The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase

Cationic triarylmethane dyes (TAM(+))s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM(+)s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25°C in 50mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K(i) values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 μM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K(i) values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.     

Inhibition of human plasma cholinesterase by malachite green and related triarylmethane dyes: mechanistic implications

The inhibitory effects of the cationic triarylmethane (TAM+) dyes, pararosaniline (PR+), malachite green (MG+), and methyl green (MeG+) on human plasma cholinesterase (BChE) were studied at 25 degrees C in 100 mM Mops, pH 8.0, with butyrylthiocholine as substrate. PR+ and MG+ caused linear mixed inhibition of enzyme activity. The respective inhibitory parameters were K(i) = 1.9 +/- 0.23 microM, alpha = 13 +/- 48, beta = 0 and K(i) = 0.28 +/- 0.037 microM, alpha = 23 +/- 7.4, beta = 0. MeG+ acted as a competitive inhibitor with K(i) = 0.12 +/- 0.017 microM (alpha, infinity, beta, not applicable). The K(i) values were within the same range reported for a number of ChE inhibitors including propidium ion, donepezil, and the phenothiazines, suggesting that TAM+s are active site ligands. On the other hand, the alpha values failed to correlate with values previously reported for a number of ChE inhibitors. It appears that mixed inhibition is the combined result of more than one type of binding and S-I interference. The impact of ligands at the choline-specific and peripheral anionic sites (or, possibly, accessory structural domains) on BChE activity needs to be studied in further detail.     

Comparative effects of cationic triarylmethane, phenoxazine and phenothiazine dyes on horse serum butyrylcholinesterase

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25 ^°C in 50 mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K_i values of 4.5 ± 0.50 μM, 0.41 ± 0.007 μM, 0.44 ± 0.086 μM and 0.050 ± 0.0074 μM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K′ values (≡[I]²_0.5 extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20 ± 0.096 μM, 0.0018 ± 0.0015 μM, 0.92 ± 0.23 μM and 0.23 ± 0.08 μM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.     

Inhibition of electric eel acetylcholinesterase by triarylmethane dyes

The effects of three cationic triarylmethane dyes - pararosaniline (PR), malachite green (MG), methyl green (MetG) - on electric eel AChE (eAChE) activity were tested at 25 degrees C, in 100mM MOPS buffer (pH 8) containing 0.125mM 5-5-dithio-bis(2-nitrobenzoic acid), 20-120muM acetylthiocholine and 0-20muM dye. All three dyes caused reversible, linear- or hyperbolic-mixed inhibition of esteratic activity. The respective inhibitory parameters for PR, MG and MetG were K(i)=8.4+/-0.67, 1.9+/-0.51 and 0.27+/-0.017muM; alpha (competitive coefficient)=5.8+/-2.0, 4.8+/-1.8 and 2.7+/-0.32; beta (noncompetitive coefficient)=0, 0 and 0.20+/-0.011. The data were consistent with ligand binding at the peripheral site and a remote effect on substrate binding and turnover.     

Decolorization of triphenylmethane dyes by wild mushrooms

Triphenylmethane dyes such as Crystal Violet (CV) and Malachite Green (MG) are common textile dyes. MG, which is toxic to humans, is widely used in aquaculture as an antifungal agent. In this study, 56 mushroom strains from 12 species of wild mushrooms were examined on dye-containing PDA plates to evaluate their potential for the bioremediation of synthetic dyes. Pycnoporus coccineus, Coriolus versicolor, and Lentinula edodes showed fair growth on CV, but only a few survived on MG. However, a decolorization experiment in an aqueous system revealed that the growth on MG-containing solid medium did not directly match the decolorization of MG in the aqueous system. C. versicolor IUM0061 grew well on both MG and CV plates, but could not decolorize MG in the reaction mixture. Conversely, HPLC analysis revealed that P. coccineus IUM0032, which could not grow on the MG plate, completely mineralized MG within 3 days. A subsequent enzyme activity assay revealed a high lignin peroxidase activity in the reaction mixture, indicating that lignin peroxidase is the key enzyme involved in degradation of MG in P. coccineus IUM0032.     

Peroxidase-mediated dealkylation of tamoxifen, detected by electrospray ionization-mass spectrometry, and activation to form DNA adducts

Tamoxifen (TAM) is extensively used for the treatment and prevention of breast cancer. Associated with TAM treatment is a two- to eightfold increase in risk of endometrial cancer. To understand the mechanisms associated with this increased risk several pathways for TAM metabolism and DNA adduct formation have been studied. The purpose of this study was to investigate the role of peroxidase enzymes in the metabolism of TAM and its activation to form DNA adducts. Using advanced tandem mass spectrometry we have investigated the peroxidase-mediated metabolism of TAM. Incubation of TAM with horseradish peroxidase (HRP) and H(2)O(2) produced multiple metabolites. Electrospray ionization-MS/MS analysis of the metabolites demonstrated a peak at 301.3m/z with daughter ions at 183.0, 166.9, 128.9, and 120.9m/z, which identified the metabolite as metabolite E (ME). The levels of ME were significantly inhibited by the addition of ascorbic acid to the incubation mixture. Co-incubation of either TAM or ME and DNA with HRP and H(2)O(2) produced three DNA adducts with a RAL of 1.97±0.01×10(-7) and 8.45±2.7×10(-7). Oxidation of ME with MnO(2) produced metabolite E quinone methide (MEQM). Furthermore, incubation of either TAM or ME with HRP and H(2)O(2) resulted in formation of MEQM. Reaction of calf thymus DNA with MEQM produced three DNA adducts with a RAL of 9.8±1.0×10(-7). Rechromatography analyses indicated that DNA adducts 1, 2, and 3 formed in the HRP activation of either TAM or ME were the same as those formed by the chemical reaction of DNA with MEQM. The results of these studies demonstrate that peroxidase enzymes can both metabolize TAM to form the primary metabolite ME and activate ME to a quinone methide intermediate, which reacts with DNA to form adducts. It is possible that peroxidase enzymes or peroxidase-like activity in endometrium could contribute to the formation of DNA damage and genotoxic effects in endometrium after TAM administration.     

Permeability of dormant spores of Bacillus subtilis to malachite green and crystal violet

The permeability of dormant spores of Bacillus subtilis to malachite green (MG) and crystal violet (CV) was examined by using potassium trichloro(eta 2-ethylene)platinum(II) (KTPt) as an electron-opaque marker for the dyes. The spores were treated with the dyes and other substances at 30 degrees C for 30 min or at 80 degrees C for 5 min. When the spores were incubated in 50 mM-MG solution at 30 degrees C and in 50 mM-CV solution at 30 degrees C or 80 degrees C, many small electron-dense precipitates, which were chemical complexes of dyes and platinum, were seen, mainly around the boundary between the inner and outer coat regions. The spores treated under the above conditions were not stained. Treatment with 50 mM-MG alone or a mixture of 25 mM-oxalic acid and 50 mM-CV at 80 degrees C made the spores stainable and dye-TPt precipitates were observed mainly in the outer pericortex region. Pretreatment with 25 mM-oxalic acid and 5% (v/v) phenol at 80 degrees C followed by 50 mM-CV treatment at 30 degrees C gave the same results as above. It was considered from these results that the inner coat itself might function as the primary permeability barrier to MG and CV, and that a secondary barrier to the dyes might exist around the cortex region.     

Phytoremediation of triphenylmethane dyes by overexpressing a Citrobacter sp. triphenylmethane reductase in transgenic Arabidopsis

Triphenylmethane dyes are extensively utilized in textile industries, medicinal products, biological stains, and food processing industries, etc. They are generally considered as xenobiotic compounds, which are very recalcitrant to biodegradation. The widespread persistence of such compounds has generated concerns with regard to remediation of them because of their potential carcinogenicity, teratogenicity, and mutagenicity. In this study, we present a system of phytoremediation by Arabidopsis plants developed on the basis of overexpression of triphenylmethane reductase (TMR) from the Citrobacter sp. The morphology and growth of TMR transgenic Arabidopsis plants showed significantly enhanced tolerances to crystal violet (CV) and malachite green (MG). Further, HPLC and HPLC–MS analyses of samples before and after dye decolorization in culture media revealed that TMR transgenic plants exhibited strikingly higher capabilities of removing CV from their media and high efficiencies of converting CV to non-toxic leucocrystal violet (LCV). This work indicates that microbial degradative gene may be transgenically exploited in plants for bioremediation of triphenylmethane dyes in the environment.     

Decolorization of synthetic dyes by basidiomycetes isolated from woods of the Atlantic Forest (PE), Brazil

In this study, the biodegradability of synthetic dyes used in the textile industry by Basidiomycetes collected in woods of the Atlantic Forest of Pernambuco, Brazil, was evaluated. Among the fungi, the following species were identified: Caripia montagnei, Datronia caperata, Earliella scabrosa, Fomitopsis feei, Ganoderma stiptatum, Hexagonia hydnoides, Pleurotus ostreatus, Pycnoporus sanguineus and Trametes membranacea. Mycelium discs (2 cm2) of each fungal isolate were transferred to 30 ml of King medium supplemented with 0.05% m/v of each of the following dyes: methyl orange, bromophenol blue (BB), methylene blue, Congo red, phenol red (PR) and methyl green (MG). All these species showed potential to decolorize the tested dyes, with decolorization efficiency varying from 7.26 to 99.2%. H. hydnoides and T. membrancea were the only species able to degrade all dyes. BB was effectively metabolized by P. sanguineus (97.4%), H. hydnoides (95.6%), E. scabrosa (96.6%) and T. membranacea (99.2%). E. scabrosa stood out among the isolates in dyes decolorization, allowing for efficiencies >90% for MG, PR, and BB.     

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.     

Proteasome-dependent degradation of cyclin D1 in 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell cycle arrest

1-Methyl-4-phenylpyridinium ion (MPP(+)), an active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, induces cell death and inhibition of cell proliferation in various cells. However, the mechanism whereby MPP(+) inhibits cell proliferation is still unclear. In this study, we found that MPP(+) suppressed the proliferation with accumulation in G(1) phase without inducing cell death in p53-deficient MG63 osteosarcoma cells. MPP(+) induced hypophosphorylation of retinoblastoma protein and rapidly down-regulated the protein but not mRNA levels of cyclin D1 in MG63 cells. The down-regulation of cyclin D1 protein was suppressed by a proteasome inhibitor, MG132. The cyclin D1 down-regulation by MPP(+) was also observed in p53-positive PC12, HeLa S3, and HeLa rho(0) cells, which are a subclone of HeLa S3 lacking mitochondrial DNA. Moreover, MPP(+) dephosphorylated Akt in PC12 cells, which was rescued by the pretreatment with nerve growth factor. In addition, the pretreatment with nerve growth factor or lithium chloride, a glycogen synthase kinase-3beta inhibitor, suppressed the cyclin D1 down-regulation caused by MPP(+). Our results demonstrate that MPP(+) induces cell cycle arrest independently of its mitochondrial toxicity or the p53 status of the target cells, but rather through the proteasome- and phosphatidylinositol 3-Akt-glycogen synthase kinase-3beta-dependent cyclin D1 degradation.     

Hepatoprotective effect of green tea (Camellia sinensis) extract against tamoxifen-induced liver injury in rats

Tamoxifen citrate (TAM), is widely used for treatment of breast cancer. It showed a degree of hepatic carcinogenesis. The purpose of this study was to elucidate the antioxidant capacity of green tea (Camellia sinensis) extract (GTE) against TAM-induced liver injury. A model of liver injury in female rats was done by intraperitoneal injection of TAM in a dose of 45mg Kg(-1) day(-1), i.p. for 7 successive days. GTE in the concentration of 1.5 %, was orally administered 4 days prior and 14 days after TAM-intoxication as a sole source of drinking water. The antioxidant flavonoid; epicatechin (a component of green tea) was not detectable in liver and blood of rats in either normal control or TAM-intoxicated group, however, TAM intoxication resulted in a significant decrease of its level in liver homogenate of tamoxifenintoxicated rats. The model of TAM-intoxication elicited significant declines in the antioxidant enzymes (glutathione-S-transferase,glutathione peroxidase, superoxide dismutase and catalase) and reduced glutathione concomitant with significant elevations in TBARS (thiobarbituric acid reactive substance) and liver transaminases; sGPT (serum glutamate pyruvate transaminase) and sGOT (serum glutamate oxaloacetate transaminase) levels. The oral administration of 1.5 % GTE to TAM-intoxicated rats, produced significant increments in the antioxidant enzymes and reduced glutathione concomitant with significant decrements in TBARS and liver transaminases levels. The data obtained from this study speculated that 1.5 % GTE has the capacity to scavenge free radical and can protect against oxidative stress induced by TAM intoxication. Supplementation of GTE could be useful in alleviating tamoxifen-induced liver injury in rats.     

Fast decolorization of cationic dyes by nano-scale zero valent iron immobilized in sycamore tree seed pod fibers: kinetics and modelling study

Abstract

In the present work, Sycamore (Platanus occidentalis) tree seed pod fibers (STSPF) and nano-scale zero valent iron particles (nZVI) immobilized in Sycamore tree seed pod fibers (nZVI?STSPF) were produced. This biosorbent has been utilized as a viable effective biosorbent in the removing of methylene blue hydrate (MB), malachite green oxalate(MG), methyl violet 2B(MV) dyes from synthetic wastewater. The biosorbents were characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FT-IR) spectroscopy. Various parameters such as contact time, solution concentration, pH and amount of biosorbent were investigated in order to evaluate the potential of the nanomaterials immobilized on natural wastes as sorbing biomaterials for the cationic dyes. Study on sorption kinetic and the sorption isotherm was carried out and best fitting models for the rate kinetics and isotherms were suggested. Langmuir isotherm was observed to be compatible with the isotherm models. The STSPF in the raw form showed the best dye sorption capacity of 43.67?mg/g for MG, 25.32?mg/g for MV, and 126.60?mg/g for MB. The magnetic nZVI?STSPF showed the best dye sorption capacity 92.59?mg/g for MG, 92.59?mg/g for MV, and 140.80?mg/g for MB. The iron nanoparticles immobilized biosorbent exhibited a higher removal capacity for all dyes compared to the raw biosorbent.     

Synthesis and biological evaluation of water-soluble progesterone-conjugated probes for magnetic resonance imaging of hormone related cancers

Progesterone receptor (PR) is strongly associated with disease prognosis and therapeutic efficacy in hormone-related diseases such as endometriosis and breast, ovarian, and uterine cancers. Receptor status is currently determined by immunohistochemistry assays. However, noninvasive PR imaging agents could improve disease detection and help elucidate pathological molecular pathways, leading to new therapies and animal disease models. A series of water-soluble PR-targeted magnetic resonance imaging (MRI) probes were synthesized using Cu(I)-catalyzed click chemistry and evaluated in vitro and in vivo. These agents demonstrated activation of PR in vitro and preferential accumulation in PR(+) compared to PR(-) human breast cancer cells with low toxicity. In xenograft tumor models, the agents demonstrated enhanced signal intensity in PR(+) tumors compared to PR(-) tumors. The results suggest that these agents may be promising MRI probes for PR(+) diseases.     

Attenuation of photobleaching in two-photon excitation fluorescence from green fluorescent protein with shaped excitation pulses

The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. Our method will provide a powerful solution to various problems confronting researchers, such as the photobleaching of dyes in two-photon excitation microscopy.     

Relationship of methyl purines produced by MNNG in adenovirus 5 DNA to viral inactivation in repair-deficient (Mer-) human tumor cell strains

Adenovirus 5 treated with MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) has greater plaque-forming ability in cell strains having the Mer+ phenotype than in strains having the Mer- phenotype. MNNG-treated Mer- strains repair the N3-methyladenine (N3MeA) but not the O6-methylguanine (O6MeG) produced in their DNA, while MNNG-treated Mer+ strains repair both of these adducts. The fate of N7-methylguanine (another DNA adduct produced by MNNG) is similar in Mer+ and Mer- strains. We show in this paper that 2.3 +/- 0.4 O6MeG and 1.4 N3MeA per adenovirus genome correlate with one lethal hit when the survival assay is done using Mer- strains as viral hosts. We suggest that O6MeG is the lesion lethal to the virus.     

Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism

In this report, we investigated the phenotypes caused by temperature-sensitive (ts) mutant alleles of dna2(+) of Schizosaccharomyces pombe, a homologue of DNA2 of budding yeast, in an attempt to further define its function in vivo with respect to lagging-strand synthesis during the S-phase of the cell cycle. At the restrictive temperature, dna2 (ts) cells arrested at late S-phase but were unaffected in bulk DNA synthesis. Moreover, they exhibited aberrant mitosis when combined with checkpoint mutations, in keeping with a role for Dna2 in Okazaki fragment maturation. Similarly, spores in which dna2(+) was disrupted duplicated their DNA content during germination and also arrested at late S-phase. Inactivation of dna2(+) led to chromosome fragmentation strikingly similar to that seen when cdc17(+), the DNA ligase I gene, is inactivated. The temperature-dependent lethality of dna2 (ts) mutants was suppressed by overexpression of genes encoding subunits of polymerase delta (cdc1(+) and cdc27(+)), DNA ligase I (cdc17(+)), and Fen-1 (rad2(+)). Each of these gene products plays a role in the elongation or maturation of Okazaki fragments. Moreover, they all interacted with S. pombe Dna2 in a yeast two-hybrid assay, albeit to different extents. On the basis of these results, we conclude that dna2(+) plays a direct role in the Okazaki fragment elongation and maturation. We propose that dna2(+) acts as a central protein to form a complex with other proteins required to coordinate the multienzyme process for Okazaki fragment elongation and maturation.     

Tumor-associated microglia/macrophages enhance the invasion of glioma stem-like cells via TGF-β1 signaling pathway

The invasion of malignant glioma cells into the surrounding normal brain tissues is crucial for causing the poor outcome of this tumor type. Recent studies suggest that glioma stem-like cells (GSLCs) mediate tumor invasion. However, it is not clear whether microenvironment factors, such as tumor-associated microglia/macrophages (TAM/Ms), also play important roles in promoting GSLC invasion. In this study, we found that in primary human gliomas and orthotopical transplanted syngeneic glioma, the number of TAM/Ms at the invasive front was correlated with the presence of CD133(+) GSLCs, and these TAM/Ms produced high levels of TGF-β1. CD133(+) GSLCs isolated from murine transplanted gliomas exhibited higher invasive potential after being cocultured with TAM/Ms, and the invasiveness was inhibited by neutralization of TGF-β1. We also found that human glioma-derived CD133(+) GSLCs became more invasive upon treatment with TGF-β1. In addition, compared with CD133(-) committed tumor cells, CD133(+) GSLCs expressed higher levels of type II TGF-β receptor (TGFBR2) mRNA and protein, and downregulation of TGFBR2 with short hairpin RNA inhibited the invasiveness of GSLCs. Mechanism studies revealed that TGF-β1 released by TAM/Ms promoted the expression of MMP-9 by GSLCs, and TGFBR2 knockdown reduced the invasiveness of these cells in vivo. These results demonstrate that TAM/Ms enhance the invasiveness of CD133(+) GSLCs via the release of TGF-β1, which increases the production of MMP-9 by GSLCs. Therefore, the TGF-β1 signaling pathway is a potential therapeutic target for limiting the invasiveness of GSLCs.     

Adsorption and decolorization of dyes using solid residues from Pleurotus ostreatus mushroom production

We investigated the use of solid residues from Pleurotus ostreatus mushroom production in adsorbing and decolorizing different dyes. The solid residue used in this study was composed of hemicellulose and cellulose (52.81 %), acid-insoluble lignin (25.42%), chitin (6.5%), and water extractives (14.82%). After incubating 14% (wt/vol) solid residue in distilled water for 4 h, laccase and manganese peroxidase (MnP) activities were 0.5 U/g and 12 mU/g, respectively. Enzymatic decolorization percentages were up to 100 for azure B (heterocyclic dye) and indigo carmine (indigoid dye), 74.5 for malachite green (MG) (triphenylmethane dye), and zero for xylidine (azoic dye). The optimum temperature for decolorization was in the range of 26 ∼ 36°C for all dyes. Data obtained on adsorption (enzymatic decolorization was prevented with sodium azide) at different dye concentrations and in a pH range of 3 ∼ 7 were used to plot Freundlich isotherms. The spent fungal substrate (SFS) displayed large differences in adsorption capacity, depending on the dye tested. The highest adsorption capacity was observed at pH 3 for MG, while xylidine was slightly adsorbed at pH 3 and 4 and not adsorbed at higher pH values. Laccase and MnP production were affected by the presence of the dyes. The highest enzyme levels were observed in the presence of MG, when laccase and MnP increased 1.39- and 2.13-fold, respectively. Decolorization and adsorption to SFS are both important processes in removing dyes from aqueous solutions. The application of this spent substrate for wastewater treatment will be able to take advantage of both of these dye removal processes. An important problem in bioremediation processes involving microorganisms is the amount of time required for their growth. In this report, we used the spent substrates from mushroom cultivation in wastewater treatment, thus solving the problem of waiting for microorganisms to grow.