Genetic toxicology and preliminary in vivo studies of nitric oxide donor tocopherol analogs as potential new class of antiatherogenic agents

Abstract

Nitric oxide donor tocopherol analogs were found to be incorporated in low-density lipoprotein to release nitric oxide into the hydrophobic core of the lipoprotein, thus inhibiting lipid oxidation processes associated with atheroma plaque formation. Previously, we studied their cytotoxicity against human and murine macrophages as first selection for in vivo studies. Herein, we examined both the in vitro mutagenic and DNA-damage effects of selected compounds to further evaluate drug potential. While the compounds of interest were nongenotoxics in both experimental tests (Ames and alkaline comet), one of the potential blood metabolites exhibited genotoxicity (alkaline comet test), and the furazan derivative was mutagenic (Ames test). Two selected (nitrooxy and furoxan) compounds were studied in long- and short-term in vivo treatment, and in these conditions, animal toxicity was not evidenced, suggesting the possibility of these compounds as potential antiatherogenic drugs.

Keywords::

• Vitamin E
• nitric oxide donor
• mutagenicity
• comet assay
• in vivo studies

Acknowledgements

M.C. and G.V.L. thank PEDECIBA (Uruguay) and ANII (Uruguay) for their doctoral scholarships. M.C. thanks Fundación Carolina for an interchange scholarship.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.

Appendix

Chemistry

6-acetyloxy-2,5,7,8-tetramethyl-N-[2-(3-phenylsulfonyl-1,2,5-oxadiazole-4-oxyl)ethyl]chroman-2-carboxamide (8)

To a stirred solution of Trolox acetate (6-acetyloxy-2,5,7,8-tetramethylchromancarboxylic acid) (280 mg, 0.96 mmol) in dry toluene (1.0 mL), thionyl chloride (0.1 mL, 1.44 mmol) and 1 drop of dry DMF were added. The mixture was heated at reflux for 90 minutes under a nitrogen atmosphere and cooled to room temperature. Solvent and thionyl chloride excess was evaporated in vacuo, and the residue was dissolved in dry dichloromethane (3.0 mL). Then, there was added dropwise a solution of 3-(2-aminoethyloxy)-4-benzenesulfonylfurazan (Boschi et al., 1997) (363 mg, 1.44 mmol) and triethylamine (0.4 mL) in dichloromethane. The reaction mixture was stirred at room temperature until reactant disappearance. It was washed with 10% sodium bicarbonate solution, 10% hydrochloric acid solution, and then with water. The organic layer was dried with sodium sulfate and solvent evaporated in vacuo. The product was purified by column chromatography (SiO₂, hexane:ethyl ether (1:1)). yellow oil, yield 42%. ¹H-NMR: δ = 8.04 (d, J = 7.6 Hz, 2H), 7.71 (t, J = 7.6 Hz, 1H), 7.58 (t, J = 7.6 Hz, 2H), 4.42 (bs, 2H), 3.79–3.67 (bs, 2H), 2.68–2.51 (m, 2H), 2.33 (s, 4H), 2.19 (s, 3H), 2.04 (s, 3H), 1.97 (s, 4H), 1.55 (s, 3H). ¹³C-RMN: δ = 175.16, 169.89, 161.65, 149.24, 148.30, 142.14, 138.35, 135.86, 130.05(2), 129.29(2), 127.93, 125.97, 123.14, 118.46, 79.07, 72.95, 38.56, 29.64–12.50 (other methylic, methylenic, and methynic carbons). Anal. (C₂₆H₂₉N₃O₈S) C, H, N.

Biology

·NO release evaluation

The rate of ·NO release was determined by measuring the oxidation of oxyhemoglobin (HbO₂) to methemoglobin (MetHb) at λ = 401 nm, at 37°C using a Shimadzu (Columbia, Maryland, USA) spectrophotometer (Gibson and Roughton, 1957). The reaction was started by adding derivative 8 (6–100 µM in 50 mM of phosphate buffer) to a 10- µM HbO₂ solution in 50 mM of phosphate buffer (pH 7.4) in the presence of a 5- to 20-fold molar excess of cysteine. The initial rate was calculated from the slope of the straight- line portion of each curve. Every NO-releasing rate is the average of at least five determinations. The molar extinction coefficient, Δϵ = ϵ_401MetHb-ϵ_401HbO2, was determined by the quantitative oxidation of five different concentrations (1–10 µM) of HbO₂ in (pH 7.4) phosphate buffer with a 20- µM solution of NOC-7 (1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene). The slope (Δϵ) of the straight line (r = 0.999) obtained from plotting the increase of the absorbance ΔA at λ = 401 nm against the HbO₂ concentrations was 57 ± 2 mM⁻¹cm⁻¹.

Vasorelaxation assays

Wistar Kyoto rats (250–300 g) were heparinized (100 U/mL intraperitoneally; i.p.) and anesthetized (40 mg/kg pentobarbital i.p.), and descending thoracic aorta was excised and cut in rings (4 mm in length) (experimental procedures approved by Comisión Honoraria de Experimentación Animal, Universidad de la República). Tissues were mounted under 2 g of passive tension in a Radnoti tissue-organ bath system containing 30 mL of Krebs-Henseleit solution, maintained at 37°C, and gassed with 95% O₂–5% CO₂, (pH 7.4). Aortic rings were allowed to equilibrate for 1 hour, and a submaximal contraction was obtained by their incubation in the presence of 1 μM of noradrenaline (NA). Compound 8 was added (final concentration, 10 μM) after plateau, using α-tocopherol and Trolox as controls. Drug vehicle (dimethyl sulfoxide) also served as a control and did not affect the experiments. Data are expressed as mean ± SEM or 95% confidence interval. Statistical comparisons were carried out with Fisher, analysis of variance, Dunnett, or Student tests.

Results

Table S1.  Biological properties of compound 8.