Tetrahydropteridines possess antioxidant roles to guard against glucose-induced oxidative stress in Dictyostelium discoideum

Abstract

Glucose effects on the vegetative growth of Dictyostelium discoideum Ax2 were studied by examining oxidative stress and tetrahydropteridine synthesis in cells cultured with different concentrations (0.5X, 7.7 g L(-1); 1X, 15.4 g L(-1); 2X, 30.8 g L(-1)) of glucose. The growth rate was optimal in 1X cells (cells grown in 1X glucose) but was impaired drastically in 2X cells, below the level of 0.5X cells. There were glucose-dependent increases in reactive oxygen species (ROS) levels and mitochondrial dysfunction in parallel with the mRNA copy numbers of the enzymes catalyzing tetrahydropteridine synthesis and regeneration. On the other hand, both the specific activities of the enzymes and tetrahydropteridine levels in 2X cells were lower than those in 1X cells, but were higher than those in 0.5X cells. Given the antioxidant function of tetrahydropteridines and both the beneficial and harmful effects of ROS, the results suggest glucose-induced oxidative stress in Dictyostelium, a process that might originate from aerobic glycolysis, as well as a protective role of tetrahydropteridines against this stress.

Fig. 1.. Cell growth and intracellular pteridines. (A) Cells grown in HL5 medium containing the indicated amounts of glucose were counted using a hemacytometer. (B) Intracellular levels of BH4 and DH4 were determined by HPLC as oxidized forms, biopterin (dark) and dictyopterin (grey), respectively.

Fig. 2.. Evaluation of oxidative stress. (A) ROS measured by DCFH-DA staining. The fluorescent cells were analysed at 525 nm using FACS. (B) Mitochondrial activity. Mitochondrial transmembrane potential measured by JC-1 staining (dark, left axis). The active red fluorescent cells were analysed at 485 nm using FACS. Quantitative real-time PCR of mtDNA and nDNA (grey, right axis). The mitochondrial COX-I gene was quantified relative to that of the nuclear GAPDH gene. (C) Spore viability was counted as plaques on an E. coli lawn (dark, left axis). Dark spores were counted using a phase contrast microscope (grey, right axis).

Fig. 3.. Specific activities of the enzymes involved in the de novo synthesis and regeneration of tetrahydropteridines. (A) GTPCH activity measured as neopterin. (B) PTPS activity measured as pterin. (C) SR activity measured as the sum of biopterin and dictyopterin. (D) DHPR activity measured as oxidized NADH.

Fig. 4.. Gene expression levels of GTPCH and DHPR. The mRNA copy numbers of GTPCH (dark) and DHPR (grey) were quantified relative to that of GAPDH by real-time PCR.