Successive transformation of benzo[a]pyrene by laccase of Trametes versicolor and pyrene-degrading Mycobacterium strains

We previously hypothesized that polycyclic aromatic hydrocarbon (PAH)-degrading bacteria that produce laccase may enhance the degree of benzo[a]pyrene mineralization. However, whether the metabolites of benzo[a]pyrene oxidized by laccase can be further transformed by PAH degraders remains unknown. In this study, pyrene-degrading mycobacteria with diverse degradation properties were isolated and employed for investigating the subsequent transformation on the metabolites of benzo[a]pyrene oxidized by fungal laccase of Trametes versicolor. The results confirm the successive transformation of benzo[a]pyrene metabolites, 6-benzo[a]pyrenyl acetate, and quinones by Mycobacterium strains, and report the discovery of the involvement of a O-methylation mediated pathway in the process. In detail, the vast majority of metabolite 6-benzo[a]pyrenyl acetate was transformed into benzo[a]pyrene quinones or methoxybenzo[a]pyrene, via two distinct steps that were controlled by the catechol-O-methyltransferase mediated O-methylation, while quinones were reduced to dihydroxybenzo[a]pyrene and further transformed into dimethoxy derivatives.     

Degradation of Benzo[a]pyrene by Mycobacterium vanbaalenii PYR-1

Metabolism of the environmental pollutant benzo[a]pyrene in the bacterium Mycobacterium vanbaalenii PYR-1 was examined. This organism initially oxidized benzo[a]pyrene with dioxygenases and monooxygenases at C-4,5, C-9,10, and C-11,12. The metabolites were separated by reversed-phase high-performance liquid chromatography (HPLC) and characterized by UV-visible, mass, nuclear magnetic resonance, and circular dichroism spectral analyses. The major intermediates of benzo[a]pyrene metabolism that had accumulated in the culture media after 96 h of incubation were cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-4,5-dihydrodiol), cis-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene cis-11,12-dihydrodiol), trans-11,12-dihydro-11,12-dihydroxybenzo[a]pyrene (benzo[a]pyrene trans-11,12-dihydrodiol), 10-oxabenzo[def]chrysen-9-one, and hydroxymethoxy and dimethoxy derivatives of benzo[a]pyrene. The ortho-ring fission products 4-formylchrysene-5-carboxylic acid and 4,5-chrysene-dicarboxylic acid and a monocarboxylated chrysene product were formed when replacement culture experiments were conducted with benzo[a]pyrene cis-4,5-dihydrodiol. Chiral stationary-phase HPLC analysis of the dihydrodiols indicated that benzo[a]pyrene cis-4,5-dihydrodiol had 30% 4S,5R and 70% 4R,5S absolute stereochemistry. Benzo[a]pyrene cis-11,12-dihydrodiol adopted an 11S,12R conformation with 100% optical purity. The enantiomeric composition of benzo[a]pyrene trans-11,12-dihydrodiol was an equal mixture of 11S,12S and 11R,12R molecules. The results of this study, in conjunction with those of previously reported studies, extend the pathways proposed for the bacterial metabolism of benzo[a]pyrene. Our study also provides evidence of the stereo- and regioselectivity of the oxygenases that catalyze the metabolism of benzo[a]pyrene in M. vanbaalenii PYR-1.     

Benzo[a]pyrene removal by Marasmiellus troyanus in soil microcosms

Benzo[a]pyrene (B[a]P) is a carcinogenic polyaromatic hydrocarbon that enters the environment as an incomplete combustion production of fossil fuels. Several species of filamentous fungi are capable of biotransforming and/or mineralizing B[a]P in liquid cultures, however there has been less success in soil habitats. In this study, the litter rot fungus Marasmiellus troyanus was encapsulated in alginate and delivered to B[a]P-spiked soil microcosms (100 μg B[a]P/g soil) for 1, 2 and 6 weeks, with and without a fertilizer solution. After 2 weeks, 32.5% of B[a]P was recovered from soil microcosms treated with M. troyanus compared to 55–70% for controls. After 6 weeks, controls demonstrated an average percent recovery of B[a]P of 54% while M. troyanus-inoculated samples gave an average percent recovery of 11%. Similar bioaugmentation of contaminated habitats with appropriately formulated fungi has potential for practical bioremediation in soil environments. Journal of Industrial Microbiology & Biotechnology (2000) 25, 116–119.     

Removal of Benzo[a]pyrene by a fungus Aspergillus sp. BAP14

A fungal strain BAP14 isolated from marine sediments of coast in Xiamen city, was found to have the ability to degrade benzo[a]pyrene (BaP), and identified as Aspergillus sp. based on 18S rRNA gene sequence. Aspergillus sp. BAP14 was able to remove about 30 and 60% of BaP with initial concentration of 10 mg l⁻¹ in 3 and 12 days of incubation, respectively. Addition of saccharides and low molecular weight polycyclic aromatic hydrocarbons appeared to have effect on the degradation ability, in particularly the addition of lactose and naphthalene. Furthermore, we demonstrated that lipidic particles could be observed in the presence of benzo[a]pyrene based on the morphologic performance of Aspergillus sp. BAP14 through scanning electronic microscopy (SEM) and atomic force microscopy (AFM), respectively.     

Benzo[a]pyrene uptake by serum lipids: correlation with triglyceride concentration

An in vitro study of the relationship between benzo[a]pyrene (BaP) association with serum lipoproteins (LP) and LP composition was conducted using human subjects. BaP partitioning into different serum LP ranged from 53 to 71% of available BaP. Efficiency of BaP partitioning was examined for the relationship with lipid components of different sera. The data indicate that triglyceride (TG) concentrations were more directly correlated with BaP uptake than were concentrations of other LP components. Adjusting sera to a uniform TG concentration (96.5 mg/dl) resulted in the same BaP uptake for each serum type, while adjusting sera to contain a uniform cholesteryl ester concentration (104.6 mg/dl) did not result in similar BaP uptake among serum types. Analysis of serum LP composition suggested that marked differences in both BaP uptake and serum TG concentrations among the subjects were due mainly to differences in serum very low density lipoprotein (VLDL) concentrations. A correlation study using 14 human subjects showed that serum TG concentration was the best predictor (r = 0.973, P less than 0.001) for BaP uptake by serum, followed by phospholipid (r = 0.658, P less than 0.01) and total cholesterol (r = 0.514, P less than 0.05) concentrations. The results indicate that serum TG concentration (typically VLDL-TG) may be the primary factor affecting BaP uptake by serum LP, and suggest that a small change in serum TG concentration could cause a significant increase in BaP uptake by serum LP, contributing to an increased level of circulating carcinogen.     

Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

Summary Laccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.     

Oxidation of phenyl compounds using strongly stable immobilized-stabilized laccase from Trametes versicolor

The hydrolysis of phenolic compounds using an immobilized and highly active and stable derivative of laccase from Trametes versicolor is presented. The enzyme was immobilized on aldehyde supports. For this, the enzyme was enriched in amino groups by chemical modification of its carboxyl groups. The aminated enzyme was immobilized with a high recovered activity (over 60%). Aldehyde derivatives were more stable than soluble or aminated-soluble enzyme and the reference derivatives after incubation in different inactivating conditions (high temperatures, different pH values or presence of organic cosolvents). The most stable derivative was obtained immobilizing the chemically aminated enzyme at pH 10 on aldehyde supports with a stabilization factor approximately 280 fold after incubation at pH 7 and 55 °C. In addition, it was possible to prepare immobilized derivatives with a maximal enzyme loading of 60 mg g^?1 of support. This derivative could be reused for 10 reaction cycles with negligible lost of activity.     

Benzo[a]pyrene exposed to solar-simulated light inhibits apoptosis and augments carcinogenicity

Polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) are widespread environmental pollutants and several lines of experimental evidence have suggested a role in carcinogenesis. PAHs in the environment are exposed to sunlight and photomodified PAHs have been detected in contaminated sediment and air particulate matter; however, the carcinogenicity of photomodified PAHs is not well understood. In this study, we found that solar-simulated light-irradiated BaP (LBaP) inhibited apoptosis, leading to cancer. LBaP suppressed apoptosis induced by cell detachment and serum depletion in a dose and light-irradiated time-dependent manner. The antiapoptotic effect was related to the production of reactive oxygen species from degraded BaP. The cells that survived apoptosis by LBaP treatment were transformed having the ability to form colonies in soft agar and tumors in nude mice. These capabilities were specific to LBaP, not BaP itself. The results suggested that the carcinogenicity of PAHs may be attributable not only to the genetic damage induced by their metabolites, but also to the antiapoptotic effects of oxidative products on exposure to sunlight.     

Benzo[a]pyrene uptake into rat liver microsomes: Effects of adsorption of benzo[a]pyrene to asbestos and non-fibrous mineral particulates

The fluorescence yield of benzo[a]pyrene (BP) increases dramatically upon its transfer from the surface of particulates to rat liver microsomes. Adsorption of BP to Canadian chrysotile, anthophyllite, hematite and silica results in greatly enhanced uptake rates into microsomes when compared to uptake from a microcrystalline dispersion of BP. The fibrous minerals chrysotile and anthophyllite were more effective than silica and hematite in enhancing BP uptake. Simple mixtures of BP microcrystals and particles did not display enhanced transport, indicating that adsorption of BP to the particulate surface is necessary for enhanced microsomal uptake. BP was not released into microsomes from carbon black.We suggest that particulate-enhanced availability of BP may be of significance in the co-carcinogenesis between particulates and polynuclear aromatic hydrocarbons. However, other mechanisms are also possible, and are not excluded by our experiments. The fluorescence methodology described in this paper provides a novel and convenient means to quantify microsomal uptake of BP and thereby investigate further the mechanisms of cocarcinogenesis.     

Factors Influencing the Photodynamic Action of Benzo[a]pyrene on Escherichia coli

Death of Escherichia coli resulted when a buffer suspension was exposed simultaneously to colloidal benzo[a]pyrene (BP) and 355-mmu illumination. Neither hydrocarbon nor illumination alone caused death; oxygen had to be present. The survival curve had a shoulder, and then death proceeded exponentially with time. Death rate was independent of temperature between 6 and 32 C. The duration of the shoulder, however, decreased slightly with increase in temperature. The shoulder was not due to delay in BP entering the cell. Death was influenced by the composition of the medium in which the cells were grown prior to illumination. The amount of BP bound to the cells was determined after three ethyl alcoholether extractions. Appreciable binding occurred in the presence of 355-mmu illumination with air, and relatively little binding occurred under nitrogen; very little binding occurred in the dark with nitrogen or air. At the outset, rate of binding under illumination with air was not temperature-dependent, but with time it became strongly temperature-dependent. Binding under illumination with nitrogen was temperature-independent. Bound BP was associated primarily with cell protein. Cells in growth medium resisted death and BP binding. At 21 and 32 C, deoxyribonucleic acid damage occurred during exponential death. No damage was detected at 21 and 32 C in the dark with BP, under illumination in absence of BP, or under illumination with BP in a nitrogen atmosphere.     

Benzo[a]pyrene metabolism and induction of enzyme-altered foci in regenerating rat liver

The metabolism of benzo[a]pyrene (BP) in regenerating rat liver and the induction of enzyme-altered foci (EAF) in the liver of partially hepatectomized rats, treated with BP and promoted with 2-acetylaminofluorene (2-AAF)/CCl4 was investigated. The aim was to examine factors that might be of importance for the tumorigenicity of BP in the regenerating rat liver, such as cytochrome P-450 activity and glutathione levels. In regenerating rat liver, obtained 18 h after partial hepatectomy (PH), the amount of microsomal cytochrome P-450 was reduced by 20% whereas the level of glutathione was elevated by 15% and the cytosolic glutathione transferase activity towards chlorodinitrobenzene and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) was unaffected. Microsomes from these animals had a reduced capacity to activate (-)-trans-7,8-dihydroxy-7,8-dihydro-BP (BPD) to DNA-binding products but the pattern of BP metabolites was similar to that observed with control rat liver microsomes. Treatment of rats with 3-methylcholanthrene (MC, 50 mg/kg body wt.) increased cytochrome P-450 levels and glutathione transferase activity towards both substrates. Regenerating livers from these animals retained their cytochrome P-450 level and enzymatic activity towards BP and BPD. Regenerating rat liver microsomes from MC-treated animals were about 35 times more efficient in activating BPD than microsomes from uninduced, partially hepatectomized animals. Intraperitoneal administration of BP (50 mg/kg body wt.) 18 h after PH induced EAF in rats subsequently promoted with 2-AAF/CCl4. Pretreatment of rats with MC 66 h before PH and 84 h before BP administration, increased the number of EAF. In accordance with results by Tsuda et al. (Cancer Res., 40 (1980) 1157-1164), these studies demonstrate that BP is tumorigenic in regenerating rat liver, despite a reduced ability of the liver to activate this compound. Furthermore, MC, an inducer of certain cytochrome P-450 species ("aryl hydrocarbon hydroxylase"), potentiates the effect of BP.     

Oxidation of benzo[a]pyrene by the filamentous fungus Cunninghamella elegans.

Cunninghamella elegans oxidized benzo[a]pyrene to several metabolic products. Compounds that were isolated and identified were: trans-9,10-dihydroxy-9,10-dihydrobenzo[a]pyrene, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, benzo[a]pyrene 1,6-quinone, benzo[a]pyrene 3,6-quinone, 9-hydroxybenz[a]pyrene, and 3-hydroxybenzo[a]pyrene. In addition, an unidentified dihydroxybenzo[a]pyrene metabolite was also formed. Experiments with [14C]benzo[a]pyrene showed that over a 96-h period, 18.4% of the hydrocarbon was converted to metabolic products. Most of the metabolites were sulfate conjugates as demonstrated by the formation of benzo[a]pyrene quinones and phenols after treatment with aryl sulfatase. Glucuronide and sulfate conjugates were also detected as water-soluble metabolites. The results show that benzo[a]pyrene is metabolized by a filamentous fungus in a manner that is remarkably similar to that observed in higher organisms.     

Benzo[a]pyrene diol-epoxides: different mutagenic efficiency in human and bacterial cells

Monolayer cultures of diploid human fibroblasts and suspensions of S. typhimurium TA100 cells were treated with [3H]-labelled enantiomeric forms of benzo[a]pyrene anti and syn 7,8-dihydrodiol 9,10-epoxides. In both cell types, all of the enantiomers induced the formation of mutant 6-thioguanine (human) or 8-azaguanine-(bacterial)resistant cells. Diol-epoxide-modified nucleosides from human and from bacterial DNA hydrolysates were characterized by HPLC and showed essentially the same adduct species for human and bacterial cells treated with the same enantiomers. There were substantial differences, however, in the efficiency with which structurally-different adduct species were converted to mutant genotypes. In human cells, the mutagenic efficiency (mutation frequency/unit modified DNA) of the respective adduct species (+ anti much greater than -anti = +/- syn) at the hprt locus was exactly the opposite of that seen at a similar gene locus (gpt) in TA100 (-anti = +/- syn greater than + anti). The results suggest that the structural configuration of adducts in genomic DNA is important in determining whether a mutant genotype will result, and likewise, that there are differences in specificity between the human and bacterial systems which process these adduct lesions.     

Degradation of Benzo[a]pyrene by the Litter-Decomposing Basidiomycete Stropharia coronilla: Role of Manganese Peroxidase

The litter-decomposing basidiomycete Stropharia coronilla, which preferably colonizes grasslands, was found to be capable of metabolizing and mineralizing benzo[a]pyrene (BaP) in liquid culture. Manganese(II) ions (Mn²⁺) supplied at a concentration of 200 μM stimulated considerably both the conversion and the mineralization of BaP; the fungus metabolized and mineralized about four and twelve times, respectively, more of the BaP in the presence of supplemental Mn²⁺ than in the basal medium. This stimulating effect could be attributed to the ligninolytic enzyme manganese peroxidase (MnP), whose activity increased after the addition of Mn²⁺. Crude and purified MnP from S. coronilla oxidized BaP efficiently in a cell-free reaction mixture (in vitro), a process which was enhanced by the surfactant Tween 80. Thus, 100 mg of BaP liter⁻¹ was converted in an in vitro reaction solution containing 1 U of MnP ml⁻¹ within 24 h. A clear indication was found that BaP-1,6-quinone was formed as a transient metabolite, which disappeared over the further course of the reaction. The treatment of a mixture of 16 different polycyclic aromatic hydrocarbons (PAHs) selected by the U.S. Environmental Protection Agency as model standards for PAH analysis (total concentration, 320 mg liter⁻¹) with MnP resulted in concentration decreases of 10 to 100% for the individual compounds, and again the stimulating effect of Tween 80 was observed. Probably due to their lower ionization potentials, poorly bioavailable, high-molecular-mass PAHs such as BaP, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene were converted to larger extents than low-molecular-mass ones (e.g., phenanthrene and fluoranthene).     

Optimization of laccase production from Trametes versicolor by solid fermentation

The regulation of culture conditions, especially the optimization of substrate constituents, is crucial for laccase production by solid fermentation. To develop an inexpensive optimized substrate formulation to produce high-activity laccase, a uniform design formulation experiment was devised. The solid fermentation of Trametes versicolor was performed with natural aeration, natural substrate pH (about 6.5), environmental humidity of 60% and two different temperature stages (at 37 degrees C for 3 days, and then at 30 degrees C for the next 17 days). From the experiment, a regression equation for laccase activity, in the form of a second-degree polynomial model, was constructed using multivariate regression analysis and solved with unconstrained optimization programming. The optimized substrate formulation for laccase production was then calculated. Tween 80 was found to have a negative effect on laccase production in solid fermentation; the optimized solid substrate formulation was 10.8% glucose, 27.7% wheat bran, 9.0% (NH4)2SO4, and 52.5% water. In a scaled-up verification of solid fermentation at a 10 kg scale, laccase activity from T. versicolor in the optimized substrate formulation reached 110.9 IU/g of dry mass.     

Comparative Sorption of Phenanthrene and Benzo[α]pyrene to Soil Humic Acids

Sorption of phenanthrene (Phen) and benzo[α]pyrene (BaP) by humic acids (HAs) extracted from four typical soils in China, including cinnamon soil, fluvo-aquic soil, red soil, and mountain meadow soil were investigated. All sorption data were fitted well by the Freundlich model, but BaP exhibited stronger and more nonlinear sorption than Phen by a given HA. For Phen isotherms, there was a positive relation between K _oc values and aliphaticity of HAs, whereas a negative correlation was observed between n values and aliphaticity. This indicated the importance of aliphatic groups in Phen sorption capacity and nonlinearity. Compared to Phen, a similar trend was obtained between n values of BaP and aliphaticity or aromatic carbon. However, no correlation existed between K _oc values for BaP and aliphaticity or aromatic carbon.     

Benzo[a]pyrene-collagen interactions

In vitro interactions of benzo[a]pyrene (BaP) with acid-soluble type I collagen from rat tail tendon have been investigated. The fluorescence of BaP increases in the presence of collagen. Bound BaP inhibits the formation of collagen fibrils in solution. When BaP-collagen complexes are irradiated in air with UV (365 nm) light, BaP rapidly undergoes photooxidation with the further inhibition of fibril formation. Viscosity and circular dichroism (CD) studies show that neither BaP nor further UV-irradiation alters the size or helical conformation of the protein. During thermal denaturation of collagen, BaP fluorescence changes. Collagen from young rat tail tendon shows a pronounced drop at about 38 degrees C, whereas that from old rat tail tendon exhibits an increase with a plateau in the same temperature range. These anomalous changes are observed when tyrosine residues, present only in the non-helical terminal telopeptides of collagen, are excited at 275 nm, but not by direct BaP excitation at 387 nm. These findings suggest that the specific hydrophobic telopeptide region, which plays an important role in fibril formation, are affected by bound BaP.