Journal: Environmental and Molecular Mutagenesis
Article Title: Bioactivation mechanisms of N‐hydroxyaristolactams: Nitroreduction metabolites of aristolochic acids
Figure Lengend Snippet: Effect of dialysis on 10 kDa MWCO and pentachlorophenol on activation of AL‐I‐NOH in AL‐DNA by HK‐2 cytosols. (A) Cytosols from HK‐2 cells (25 ng/100 μL) were incubated with 1 (filled circles), 10 (empty circles), 25 (filled triangles), or 50 μM (empty triangles) AL‐I‐NOH in the presence of ssDNA for 15–60 min at 37°C. DNA was extracted and five micrograms from each sample was subjected to adduct analysis as described in the section Materials and Methods. Results are presented as the dependence of AL‐DNA (dG‐AL and dA‐AL adducts) levels on the reaction time. (B) HK‐2 cytosols were incubated with AL‐I‐NOH and ssDNA in the presence and absence of pentachlorophenol (PCP) for 30 min as indicated. Each reaction with PCP was conducted in duplicate, and reaction without PCP was set in triplicate. A fragment of representative PAGE with DNA adduct analysis for five microgram of DNA is shown. AL‐DNA levels without PCP were 58 ± 4 adducts per 10 7 nucleotides, and 60 ± 4 with PCP, combined across all concentrations of PCP. c1‐ssDNA incubated with AL‐I‐NOH. C. HK‐2 cytosol was dialyzed on 10 MWCO membrane against Tris–HCl buffer (pH 7.5) overnight at 4°C. The protein amount was quantified by Bradford assay, and various amount of protein (25 ng, 50 ng, 200 ng, 1000, 5000, 10,000 and 25,000/100 μL) was incubated with ssDNA and AL‐I‐NOH with or without PAPS for 30 min. c2—DNA; c3—DNA, AL‐I‐NOH; c4—DNA, AL‐I‐NOH, PAPS. St‐ standard mixture of oligonucleotides containing dG‐AL‐II and dA‐AL‐II adducts, 15 and 30 fmol each. AL‐DNA levels were 0.3 (25 ng protein) and 8 (10,000 ng protein) adducts per 10 7 nucleotides, which is ~500‐times less than before dialysis. Reactions in (B) and (C) were conducted in parallel and analyzed in the same digestion assay and resolved on the same gel. The vertical line was introduced manually to indicate the border between two different experiments. Upper band is dG‐AL, lower band is dA‐AL.
Article Snippet: DMSO, 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) (70% purity), salmon sperm DNA (ssDNA), pentachlorophenol (PCP), quercetin, β‐estradiol, 4‐nitrophenol were from Sigma‐Aldrich.
Techniques: Activation Assay, Incubation, Polyacrylamide Gel Electrophoresis, Bradford Assay, Papanicolaou Stain