Tetrahydrobiopterin as a rheostat of cell resistance to oxidant injury

Abstract

Tetrahydrobiopterin (BH4) deficiency is caused by genetic abnormalities that impair its biosynthesis and recycling, which trigger neurochemical, metabolic, and redox imbalances. Low BH4 levels are also associated with hypoxia, reperfusion reoxygenation, endothelial dysfunction, and other conditions that are not genetically determined. The exact cause of changes in BH4 in nongenetic disorders is not entirely understood, but a role for oxidant species has been implicated. The oxidation of BH4 generates several products, including 7,8-dihydrobiopterin (BH2), the accumulation of which is predicted in cells with low dihydrofolate reductase activity. The relative efficiency of oxidant species at causing variations in BH4/BH2 levels in cells furnished with several antioxidant enzymes has not yet been systematically analyzed. This study examined the quantitative changes of BH4/BH2 in cells challenged with several oxidants. We showed that BH2 is not a major product of treatments with hydrogen peroxide or RSL3, as indicated by the moderate effect of dihydrofolate reductase-inhibitor methotrexate on the accumulation of BH2. However, we found a net loss in BH4/BH2, suggesting that products other than BH2 were generated. These reactions were further examined in NOX4-expressing HEK cells producing hydrogen peroxide. These cells showed slightly decreased BH4/BH2 ratios compared with HEK wild-type cells, and, again, methotrexate treatment moderately increased BH2 levels. In contrast, peroxynitrite-producing RAW 264.7 cells showed dramatically decreased BH4 levels without BH2 accumulation. Following the activation of peroxynitrite production with PMA in lipopolysaccharide-treated cells, we also found a significant time-dependent decline in GTPCH-I protein levels. We conclude that hydrogen peroxide is the least effective oxidant species at decreasing intracellular BH4 levels, while peroxynitrite is highly effective by targeting GTPCH-I and BH4 directly. Moreover, we conclude that BH4/BH2 levels are not a determinant of RSL3 cytotoxicity.

Keywords: Antioxidants; BH2; Cytochrome c; Hydrogen peroxide; NOX4; Peroxynitrite; RSL3.

Graphical abstract

Fig. 1

Oxidation of BH4 by cytochrome c and peroxynitrite in solution. A. Scheme representing the preparation of BH4 stock and working solutions for the in vitro experiments. (BH4 ac sol) 10 mM BH4 in 25 mM phosphate buffer pH 2.6; (BH4 pH 7.4) 100 nM BH4 diluted in phosphate buffer 100 mM pH 7.4. B. chromatographic separation and electrochemical detection (ECD) of a mixture of authentic BH4 and BH2 standards at 0, 150, 280 and 365 mV applied electrode potentials. C. Cytochrome c dose-dependent oxidation of BH4 increasing BH2 (n = 5) D. Peroxynitrite dose-dependent oxidation of BH4 increasing BH2 (n = 5 except for ONOO- 1000 nM n = 3 independent experiments). Controls included decomposed (d.ONOO⁻) (1000 nM ONOO⁻ diluted in 0.1 M phosphate buffer pH 7.4 and incubated at room temperature for 10 min (n = 1); NaOH, an equivalent volume of NaOH was added instead of ONOO⁻ at high dose (1000 nM) to control for possible pH change (n = 4 independent experiments).

Fig. 2

Dose-dependent oxidation of BH4 by peroxides. A. Cumene hydroperoxide (COOH) (n = 5 independent experiments) B. hydrogen peroxide (H₂O₂) (n = 5 buffer, n = 3 for other conditions); (1000+cat), catalase added before the bolus addition of H₂O₂. Incubations were performed at room temperature for 30 min and analyzed immediately after.

Fig. 3

BH4/BH2 changes in HEK cells treated with an extracellular flux of hydrogen peroxide. A. Quantification of H₂O₂ in incubations of GO/glucose with and without cells. The difference between these two curves was taken as the amount of H₂O₂ consumed by cells (Fig. 1A, arrow). B. BH4/BH2 after treatment with MTX (5 μM) or PEG-catalase 2h before GO-5 or 10 mU/ml addition. C. BH4/BH2 levels after treatments with GO or RSL3. D. GTPCH-1, SPR, and DHFR expression levels in HEK cells do not change after GO-5 mU/ml treatment and 10 μM RSL3 for 2h. The experimental data represent n ≥ 3 independent experiments.

Fig. 4

RSL3 decreases cell viability independently of BH4 availability. A. BH4/BH2 changes in HEK cells incubated with increased concentrations of RSL for 2 h. B. BH4/BH2 changes by RSL3 are enhanced by co-treatment with MTX and MTX + GO. The experimental data represent n ≥ 3 independent experiments. C. Experimental scheme to assess cell viability post-RSL3 and GO-5 mU/ml treatment using a label-free cell assay (Incucyte). Post-treatment increase in cell number (Δ confluency per well) after 30h culture. The experimental data represent n ≥ 3 independent experiments.

Fig. 5

BH4/BH2 changes in HEK-NOX4 cells A. BH4/BH2 levels in HEK wild type (WT); HEK-NOX4 cells untreated and (NOX4-MTX) after 2h treatment with 10 μM MTX. B. HEK-NOX4 cells after 24h treatment with NAMPT-inhibitor FK866 (10 nM), after 2h treatment with DPI (5 μM), and RSL3, (10 μM) with and (RSL3+MTX) MTX for 2h before addition RSL3 (10 μM). The experimental data represent n ≥ 3 independent experiments. C. NOX4 mRNA expression in HEK-WT and HEK-NOX4 mRNA(inset) C. Expression of BH4-pathway enzymes GTPCH-I, SPR and DHFR in HEK-WT and HEK-NOX4. D. Inhibition of H₂O₂ released from HEK-NOX4 by FK866 (10 nM, 24h), MTX(10 μM, 2h), DPI (1 mM, 2h). Amplex red controls (No-HRP) no-HPR initiated changes in fluorescence and catalase (200U/ml); The experimental data represent n = 6 independent experiments.

Fig. 6

BH4/BH2 levels in RAW264.7 cells following NOX2 and iNOS activation. A. NOX2 activation with PMA (200 ng/ml, 2h) increasing H₂O₂ formation and inhibited by SOD (10 μg/ml) and catalase (200U/ml). LPS (1 μg/ml) treatment for 24 h decreases hydrogen) and PMA + LPS treatments. The experimental data represent n = 6 independent experiments. B. Nitrite accumulation in untreated RAW264.7 cell culture media (UNT) and after PMA (2h), LPS (24h) and a combination of PMA + LPS treatment. The experimental data represent n = 6 independent experiments C. Representative Western blot of iNOS and GTPCH-I expression levels in untreated (UNT) and LPS-stimulated cells; D. Peroxynitrite detection in RAW cells stimulated or not (UNT) with PMA, LPS, and LPS + PMA. The experimental data represent n = 6 independent experiments E. Representative PRX1 reduced and oxidized ratios in untreated (UNT) and after PMA, LPS, PMA + LPS treatments; F. HPLC-ECD detection of BH4/BH2 in untreated (UNT) and after 2h activation with PMA, 24h stimulation with LPS (LPS) and LPS and 2h PMA (LPS + PMA); experimental data represent n = 3 independent experiments. G. GTPCH-I levels upon LPS (1 μg, 24h) and PMA (P, 200 ng/ml, 6h) and LPS + PMA, 2h (L/P-2), LPS + PMA,4h (L/P-4) and LPS + PMA, 6h (L/P-6). The experimental data represent n = 3 independent experiments.