Prooxidant effect of uric acid on human leukocytic DNA: An in vitro and ex vivo study

Abstract

Background/aim: Uric acid is a major contributor to the total antioxidant capacity of human plasma. However, this endogenous substance's antioxidant and prooxidant properties have not yet been reported.

Materials and methods: In this study, the comet assay was employed in vitro to determine the effect of uric acid on DNA damage in human lymphocytes and leukocytic DNA damage in hyperuricemia patients with and without renal failure.

Results: DNA damage in lymphocytes occurred at uric acid concentrations of ≥600 μM. Adding catalase to the uric acid solution diminished the damaging effect, indicating that hydrogen peroxide mediated the prooxidant activity. Moreover, adding Fe²⁺ did not enhance the DNA damage, suggesting that the urate's prooxidant activity is independent of the Fenton reaction. The unstable nature of uric acid at nearly neutral and acidic pH levels resulted in autooxidation and the generation of hydrogen peroxide. Maintaining the stability of uric acid in vivo may lead to the consumption of antioxidants in the body and affect the antioxidant status. Hyperuricemia patients with and without renal failure had higher levels of leukocytic DNA damage compared to healthy individuals. However, there was no significant difference in leukocytic DNA damage between hyperuricemia patients with and without renal failure, which showed that the damaging effect was not due to renal failure. A correlation study suggested that serum uric acid level had a stronger correlation with DNA damage than the severity of renal failure as indicated by serum creatinine or urea.

Conclusion: Uric acid demonstrated prooxidant activity in both in vitro and in vivo studies, which was mediated by the production of hydrogen peroxide and independent of both the Fenton reaction and renal failure.

Keywords: Antioxidant; DNA; comet assay; prooxidant; urate.

Figure 1

Effect of fresh and aged uric acid solution pretreatment on lymphocytes. Results were obtained using samples taken from three healthy participants and tested in two individual experiments. Each point represents the mean of tail %DNA content with ±1 SD error bars shown. DNA damage was positively related to concentration in fresh uric acid solution. A statistically significant difference was found at concentrations of ≥800 μM compared to 0 μM (Dunnett’s test, *: p < 0.05). No DNA damage was observed in cells incubated with aged uric acid solution at various concentrations (Dunnett’s test, p > 0.05). White circles represent fresh uric acid; black squares represent aged uric acid.

Figure 2

Effect of uric acid (UA) on lymphocytes with and without treatment by endonuclease III (Endo III). Results were obtained using samples taken from three healthy participants and tested in two individual experiments. Each bar represents the mean of tail %DNA content with +1 SD error bars shown. No statistically significant difference was found between cells treated with uric acid with or without the addition of treatment by endonuclease III (unpaired-t test, p > 0.05). White bars denote the lack of endonuclease III pretreatment; black bars denote the presence of endonuclease III pretreatment.

Figure 3

Effect of uric acid on lymphocytes with and without the presence of Fe²⁺. Results were obtained using samples taken from three healthy participants and tested in two individual experiments. Each point represents the mean of tail %DNA content with ±1 SD error bars shown. No statistically significant difference was found between cells treated with uric acid with and without adding Fe²⁺ (unpaired-t test, p > 0.05). White circles represent lack of Fe²⁺; black squares represent presence of 50 μM Fe²⁺ (final concentration).

Figure 4

Effect of uric acid on lymphocytes at different pH levels. Results were obtained using samples taken from two healthy participants and tested in three individual experiments. Each bar represents the mean of tail %DNA content with +1 SD error bars shown. A statistically significant difference was found between cell pretreatment with uric acid at various pH levels compared to PBS at 7.4 (Dunnett’s test, *: p < 0.05).

Figure 5

DNA damage according to comet scores for participants with hyperuricemia and renal failure (↑UA+RF; 202.0 ± 64.7, n = 15), participants with only hyperuricemia (↑UA; 207.0 ± 33.3, n = 20), and healthy participants (Normal UA; 125.0 ± 23.8, n = 20). ***: p < 0.05 (size effects from 1.7 to 2.9), with the values of healthy participants being statistically significantly lower than those of both hyperuricemia groups.

Figure 6

Linear regression studies demonstrated significant associations of DNA damage with (a) uric acid (r² = 0.3939, p < 0.0001), (b) creatinine (r² = 0.1107; p = 0.0159), and (c) urea levels (r² = 0.0893; p = 0.0314).