D, which reacts gradually with DNA in vitro, resulting in formation
D, which reacts gradually with DNA in vitro, resulting in formation of 7-CEGua [20].NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript2. Materials and Methods2.1 Chemical IKKε manufacturer substances DHU, NaNO2, and acrylic acid had been purchased from Sigma-Aldrich (St. Louis, MO). UPA was obtained from Chem-Impex International Inc (Wooddale, IL). two.2 Animal study This study was authorized by the University of Minnesota Institutional Animal Care and Use Committee. Male F-344 rats, age six weeks, had been obtained from Charles River Laboratories (Kingston, NY), housed two per cage with Harlan irradiated corncob bedding (Harlan, Indianapolis IN), and allowed to acclimate to the Study Animal Resources facility, University of Minnesota, for one particular week. The rats were maintained below normal circumstances (204 temperature, 292 relative humidity and 1410 lightdark cycle). They had been fed Harlan Teklad 7022 (NIH-07) eating plan. Water and diet plan Caspase 6 MedChemExpress consumption have been measured three occasions per week. In Study 1, 4 rats per group, except as noted, have been treated for two weeks as follows: 1, NaNO2 1500 ppm in drinking water; two, DHU 2480 ppm in powdered diet plan; three, NaNO2 1500 ppm DHU 2480 ppm; four, -UPA 2870 ppm in powdered eating plan; five, NaNO2 1500 ppm -UPA 2870 ppm; 6, acrylic acid 1565 ppm in drinking water, 6 rats; 7, untreated manage, 6 rats. The rats had been humanely sacrificed by CO2 overdose, and tissues have been collected and frozen at -20 until analysis. In Study two, 4 rats per treatment group had been offered the compounds for 4 weeks exactly as in Study 1, except that one particular additional group was added using a greater dose of acrylic acid (3130 ppm). There were 6 rats inside the control group. The rats had been sacrificed and tissues collected and frozen as in Study 1. two.three Analysis of rat hepatic DNA hydrolysates for 7-CEGua by liquid chromatographyelectrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC-ESIMSMS-SRM) DNA isolation and enzymatic hydrolysis had been carried out primarily as described [11]. In short, for hydrolysis, DNA (1 mg) was dissolved in 1 mL of ten mM Tris-HCl5 mM MgCl2 buffer (pH 7.0) containing [15N5]7-CEGua (1300 fmol). The DNA was enzymatically hydrolyzed for 70 min at 37 with 1060 units of DNase I (variety II, bovine pancreas), 0.05 units of phosphodiesterase I (form II, Crotalus adamanteus venom), and 300 units of alkaline phosphatase (calf intestine). The hydrolysate, right after removal of a ten .. L aliquot for dGuo quantitation, was purified making use of a mixed mode cation exchange cartridge [MCX Vac RC, 60 mg (Waters Corp, Milford, MA)]. The cartridge was conditioned with two mL of CH3OH and 2 mL two H3PO4. The sample was acidified with 10 .. L of 86 H3PO4. Right after the sample was applied, the cartridge was washed with 2 mL 0.1 H3PO4 and two mL of CH3OH, and also the analyte was eluted with two mL 3 NH4OH in CH3OH. This fraction was collected and concentrated to dryness. A single mL of a freshly ready ten CH3COCl option in CH3OH was added to the vial. The mixture was then heated for 1 h at 50 to convert 7-CEGua to its methyl ester, thenChem Biol Interact. Author manuscript; offered in PMC 2014 October 25.Wang et al.Pageconcentrated to dryness. The residue was dissolved in 1 mL 15 mM NH4OAc buffer (pH six.six) and purified applying a Strata-X solid-phase extraction cartridge [33 .. m, 30 mg1 mL (Phenomenex, Torrance, CA)]. The cartridge was conditioned with 1 mL CH3OH, 1 mL H2O and 1 mL 15 mM NH4OAc buffer (pH 6.6). Just after the sample was applied, the cartridge was washed with 1 mL 15 m.