2.3. Biological Toxicity Studies

The human embryonic kidney cell line HEK-293 (obtained from ATCC) as well as three human cancer cell cultures, the epidermal carcinoma-derived cell KB-3-1 (generously donated by Dr. Shen, Bethesda, MD, USA), the colon carcinoma cell line CaCo-2 and the adenocarcinomic human alveolar basal epithelial cell A549 (obtained from ATCC) were used in this study. Cells were cultivated in Dulbecco’s modified Eagle medium (DMEM, Gibco by Life Technologies, LifeTech Austria, Vienna, Austria), supplemented with 5% fetal bovine serum (FBS, Gibco by Life Technologies, LifeTech Austria) and 1% penicillin-streptomycin (Sigma-Aldrich, Vienna, Austria) at 37 °C with 5% CO₂ in a humidified incubator. Cultures were periodically checked for Mycoplasma contamination. Cells were seeded in 96 well plates at a density of 2 × 10⁴ cells/mL in 100 µL per well and allowed to attach for 24 h. Afterward, cells were incubated with 100 µL of LQOF-G6 diluted in DMEM at concentrations ranging from 1.56 to 400 µM. Stock solutions were prepared in water with 10% (v/v) dimethyl sulfoxide (DMSO) and stored at 4 °C. The proportion of viable cells was determined after 72 h exposure to LQOF-G6 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based vitality assay kit (EZ4U, Biomedica, Vienna, Austria) [30,31]. Briefly, 20 µL of the EZ4U assay solution was added to each well. After 2 h of incubation in the dark, the absorbance was measured by a microplate reader, at 450 nm with 620 nm as a reference to reduce unspecific background values. All experiments were performed three times in triplicates.

Guinea pigs of either sex weighing 250–380 g have been used. Animals were kept in an air-conditioned room at a temperature of 22–24 °C and relative humidity of 50–60% with a 12-h photo period. On the day of the experiments, the animal was sacrificed by cervical dislocation. The heart, aorta, pulmonary artery, and ileum were surgically excised and kept in a Krebs-Henseleit solution (NaCl 144.9 mM, KCl 4.73 mM, CaCl₂ 3.2 mM, MgSO₄ 1.18 mM, NaHCO₃ 24.9 mM, KH₂PO₄ 1.18 mM and glucose 10 mM; pH 7.2–7.4), continually aerated with 95% O₂ and 5% CO₂. Papillary muscles were dissected from the right ventricle of the heart and cleared from Purkinje fibers to avoid spontaneous activity. We used muscles having a diameter of less than 0.87 mm to ensure proper oxygen supply. The right atrium was also dissected to check the chronotropic activity. Both the aorta and the pulmonary artery were cleaned, and rings of 5 mm were cut while the ileum was cut from the terminal portion into pieces of 1–2 cm. One end of the dissected tissues was tied with silver wire for attachment to the tissue holder while the other end was attached to the force transducer (Transbridge, 4-Channel Transducer Amplifier, World Precision Instruments, Sarasota, FL, USA). The terminal ileum was contracted with 60 mM KCl while a 90 mM KCl solution was used for the pulmonary artery and aorta rings. Papillary muscles were electrically stimulated by an Anapulse Stimulator model 301-T and an Isolation UnitModel 305-1 (WPI, Hamden, CT, USA) with rectangular pulses of 3 ms at a frequency of 1 Hz. The amplitude of the stimulation pulse was kept 10% above the threshold level. To obtain maximum contractility from the respective tissues, a constant resting tension of 3.9 mN for papillary muscle, 4.9 mN for terminal ileum, 10.4 mN for the right atrium, and 19.6 mN for aorta and pulmonary artery rings was applied throughout the experiment. After a control period of 30 min, different concentrations (3, 10, 30, and 100 µM) of test compounds were applied cumulatively in a bath solution every 45 min until a steady effect was obtained. The responses were recorded by a chart recorder (BD 112 Dual Channel, Kipp & Zonen) and evaluated later. Stock concentrations for the test compounds were made with DMSO. To exclude the effect of DMSO, experiments were performed with solvent only, and the observed effect was subtracted from the response of the test compounds. Data are presented as means ± SEM and were statistically evaluated using Student’s t-test for paired observations. A p-value of less than 0.05 was considered to indicate a significant change.