LT1A1 or SULT1B1 (24). In contrast to these findings, Stiborova et al., applying somewhat various procedures, reported that SULT1A enzymes do `not’ stimulate reactivity of AAs with DNA in the presence of NQO1 (25). Our research are designed to resolve this apparent discrepancy. We offer evidence of the direct involvement of SULTs in converting AL-NOHs into forms that bind efficiently to DNA. Moreover, these research demonstrate that sulfonation following nitroreduction further increases the mutagenic and cytotoxic possible of AAs. Supplies and methodsEthics statement Animal protocols have been reviewed and authorized by the Stony Brook Institutional Animal Care and Use Committee. Chemical compounds -32P-ATP (6000 Ci/mmol) was obtained from PerkinElmer (Boston, MA). liquid chromatography/mass spectrometry (LC/MS) grade acetonitrile and?The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupBioactivation of the human carcinogen aristolochic acidFig. 1. Proposed route for bioactivation of AAs. AA-I and AA-II undergo four electron nitroreduction to type AL-I-NOH and AL-II-NOH followed by N-acetylation or N-O-sulfonation catalyzed by NATs and SULTs, respectively. AL-N-oxyesters (AL-I-N-OAc, AL-II-N-OAc, AL-I-N-OSO3H and AL-II-NOSO3H) solvolize in aqueous solution, leading to cyclic nitrenium ion formation, which, in turn, reacts with DNA to kind AL-DNA adducts. aqueous ammonium hydroxide (28 ) have been bought from Fisher Scientific. AA-I, AA-II, N-hydroxyaristolactam I (AL-I-NOH) and N-hydroxyaristolactam II (AL-II-NOH) and its sulfated and acetylated analogs, having a purity of 97 , had been synthesized in our laboratory (Attaluri, unpublished data). AAs and their metabolites were dissolved, at five?0 mM, in dimethyl sulfoxide and stored at -20 . Concentrations of AAs have been established by UV absorption at 250 nm (26). Enzymes utilized for 32P-post-labeling analysis were obtained from Worthington (Newark, NJ) and Sigma ldrich (St Louis, MO). Zinc powder, dimethyl sulfoxide, salmon sperm DNA (ssDNA), PAPS (60 purity) and acetyl-CoA were purchased from Sigma ldrich. 7-(deoxyguanosin-N2-yl)aristolactam II (dG-AL-II) and dA-AL-II containing oligonucleotides have been synthesized as described earlier (12).Buy114932-60-4 Human histidine-tagged SULT1A1, SULT1A2 and SULT1A3, expressed in Escherichia coli and purified with all the specific activity of 15 pmoles/min/ g, as defined by transfer of sulfonate groups from PAPS to 1-naphtol, have been purchased from US Biological (Swampscott, MA).Oxetan-3-yl trifluoromethanesulfonate supplier Recombinant human SULT1B1 was purchased from MyBioSource (San Diego, CA).PMID:35227773 Cytosols from insect cells infected with NAT1 and NAT2 baculovirus expressing vectors have been obtained from BD Biosciences (Woburn, MA). Human NQO1 was bought from Sigma ldrich. Stability of AA-I metabolites AL-I-NOH, aristolactam-I-N-acetoxy ester (AL-I-N-OAc) and aristolactam-IN-sulfate (AL-I-N-OSO3H) (50 M) were ready in 50 mM Tris-HCl buffer (pH 7.5) and incubated for various periods of time at 37 . Aliquots (100 ) had been withdrawn in the reaction at intervals for analysis on a Waters high-performance liquid chromatography (HPLC) method equipped with an XTerraTM MS C18 column (five , 4.6 ?250 mm), a 2996 Photodiode Array detector and Waters Empower software program. Samples were eluted over 40 min at 1 ml/min having a linear gradient beginning at 20 solvent B and 80 solvent A and ending with one hundred solvent B. Solvent A was 5 mM ammonium acetate dissolved in a 1:9 acetonitrile/.
