Intracellular Enzymes Contribution to the Biocatalytic Removal of

Article pubs.acs.org/est

Intracellular Enzymes Contribution to the Biocatalytic Removal of Pharmaceuticals by Trametes hirsuta Lounès Haroune,† Sabrina Saibi,‡ Hubert Cabana,‡ and Jean-Philippe Bellenger*,† †

Department of Chemistry, Université de Sherbrooke, 2500 Boul. de l’Université, Sherbrooke, Quebec J1K 2R1, Canada Department of Civil Engineering, Université de Sherbrooke, 2500 Boul. de l’Université, Sherbrooke, Quebec J1K 2R1, Canada

‡

S Supporting Information *

ABSTRACT: The use of white rot fungi (WRF) for bioremediation of recalcitrant trace organic contaminants (TrOCs) is becoming greatly popular. Biosorption and lignin modifying enzymes (LMEs) are the most often reported mechanisms of action. Intracellular enzymes, such as cytochrome P450 (CYP450), have also been suggested to contribute. However, direct evidence of TrOCs uptake and intracellular transformation is lacking. The aim of this study was to evaluate the relative contribution of biosorption, extracellular LMEs activity, TrOCs uptake, and intracellular CYP450 on the removal of six nonsteroidal anti-inflammatories (NSAIs) by Trametes hirsuta. Results show that for most tested NSAIs, LMEs activity and biosorption failed to explain the observed removal. Most tested TrOCs are quickly taken up and intracellularly transformed. Fine characterization of intracellular transformation using ketoprofen showed that CYP450 is not the sole intracellular enzyme responsible for intracellular transformation. The contribution of CYP450 in further transformation of ketoprofen byproducts is also reported. These results illustrate that TrOCs transformation by WRF is a more complex process than previously reported. Rapid uptake of TrOCs and intracellular transformation through diverse enzymatic systems appears to be important components of WRF efficiency toward TrOCs.

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INTRODUCTION Nowadays, the presence of multiple trace organic contaminants (TrOCs), such as pharmaceuticals, pesticides and personal care products in many compartments of the environment is well documented.1 This highlights the inefficiency of traditional wastewater treatment plants to remove these molecules and the urgency to implement new processes to address the challenge of TrOCs.2 Among the new avenues explored to upgrade water treatment systems, advanced oxidation processes bear great potential due to their high efficiency to remove most recalcitrant compounds.3−5 However, the high-energy consumption and the use of hazardous materials (i.e., reagents and catalysts) could limit their implementation.3,5−7 Thus, bioengineering approaches are also explored as ecofriendly alternatives. These processes exploit the ability of various microorganisms, such as fungi and bacteria, or pure enzymes to remove TrOCs. Over the past few years, the use of white rot fungi (WRF) for bioremediation purposes has gained interest in the scientific community.8−10 The diversity of transformation products identified after TrOCs treatment by WRF suggests the involvement of multiple enzymatic systems in TrOCs transformation (Supporting Information (SI) Table S6). Fungal extracellular lignin modifying enzymes (LMEs) have been reported to be particularly efficient at removing TrOCs.11 WRF secrete mainly three enzymes of interest; the © XXXX American Chemical Society

laccases (LAC; EC 1.10.3.2), the lignin peroxidase (LiP; EC 1.11.1.14) and manganese-dependent peroxidase (MnP; EC 1.11.1.13).12 The low specificity of these LMEs makes fungi suitable for several applications in environmental biotechnology.13 However, the exact mechanisms involved in TrOCs removal by WRF remain elusive. Several studies comparing the removal efficiency of crude enzyme extracts (extracellular content without fungus cells) and whole cell cultures (including living fungus cells) suggested that the removal of TrOCs does not solely rely on the activity of extracellular LMEs.14−17 Several authors have demonstrated the contribution of biosorption to the observed removal.14−17 However, for several TrOCs (e.g., ibuprofen, indomethacin, naproxen, and carbamazepine) biosorption was insufficient to explain the higher efficiency of whole cultures, as compared to crude extract.9,18−20 Recent studies suggested that intracellular enzymes (i.e., cytochrome P450) or mycelium-associated enzymes, as well as interactions between intracellular CYP450 and LMEs, could play a significant role in the removal of TrOCs.8−10,14,18 The role of Received: Revised: Accepted: Published: A

August 31, 2016 November 16, 2016 December 1, 2016 December 1, 2016 DOI: 10.1021/acs.est.6b04409 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

NSAIs Degradation Experiments. Whole Cell Cultures. Biodegradation studies were performed in 250 mL Erlenmeyer flasks equipped with foam stoppers that allow gas exchange. Each flask containing 50 mL of sterile medium was inoculated with 0.5 mL (≈ 400 mg in dry weight of WRF) of the mycelia solution. After 3 days of fungal growth (26 °C, 135 rpm, pH 4.6) homogeneous pellets appeared in the flask. Then 50 μL of a solution containing 6 NSAIs (acetaminophen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, and naproxen) at 100 mg L−1 were added for a final concentration of 100 μg L−1. After 48 h of treatment, one triplicate (whole flasks) was sacrificed, and filtered through a 0.22 μm syringe filter. NSAIs concentration (see below), LAC activity, pH, and dry weight were measured in the supernatant. Crude Enzyme Extracts. Two-week-old cultures of T. hirsuta were filtered at 0.22 μm under sterile conditions and the pH was adjusted to pH 4.6. 50 mL of the crude extract was placed in 250 mL Erlenmeyer flasks. The measured residual LAC activity was 30 U L−1 (see below). Then 50 μL of a solution containing six NSAIs (acetaminophen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, and naproxen) at 100 mg L−1 were added for a final concentration of 100 μg L−1. After 48 h of treatment, one triplicate (whole flasks) was sacrificed and samples were centrifuged at 4600 rpm for 15 min at 4 °C. Then, NSAIs concentration, LAC activity and pH were measured. Commercial Laccase Solutions. The commercial LAC enzymatic degradation experiments were performed in triplicate using commercial T. versicolor LAC in 250 mL Erlenmeyer flasks of a commercial LAC solution, prepared in miliQ water, at a final enzyme activity of 30 U L−1 at pH 4.6. This activity was selected to make it equal to the residual LAC activity measured in the crude extracts. Then 50 μL of a solution containing 6 NSAIs (acetaminophen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, and naproxen) at 100 mg L−1 were added for a final concentration of 100 μg L−1. A second set of experiments was carried out in the growth media (commercial LAC + sterile growth medium at pH 4.6) in order to account for the contribution of the culture medium components to the observed degradation. After 48 h of treatment, one triplicate (whole flasks) was sacrificed and samples were centrifuged at 4600 rpm for 15 min at 4 °C. Then, NSAIs concentration, LAC activity and pH were measured. Biosorption Experiments. Fungal cultures were inactivated with 10 mM sodium azide after 3 days of cultivation. After 60 min, 50 μL of a solution containing 6 NSAIs (acetaminophen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, and naproxen) at 100 mg L−1 were added for a final concentration of 100 μg L−1. After 48 h of treatment, one triplicate (whole flasks) was sacrificed, prepared and analyzed as described above (whole cultures). NSAIs Uptake Experiments. NSAIs Uptake experiments were performed in 250 mL Erlenmeyer flasks as described above. 50 μL of a solution containing the six NSAIs at 500 mg L−1 was added for a final concentration of 500 μg L−1. At selected time intervals (1, 3, 6, 9, 12, 24, 48, and 72 h), one triplicate (whole flasks) was sacrificed, and filtered through a 0.22 μm syringe filter. NSAIs concentration, LAC activity and pH were measured in the supernatant and in the biomass. Cytochrome P450 Inhibition. The contribution of the internal enzyme CYP450 was evaluated by adding 10 mM of 1aminobenzotriazole (ABT) or 10 mM of piperonyl butoxide (PB) in whole cultures (see above) and a mix of both. After 60

biosorption and the interactions between extra- and intracellular enzymes on the efficiency of WRF to remove TrOCs remains to be fully explored. It is critical to fill this gap in order to extend the performance and biotechnological potential of WRF for TrOCs removal. The present work aims to provide new insights on the specific mechanisms involved in the removal of multiple TrOCs by the WRF Trametes hirsuta. We hypothesized that the transformation of TrOCs by T. hirsuta is a multistep process involving uptake of the target molecule and transformation by intracellular (e.g., CYP450) enzymes. To test this hypothesis, we evaluated the removal of a mixture of six nonsteroidal antiinflammatories (NSAIs: acetaminophen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, and naproxen) by the WRF T. hirsuta. These contaminants have been chosen because they are ubiquitous recalcitrant TrOCs in wastewaters and sludges.2 In addition, NSAIs are efficiently removed by WRF,8−10,20 but are poor subtracts to LMEs21 making these molecules perfect candidates to investigate the role of uptake and intracellular enzymes activity on transformation. We specifically evaluated the contribution of LMEs and non-LMEs (crude extract), biosorption and intracellular enzymes (i.e., CYP450) on the removal of the selected NSAIs. All selected NSAIs were treated with (i) a solution containing commercial LAC, (ii) crude extracts of T. hirsuta, (iii) whole cultures containing living fungal mycelia, and (iv) cultures containing chemically inactivated fungi (using sodium azide (NaN3)). Extra and intracellular concentrations of the six selected NSAIs during treatment were monitored by UPLC/MS−MS. The contribution of cytochrome P450 (CYP450) to the removal of ketoprofen was specifically tested using CYP450 inhibitors. Transformation products of ketoprofen during treatments were also investigated.

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EXPERIMENTAL SECTION Chemicals and Reagents. All chemicals were analytical grades. Formic acid, methanol and acetonitrile (Optima grade for LC/MS) were purchased from Fisher Scientific (Ottawa, ON, Canada). Pharmaceutical compounds (acetaminophen, ibuprofen, indomethacin, ketoprofen, mefenamic acid and naproxen), malt extract, yeast extract, D-glucose and commercial laccase from T. versicolor (LAC, EC 1.10.3.2; 13.6 U mg−1) were purchased from Sigma−Aldrich (Saint-Louis, MO). Fungal Strain, Medium, Culture Conditions and Glass Passivation. The fungal strain of T. hirsuta (IBB 450) was obtained from the culture collection of the institute of biochemistry and biotechnology (Tbilisi, Georgia). All biodegradation experiments were carried out under pelleted mycelia forms as previously described.20 Briefly, blended mycelia of T. hirsuta were grown on a rotary shaker at 135 rpm at 26 °C in 250 mL Erlenmeyer flasks containing 50 mL of sterile medium: 0.4% glucose, 0.4% yeast, 1% malt. The pH of the growth medium was 4.6 for all conditions and did not significantly vary during the time of the experiments (data not shown). This pH was selected based on LAC activity (maximum activity)22 and fungal growth performance. All experiments were performed in the dark. In order to minimize the sorption of target TrOCs on glassware during experiments, all the glassware was deactivated using 5% dimethyldichlorosilane in toluene soaked 1 h. Glassware was then rinsed with two volumes of toluene and rinsed with three volumes of methanol and water until reaching a neutral pH. B

DOI: 10.1021/acs.est.6b04409 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article


Figure 1. Percentage of NSAIs removal at 100 μg L−1 after 48 h of treatment by the living WRF (A) and by extracellular enzymes (pure laccase and crude extracts) (B). Values plotted are the means and the error bars represent the standard deviation of triplicate cultures.

min, 100 μg L−1 of ketoprofen was added to the fungal culture. At selected time intervals (0.5, 3, 24, and 30 h), one duplicate (whole flasks) was sacrificed, prepared and analyzed as described above (whole culture). Analytical Methodologies. Extracellular Enzymatic Activity Assay. Laccase activity was measured according to Touahar et al. (2014)21 by following the conversion of 0.5 mM 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to its radical cation (ABTS•+) at 420 nm (εmax = 36 000 M−1 cm−1) in 0.1 M citric acid/0.1 M disodium hydrogen phosphate buffer at pH 3. One unit of activity (U) was defined as the amount of enzyme that forms 1 μmol of products per min. Activity measurements were carried on a 96-well plate using a double-beam UV−Vis spectrophotometer (SpectraMax Plus 3250, Molecular Devices Corp., Sunnyvale, CA). Results report the mean of triplicates. NSAIs Quantification. The analyses of NSAIs were performed using a positive electrospray ionization (ESI+) source in Multi-Reaction-Monitoring mode on an Acquity UPLC XEVO TQ mass spectrometer (Waters Corporation, Milford, MA) equipped with an Acquity UPLC HSS-T3 column (100 mm × 2.1 mm, 1.8 μm) and a fritted 0.2 μm prefilter. The solvent flow rate was set to 0.40 mL min−1 and the column temperature was kept at 35 °C. The sample volume injected was 5 μL. The mobile phase was 0.20% formic acid/ water (A) and 0.20% formic acid/methanol-acetonitrile (80−20 v+v) (B). The elution gradient started with 5% of eluent B, increasing to 90% in 8 min and then back to initial conditions in 4 min. The optimized parameters were obtained by direct infusion of analytical standard solutions at 10 μg mL−1 as follows: desolvation gas (nitrogen) at 800 L h−1; cone gas (nitrogen) at 50 L h−1; collision gas (nitrogen) at 0.22 mL min−1; capillary voltage 2.5 kV; source temperature, 150 °C and desolvation temperature 550 °C. Two daughter traces (transitions) were used. The most abundant transition was used for quantification, whereas the second most abundant was used for confirmation. MS parameters for each molecule are available in SI Table S1, S2, and S3. NSAIs in the supernatant were analyzed after a sample purification (see SI Figure S1A). Briefly, 1 mL of the supernatant was passed through an Oasis HLB solid phase extraction cartridge and then reconstituted in 1 mL of methanol. The biomass (whole cell fungi) was withdrawn and washed with 10 mL of miliQ water and dried under vacuum. The biomass was weighted and 1 g of NaCl and

10 mL of a mixture of methanol and acetonitrile (1:1 v/v) was added to the sample. After three times 5 min of ultrasonic and vortex agitation, samples were frozen at −80 °C for 24 h. The supernatant was then centrifuged at 4600 rpm for 15 min at 4 °C and 1 mL of the supernatant was filtered through a 0.22 μm PTFE filter before analysis (see SI Figure S1B). Monitoring of Ketoprofen Transformation Products. The analysis of ketoprofen transformation product candidates was carried out using a Waters alliance 2695 chromatographic HPLC coupled to a Waters Synapt QTOF (Waters Corp., Milford, MA) in positive electrospray ionization (ESI+) source. MS data were acquired over a scan range between 50 and 1200 m/z. The conditions were the following: capillary 3200 V,, dry gas (N2) flow of 24 L min−1, and dry temperature 450 °C. More details on MS parameters are available in SI Tables S4, and S5. Subsequent analysis of MS data were performed using MetaboLynx (Waters, Ltd.) software as previously described.23−25 Briefly, Metabolynx processing algorithms predicts expected (e.g., hydroxylation, hydrolysis, dealkylation) and unexpected transformation products. Known ketoprofen metabolites, found in the literature, were also included in the screening method. Transformation product candidates were searched in the range of 50−800 m/z and from 0.5 to 18 at 0.05 min peak separation. Mass defect over 20 mDa were discarded in order to obtain only one proposed molecular formula for each candidat. Peaks with less than 10 area units were discarded. Statistics. The statistics treatment was performed by an analysis of variance (ANOVA), using a Holm−Sidak test. The levels of significance are expressed as a p value < 0.05.

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RESULTS AND DISCUSSION Contribution of Whole Cells, Biosorption and Extracellular LAC to NSAIs Removal. All tested NSAIs were efficiently removed (>90%) by the whole cell cultures (living fungi) within 48h of treatment (Figure 1A). Experiments in the presence of biocide (NaN3) showed that, biosorption played a limited role in the observed removal (90%). Similar conclusions can be drawn from experiments with crude extracts, which allow accounting for extracellular enzymes not assayed in this study; crude extracts failed to explain the observed removal for four of the six tested NSAIs (compare Figure 1A and B). The culture medium had no significant effect on the efficiency of LAC toward the tested NSAIs (Figure 1B). These results with commercial LAC are in agreement with results in the literature; LAC is known to efficiently target phenolic compounds such as acetaminophen, the only phenolic molecule in the selected NSAIs.21,27,28 The sensibility of acetaminophen and mefenamic acid to LAC oxidation could also be attributed to the strong electron donating groups (−OH, -NH2) of these two molecules, which are efficient substrate for LAC. On the contrary, electron withdrawing groups characterizing ibuprofen, indomethacin, ketoprofen, and naproxen are poor substrate for LAC. It is also important to mention that. In this study, as in many others, crude extracts were collected before TrOCs additions. The type, concentration and activity of extracellular enzymes produced by WRF could be significantly affected by the presence of TrOCs. Further research on the extracellular enzymatic profile, including non-LMEs, before and after exposure to TrOCs are required to provide a better understanding of the extracellular enzymes of interest for TrOCs removal. Nonetheless, for four out of the six selected NSAIs (ibuprofen, indomethacin, ketoprofen and naproxen) the removal observed in the presence of whole cells could not be explained by extracellular enzyme activity (e.g., LMEs and others; commercial LAC and crude extracts) nor biosorption. This suggests that other mechanisms, such as uptake and intracellular enzyme activity, could contribute to the observed removal. The poor removal of ibuprofen, indomethacin, ketoprofen and naproxen in the presence of biocides (NaN3) and commercial LAC suggests that some ATP-dependent mechanisms could be involved in the observed removal.9,29 The intracellular cytochrome P450 was recently proposed as a potential important enzyme for TrOCs degradation.8−10,14,18 Uptake of NSAIs by T. hirsuta and the Role of Intracellular Enzymes. In the presence of whole cells, the removal rate (expressed as the remaining concentration in the extracellular medium), was rapid for ibuprofen, indomethacin, mefenamic acid, and naproxen (100% within 3 h) and slightly slower for acetaminophen and ketoprofen (≈90% within 48 h) (Figure 2A). Analysis of the intracellular NSAIs contents D


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Figure 3. Effect of CYP450 inhibitors on the extracellular and intracellular amounts of ketoprofen over a 30 h treatment period. Percentage of ketoprofen removal in the supernatant in the presence and absence of CYP450 inhibitors (ABT at 10 mM) (A). Amount of intracellular ketoprofen in the presence and absence of CYP450 inhibitors (ABT at 10 mM) (B). The presented values plotted are the means and the error bar represent the standard deviation of duplicate cultures. Similar letters indicate no significant difference according to one-way analysis of variance (ANOVA), Holm−Sidak, P < 0.05.

Table 1. Proposed Formula and Structure of Principal Detected Transformation Products Formed During Ketoprofen Degradation by T. hirsuta with and without Addition of a CYP450 Inhibitor

specifically evaluated the contribution of CYP450 to NSAIs transformation using ketoprofen as model. Ketoprofen was selected because its degradation primarily relies on uptake and intracellular enzyme activity (see above). In addition, intracellular degradation kinetics are slow, as compared to other tested NSAIs, facilitating the fine characterization of the intracellular degradation process. We tested two CYP450 inhibitors, (1-aminobenzotriazol (ABT) and piperonyl butoxide (PB). No significant difference, on ketoprofen removal, was

observed between the two inhibitors, and we only report the results obtained with the ABT. In the presence of inhibitors, the reduction of ketoprofen concentration in the external medium was slightly more rapid than in control cultures (Figure 3A). An increase in the removal rate of ibuprofen, potentially reflecting a faster uptake, by T. versicolor in the presence of PB used as CYP450 inhibitor was also observed by Marco-Urrea et al. (2009).8 The complex physiological feedbacks leading to improved uptake of NSAIs, and potentially other TrOCs, in E


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Table 2. Presence (+ Signs) And Absence (− Signs) Of Identified Ketoprofen Transformation Products in the Intracellular and Extracellular Matrices of T. hirsuta in the Presence and Absence of CYP450 Inhibitor (ABT)a intracellular

-ABT

+ABT

extracellular

-ABT

+ABT

time (h)

TP 1

TP2

TP 3

TP 4

TP 5

TP 6

TP 7

TP 8

TP 9

TP 10

0 0.5 3 24 30 0 0.5 3 24 30 0 0.5 3 24 30 0 0.5 3 24 30

− − + + ++ − + + + − − − − − − − − − − −

− + + + + − − − − − − − + + − − − − − −

− − − − − − − − − − − − − − + − − − − −

− − − − − − − − + + − − − − − − − − − −

− ++ +++ ++++ ++++ − ++ +++ ++++ ++++ − − +++ ++++ ++++ − − +++ ++++ ++++

− − − − − − − − − − − − − − + − − − − −

− − − − − − − − − − − − − − − − − − − ++

− − − − − − − − − − − − − − − − − ++ ++

− − − − − − − − − − − − − − − − − ++ ++

− − − − − − − − − − − − + ++ − − − + ++ ++

a The number of “+” signs represent the relative abundance (absolute peak area, Area unit UA); 20 UA < + < 100 UA, 101 UA < + + < 1000 UA, 1001 UA < + ++ < 3000 UA and + +++ > 3001 UA.

low concentrations of ketoprofen by T. hirsuta and that intracellular enzymes, other than cytochrome P450, are involved in NSAIs transformation. The involvement of cytochromes other than CYP450 on the observed removal cannot be ruled out.30 Characterization of Ketoprofen Intra and Extracellular Transformation Products. Based on our criteria, we propose 10 candidates for ketoprofen transformation products formed during its removal by T. hirsuta. The proposed formula and structures are presented in Table 1. Considering that further structural elucidation technics are required to confirm the structure of the proposed transformation products,31,32 the results presented and discussed below must be taken with caution. Data on the presence of these transformation product candidates in the intracellular and extracellular matrices over time and in the presence and absence of CYP450 inhibitor (ABT) are presented in Table 2. The degradation of xenobiotic by WRF can result from diverse enzymatic reactions (see SI Table S6). Proposed enzymatic reactions (CYP450 dependent versus independent) for the 10 reported transformation product candidates are presented in SI Figure S2. No ketoprofen transformation product candidates were founds in NaN3 (biocide) treated fungal cultures (data not shown), confirming that the elimination of ketoprofen requires living fungi cells. In the intracellular matrix, some transformation products (TP1 and TP5) were found in both the presence and absence of CYP450 inhibitor (Table 2), suggesting that their formation is CYP450 independent (see SI Figure S2). On the contrary, compound TP2 present in untreated cells, is absent in ABT treated cultures, suggesting a role of CYP450 in its formation. This compound can result from an dealkylation followed by an oxidation which are known reactions catalyzed by CYP450.33,34 Another compound, TP4, was only present in CYP450 inhibited cells, suggesting that this compound is not formed by CYP450 but might require CYP450 for further trans-

the presence of CYP450 inhibitor have not been characterized. The first pKa of Ketoprofen is 4.45, meaning that under our experimental conditions (pH = 4.6 0.1) protonated and deprotonated ketoprofen species might be present. Variation in pH, between CYP450 inhibited and control cultures, impacting this speciation could influence uptake kinetics, at least for passive uptake. However, as mentioned above, passive uptake is likely negligible. In addition, the WRF are known to control the pH of their medium and no significant variation in pH was observed over time or between treatments (pH 4.6 ± 0.1, data not shown). No significant effect on the evolution of intracellular ketoprofen concentration was observed showing that the overall removal efficiency and kinetic of intracellular ketoprofen transformation was not significantly affected (Figure 3B). Similarly to ketoprofen, the addition of CYP450 inhibitor had no significant effect on the removal of the 6 NSAIs tested in mixture; after 72 h all NSAIs were completely removed in both -ABT and + ABT cultures (data not shown). These results contrast with the study of Marco-Urrea et al. (2010)9 showing significant decrease in ketoprofen removal after CYP450 inhibitor addition in T. versicolor. However, these two studies (Marco-Urrea et al. (2010)9 and the present study) used two different WRF strains (T. versicolor and T. hirsuta), growth media, and experimental conditions, concentration of inhibitors (5 mM versus 10 mM here) and concentration of ketoprofen (10 mg L−1 versus 0.1 mg L−1 here) making direct comparison hazardous. Since uptake and intracellular transformation were not monitored by Marco-Urrea et al. (2010),9 it is not clear whether the uptake, the intracellular removal or both were inhibited by the addition of the CYP450 inhibitor. More work on the effect of TrOCs and CYP450 inhibitor concentrations on extracellular enzymes activity (e.g., LMEs), TrOCs uptake and intracellular transformation is required in order to better interpret data from contrasted experimental designs. Nonetheless, data from Figure 3 suggest that CYP450 is not the primary enzyme responsible for the transformation of F


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Environmental Science & Technology Notes

formation, preventing its accumulation in cells. These data strongly suggest that the intracellular transformation of ketoprofen do not solely relies on CYP450, as previously suggested.9,35 It likely results from the activity of a cohort of enzymes, including CYP450, catalyzing various reactions. The comparison of transformation product candidates found in both the intra and extracellular matrices, shows that ketoprofen transformation products formed within cells can be excreted back to the external medium (Table 2). For instance, compounds TP2 and TP5 are present in the intracellular medium after 0.5 h but only after 3 h in the extracellular matrix. In the extracellular medium these compounds can be subject to further transformations. Regarding transformation product candidates only found in the external medium, the inhibition of CYP450 had also contrasted effects. Compound TP10 was not affected by the inhibition of CYP450 (Table 2). This proposed conjugate only found in the external medium is consistent with the action of a glutathione S-transferase, present in WRF, which function is to reduce intracellular toxicity by promoting the excretion of the target compound.36−38 On the contrary, compounds TP3 and TP6 were only found in the extracellular matrix of CYP450 inhibitor free cultures (- ABT), while compounds TP7, TP8, and TP9 were only founds in the extracellular medium of inhibited cultures (+ ABT). This suggests that the transformation of ketoprofen by T. hirsuta can involve interactions between intracellular (CYP450 and others) and extracellular enzymatic systems, which provides substrates (transformation products) to one another or compete for similar substrates. These complex intra and extracellular transformation pathways of ketoprofen and other TrOCs, (e.g., enzymes involved, their efficiency and the fate of the generated byproducts) by WRF remains to be fully characterized. Nonetheless, this is one of the first studies reporting the importance of compound exchanges between the extra and intracellular media, and interaction between intracellular and extracellular enzymes activity on the removal of NSAIs by the WRF T. hirsuta. In many cases, extracellular enzymes and biosorption were insufficient at explaining the observed removal. Data show that the removal of several tested TrOCs is a multistep process involving a fast and active uptake of TrOCs, followed by an intracellular degradation through complex enzymatic pathways (including CYP450). Intracellular degradation products can then be excreted back to the external medium for further transformations by extracellular enzymes.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by two discovery grants from the Natural Sciences and Engineering Research Council of Canada (JPB: RGPIN-386963-2011 and HC: 371681-2014) as well as research funds from the Faculty of Science, Université de Sherbrooke.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.6b04409. Supplementary methods (Figure. S1), MS parameters (Tables S1−S5), summary of reported reactions involved in xenobiotics transformation in white rot fungi (Table S6), and proposed ketoprofen transformation pathways (Figure. S2) (PDF)

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Lounès Haroune: 0000-0001-5420-6619 G


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