Phanerochaete sordida YK-624 (ATCC 90872) from rotten wood (Hirai et al., 1994) was used in this study. The fungus was maintained on potato dextrose agar slants at 4 °C. AFB1 was purchased from Wako Pure Chemical Industries (Japan). The umu test with umulac AT (Protein
Purify Ltd, Japan) was used to assay mutagenic activity. All other chemicals were extra-pure grade JNK inhibitor and were used without further purification. MnP from P. sordida YK-624 was prepared and purified using the modified method described by Kondo et al. (1994). The MnP solution did not contain LiP activity, and has been purified to homogeneity in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The purified MnP on isoelectric focusing showed one isoform (data not shown). MnP activity
was measured by monitoring the oxidation of 2,6-dimethoxyphenol to coerulignone (ɛ470=49.6 mM−1 cm−1) (Pèriè & Gold, 1991). The reaction mixture (1 mL) contained 2,6-dimethoxyphenol (1 mM), MnSO4 (1 mM), and H2O2 (0.2 mM) in 50 mM malonate, pH 4.5. One katal (kat) was defined as the amount of enzyme producing 1 mol product s−1. MnP reactions were performed in 1 mL of reaction mixture containing 5 nkat MnP, 10 μL of 1 mM AFB1 in 10% dimethylsulfoxide, 1 mM MnSO4, 0.1% Tween 80, 4 nkat glucose oxidase, and 2.5 mM glucose in 50 mM mTOR inhibitor malonate, pH 4.5. Reactions were performed in triplicate for 24 h at 30 °C and mixing at 150 r.p.m. In some experiments, the amount of MnP (1–20 nkat) and the reaction time (1–48 h) were changed, and Tween 80 was excluded. The amount of AFB1 was determined by HPLC under the following conditions: column, Wakosil-II 5C18HG (4.6 mm × 150 mm, Wako Pure Chemical Industries); mobile phase, 40% aqueous methanol; flow rate, 0.5 mL min−1; and detection wavelength, 365 nm. The umu test with umulac AT was used to assay the mutagenic activity of AFB1 (Oda et al., 1995). The test was performed with Salmonella typhimurium TA1535 and S9 liver homogenate. The TA1535 strain was constructed by subcloning the bacterial O-acetyltransferase gene into a plasmid vector pACYC184 and introducing the plasmid into the original S. typhimurium TA1535/pSK1002 strain harboring
an umuC‘–’lacZ fusion gene. Assays were mafosfamide carried out in triplicate using 10 μL of test sample, 10 μL of S9mix (a metabolic activation system based on S9 liver homogenate), and 100 μL of bacterial culture. After incubation for 2 h at 37 °C, 100 μL of X-Gal solution was added to each well, and after 1 h at 37 °C, the reaction was stopped by the addition of SDS/dimethylsulfoxide solution. The absorbance of the mixture was read at 600 nm. The relative mutagenic activity (%) was defined as the percentage of β-galactosidase activity of the AFB1-containing reaction mixture (with 5, 10, or 20 nkat MnP) divided by the activity of the AFB1-containing reaction mixture without MnP. AFB1 (final concentration 160 μM) was incubated at 30 °C for 48 h in a 100-mL reaction mixture containing 750 nkat MnP, 1 mM MnSO4, 0.
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