Detoxification of aflatoxin B₁ by manganese peroxidase from the white-rot fungus Phanerochaete sordida YK-624

Abstract

Aflatoxin B₁ (AFB₁) is a potent mycotoxin with mutagenic, carcinogenic, teratogenic, hepatotoxic, and immunosuppressive properties. In order to develop a bioremediation system for AFB₁-contaminated foods by white-rot fungi or ligninolytic enzymes, AFB₁ was treated with manganese peroxidase (MnP) from the white-rot fungus Phanerochaete sordida YK-624. AFB₁ was eliminated by MnP. The maximum elimination (86.0%) of AFB₁ was observed after 48 h in a reaction mixture containing 5 nkat of MnP. The addition of Tween 80 enhanced AFB₁ elimination. The elimination of AFB₁ by MnP considerably reduced its mutagenic activity in an umu test, and the treatment of AFB₁ by 20 nkat MnP reduced the mutagenic activity by 69.2%. ¹H-NMR and HR-ESI-MS analysis suggested that AFB₁ is first oxidized to AFB₁-8,9-epoxide by MnP and then hydrolyzed to AFB₁-8,9-dihydrodiol. This is the first report that MnP can effectively remove the mutagenic activity of AFB₁ by converting it into AFB₁-8,9-dihydrodiol.

manganese peroxidase, aflatoxin B₁, Phanerochaete sordida YK-624, mutagenic activity, detoxification

Introduction

The human diet can contain a wide variety of natural carcinogens due to the contamination of raw materials or the production of metabolites during food processing or cooking (Osowski et al., 2010). Aflatoxins, a group of potent mycotoxins with mutagenic, carcinogenic, teratogenic, hepatotoxic, and immunosuppressive properties, are of particular importance because of their adverse effects on animal and human health (Lewis et al., 2005). Aflatoxins are produced as secondary metabolites of fungal strains (Aspergillus flavus Link:Fries, Aspergillus parasiticus Speare, and Aspergillus nomius Kurtzman et al.) that grow on a variety of food and feed commodities (Peltonen et al., 2001; Jiang et al., 2005). Aflatoxin B₁ (AFB₁), which is the most toxic aflatoxin, is of particular interest because it is a frequent contaminant of many food products and one of the most potent naturally occurring mutagens and carcinogens known (Teniola et al., 2005).

White-rot fungi have the apparently unique ability to degrade lignin to the level of CO₂ (Kirk & Farrell, 1987). Lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase are the major extracellular ligninolytic enzymes of white-rot fungi involved in lignin biodegradation (Kirk & Farrell, 1987). There is a great interest in lignin-degrading white-rot fungi and their ligninolytic enzymes because of their potential to degrade recalcitrant environmental pollutants, such as polychlorinated dibenzodioxin (Kamei et al., 2005), lindene (Bumpus et al., 1985), chlorophenols (Joshi & Gold, 1993), and polycyclic aromatic carbons (Bezalel et al., 1996; Collins et al., 1996). Recently, ligninolytic enzymes such as MnP and laccase were shown to be effective in degrading methoxychlor (Hirai et al., 2004) and Irgarol 1051 (Ogawa et al., 2004) and in removing the estrogenic activities of bisphenol A, nonylphenol (Tsutsumi et al., 2001), 4-tert-octylphenol (Tamagawa et al., 2007), butylparabens (Mizuno et al., 2009), genistein (Tamagawa et al., 2005), and steroidal hormones (Suzuki et al., 2003; Tamagawa et al., 2006). More recently, the degradation of AFB₁ by fungal laccases has been reported (Alberts et al., 2009). However, a degradation product was not detected and the mechanism of degradation remains unclear.

In the present study, we demonstrate the detoxification of AFB₁ by MnP from the white-rot fungus Phanerochaete sordida YK-624, which produces LiPs (Sugiura et al., 2003; Hirai et al., 2005) and MnP (Hirai et al., 1994; Kondo et al., 1994) as ligninolytic enzymes. We also detected the metabolites and, on their basis, developed a possible mechanism for their production.

Materials and methods

Fungus

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.

Chemicals

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 and were used without further purification.

MnP preparation and determination of MnP activity

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 treatment of AFB₁

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 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.

Mutagenic activity of AFB₁

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 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.

Metabolism experiments

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, 0.1% Tween 80, 600 nkat glucose oxidase, and 2.5 mM glucose in 50 mM malonate buffer, pH 4.5. The reaction mixture was extracted twice with 100 mL ethyl acetate. The extract was dried over anhydrous sodium sulfate and then evaporated to dryness. The concentrate was separated by HPLC to isolate the AFB₁ metabolite. The purified metabolite was then analyzed by HR-ESI-MS (JMS-T100LC, JEOL, Japan) and ¹H-NMR (Jeol lambda-500, 500 MHz, JEOL). Chemical shifts are expressed in δ relative to the external standard, sodium 3-(trimethylsilyl) propionate.

Results

Elimination of AFB₁ by MnP from P. sordida YK-624

We showed previously that ligninolytic enzymes from white-rot fungi can degrade a wide range of aromatic compounds (Tsutsumi et al., 2001; Suzuki et al., 2003; Hirai et al., 2004; Tamagawa et al., 2005, 2006, 2007; Mizuno et al., 2009). In the current study, we examined whether MnP from P. sordida YK-624 can oxidize AFB₁, which is a difuranocoumarin derivate.

After a 24-h reaction using 5 nkat MnP, the level of AFB₁ was reduced by 73.3% (Fig. 1). Further examination of the dose dependence showed that the maximum elimination was obtained at 5 nkat of enzyme. Tween 80, an unsaturated fatty acid that allows MnP to oxidize nonphenolic compounds (Bao et al., 1994), enhanced the elimination of AFB₁ (Fig. 1). Analysis of the time course of AFB₁ elimination by MnP in the presence of Tween 80 (Fig. 2) reveals that AFB₁ was drastically decreased after a 4-h treatment, and that 86.0% of AFB₁ was eliminated after a 48-h treatment.

1

Elimination of AFB₁ in the presence of different activities of MnP. Closed circles, with Tween 80; open circles, without Tween 80. MnP reactions were performed in 1 mL of reaction mixture containing 1–20 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 malonate, pH 4.5. Reactions were performed for 24 h at 30°C and mixing at 150 r.p.m. Values are means±SD of triplicate samples.

2

Time course for AFB₁ elimination by MnP. Reactions contained 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 malonate, pH 4.5. Reactions were performed for 24 h at 30°C and mixing at 150 r.p.m. Values are means±SD of triplicate samples.

Removal of the mutagenic activity of AFB₁

Because the removal of toxicity is essential for the biodegradation of environmental pollutants, we examined the mutagenic activity of the metabolites of AFB₁ generated by MnP. Mutagenic activity was measured using the umu test following the treatment of AFB₁ by a metabolic activation system (S9mix) because, in animals, the toxicity of AFB₁ is activated by cytochrome P450 in the liver (Eaton & Gallagher, 1994). AFB₁ (100 μM) had approximately sevenfold higher mutagenic activity than 2-aminoanthracene (100 μM), a well-known mutagen (Fig. 3). The treatment of AFB₁ by 5 and 20 nkat MnP reduced the mutagenic activity by 49.4% and 69.2%, respectively (Fig. 4).

3

Mutagenic activity of AFB₁ in the umu test. Closed circles, AFB₁; open circles, 2-aminoanthracene. Experimental details were described in Materials and methods. Values are means±SD of triplicate samples.

4

MnP decreases the mutagenic activity of AFB₁. MnP reactions were performed in 1 mL of reaction mixture containing 5–20 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 malonate, pH 4.5. Reactions were performed for 24 h at 30°C and mixing at 150 r.p.m. Values are means±SD of triplicate samples.

Identification of an AFB₁ metabolite generated by MnP

HPLC detected a metabolite generated by MnP from AFB₁ with a retention time of 10.5 min, whereas AFB₁ has a retention time of 32.8 min (Fig. 5). The metabolite was fractionated and purified by HPLC and then analyzed using ¹H-NMR and HR-ESI-MS. The ¹H-NMR spectrum in the presence of CD₃OD yielded strong C8 and C9 proton signals (δ_H 4.54 and 3.44, respectively) in the upper field compared with AFB₁ (AFB₁ H8 [δ_H 6.78], AFB₁ H9 [δ_H 6.44]). HR-ESI-MS, which yielded an m/z of 345.06229 [M-H]⁻ (calculated for C₁₇H₁₃O₈, 345.06104), indicated a molecular formula of C₁₇H₁₄O₈, suggesting a molecular mass of 346. The metabolite had a mass 34 greater than the molecular ion of AFB₁. These results indicate that AFB₁ was converted to AFB₁-8,9-dihydrodiol by MnP.

5

Detection of the AFB₁ metabolite by HPLC (a) and ESI-MS spectra of AFB₁ metabolite (b) and AFB₁ (c). These compounds were detected by HPLC under the following conditions: column, Wakosil-II 5C18HG; mobile phase, 40% aqueous methanol; flow rate, 0.5 mL min⁻¹; detection wavelength, 365 nm.

Discussion

The extracellular ligninolytic enzymes produced by white-rot fungi are nonspecific and nonstereoselective enzymes that can degrade not only lignin but also a range of recalcitrant pollutants, making them of great interest for the removal of environmental contamination (Asgher et al., 2008). In the present study, we showed that AFB₁, which is a nonphenolic, difuranocoumarin derivate, can be oxidized by MnP from P. sordida YK-624.

MnP removed approximately 70% of AFB₁ after 24 h and was capable of removing AFB₁ even in the absence of Tween 80. Although the complete elimination of AFB₁ was not observed in the present study, it is thought that AFB₁ is completely eliminated by the multitreatment with MnP. Mn(III), which is produced by MnP, could not oxidize AFB₁ directly (data not shown). In the presence of Tween 80, lipid-derived peroxy radicals are produced (Bao et al., 1994) that may directly oxidize AFB₁. On the other hand, formate and superoxide anion radicals, which are generated in the MnP reaction mixture in the absence of Tween 80 (Khindaria et al., 1994), may mediate the oxidation of AFB₁ by MnP alone.

AFB₁-8,9-dihydrodiol was generated as a metabolite generated from AFB₁ by MnP. This metabolite has also been detected in some animals treated with AFB₁ (Wu et al., 2009). AFB₁-8,9-dihydrodiol is produced in some animals by the hydrolysis of AFB₁-8,9-epoxide, which is formed when the 8,9-vinyl bond is oxidized by the microsomal cytochrome P450 system (Kuilman et al., 2000). Our current results suggest that similar reactions, namely the epoxidation of AFB₁, followed by hydrolysis of AFB₁-8,9-epoxide, occur when AFB₁ is oxidized by MnP. As detailed in Fig. 6, we propose that the 8,9-vinyl bond of AFB₁ can be oxidized by the peroxy radicals of Tween 80, formate radical, superoxide anion radical, or MnP directly (Tuynman et al., 2000) and that the epoxide thus generated is hydrolyzed spontaneously to AFB₁-8,9-dihydrodiol (Guengerich et al., 1996).

6

Proposed mechanism of AFB₁ oxidation by MnP.

The removal of toxicity is the most important goal for the biodegradation of environmental pollutions. Here, we showed that MnP not only removes but also detoxifies AFB₁. The metabolite generated from AFB₁ by MnP, AFB₁-8,9-dihydrodiol, is less toxic than AFB₁ because AFB₁-8,9-dihydrodiol can rearrange and form a reactive dialdehyde that can react with primary amine groups in proteins by Schiff base reactions (Sabbioni et al., 1987). This prevents the formation of DNA adducts, which can cause mutations. Although AFB₁ eliminations by MnP (5–20 nkat) were almost the same, the decrease in mutagenic activity was higher with 20 nkat MnP (69.2%) than with 5 nkat MnP (49.4%), as shown in Fig. 4. It is thought that the amount of AFB₁-8,9-epoxide in the reaction mixture containing 5 nkat MnP was higher than that in the reaction mixture containing 20 nkat MnP.

In summary, we show for the first time that MnP can remove the mutagenic activity of AFB₁ by converting it to AFB₁-8,9-dihydrodiol. This system should therefore be useful in the bioremediation of AFB₁-contaminated foods.

Author notes

Editor: Andreas Stolz