic conditions and support antioxidant strategies as potential complementary approaches to mitigate genomic stress in UCDs.