Oxidative damage of DNA induced by the reaction of methylglyoxal with lysine in the presence of ferritin

Abstract

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin.

Fig. 1.. DNA strand breakage by the ferritin/MG/lysine system. (A) pUC19 (2 μg) was incubated at 37℃ for 2 h : lane 1, pUC19 DNA control; lane 2, pUC19 DNA plus 1 μM ferritin; lane 3, pUC19 DNA plus 20 mM MG; lane 4, pUC19 DNA plus 20 mM lysine; lane 5, pUC19 DNA plus 1 μM ferritin plus 20 mM MG; lane 6, pUC19 DNA plus 1 μM ferritin plus 20 mM lysine; lane 7, pUC19 DNA plus 20 mM MG plus 20 mM lysine; lane 8, pUC19 DNA plus 1 μM ferritin plus 20 mM MG plus 20 mM lysine. (B) pUC19 (2 μg) was incubated with 0-1 μM ferritin and 20 mM MG and 20 mM lysine at 37℃ for 2 h. The reaction was stopped by freezing at −80℃. The loading buffer was added to samples and analyzed by electrophoresis on 1% agarose gel. I and II indicate the position of the supercoiled and nicked circular DNA plasmid forms, respectively.

Fig. 2.. Effects of ROS scavengers on DNA strand breakage and the generation of ROS in the ferritin/MG/lysine system. (A) pUC19 (2 μg) was incubated with 1 μM ferritin, 20 mM MG and 20 mM lysine in the presence of the ROS scavenger. Lane 1, DNA alone; lane 2, no addition of effector; lane 3, 20 mM urate; lane 4, 20 mM N-acetyl-_L-cysteine (NAC); lane 5, 20 mM thiourea. (B) Ferritin (1 mg/ml) was incubated with various concentrations of MG/lysine in a 10 mM potassium phosphate buffer (pH 7.4) at 37℃ for 72 h. After incubation, the detection of ROS formation was determined by measuring thiobarbituric acid reactive 2-deoxy-D-ribose oxidation products.

Fig. 3.. Effects of deferoxamine (DFX) on DNA strand breakage and the release of iron from ferritin. (A) pUC19 (2 μg) was incubated with 1 μM ferritin, 20 mM MG and 20 mM lysine in the presence of various concentrations of DFX. Lane 1, DNA alone; lane 2, no addition of effector; lane 3, 0.1 mM DFX; lane 4, 0.5 mM DFX; lane 5, 1 mM DFX. (B) Ferritin (1 mg/ml) was incubated with various concentrations of MG/lysine in a 10 mM potassium phosphate buffer (pH 7.4) at 37℃ for 72 h.

Fig. 4.. Effects of carnosine and anserine on DNA cleavage induced by the ferritin/MG/lysine system. pUC19 DNA was incubated with 1 μM ferritin, 20 mM MG and 20 mM lysine in the presence of carnosine or anserine. (A) Lane 1, DNA alone; lane 2, no addition of effector; lane 3, 1 mM carnosine; lane 4, 10 mM carnosine; lane 5, 20 mM carnosine; lane 6, 50 mM carnosine. (C) Lane 1, DNA alone; lane 2, no addition of effector; lane 3, 1 mM anserine; lane 4, 10 mM anserine; lane 5, 20 mM anserine; lane 6, 50 mM anserine. (B and D) The relative staining intensity of agarose gel was analyzed by densitometric scanning. I and II indicate the position of the supercoiled and nicked circular DNA plasmid forms, respectively.