Ligninase-Mediated Removal of Natural and Synthetic Estrogens from

Mar 15, 2010 - University of Georgia, Griffin, Georgia 30223, College of. Resource and Environmental Sciences, Nanjing Agricultural. University, Nanji...
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Environ. Sci. Technol. 2010, 44, 2599–2604

Ligninase-Mediated Removal of Natural and Synthetic Estrogens from Water: II. Reactions of 17β-Estradiol L I A N G M A O , †,‡ J U N H E L U , ‡,§ MUSSIE HABTESELASSIE,‡ QI LUO,‡ SHIXIANG GAO,† MIGUEL CABRERA,| A N D Q I N G G U O H U A N G * ,‡ State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210093, P. R. China, Department of Crop and Soil Sciences, University of Georgia, Griffin, Georgia 30223, College of Resource and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, P. R. China, and Department of Crop and Soil Sciences, University of Georgia, Athens, Georgia 30602

Received October 7, 2009. Revised manuscript received February 16, 2010. Accepted March 3, 2010.

We have demonstrated in our earlier work that a few natural and synthetic estrogens can be effectively transformed through reactions mediated by lignin peroxidase (LiP). The behaviors of such reactions are variously influenced by the presence of natural organic matter (NOM) and/or veratryl alcohol (VA).Certainwhiterotfungi,e.g.Phanerochaete chrysosporium, produce VA as a secondary metabolite along with LiP in nature where NOM is ubiquitously present. Herein, we report a study on the products resulting from LiP-mediated oxidative coupling reactions of a representative estrogen, 17β-estradiol (E2), and how the presence of NOM and/or VA impacts the formation and distribution of the products. A total of six products were found, and the major products appeared to be oligomers resulting from E2 coupling. Our experiments revealed that these products likely formed colloidal solids in water that can be removed via ultrafiltration or settled during ultracentrifugation. Such a colloidal nature of the products could have important implications in their treatability and environmental transport. In the presence of VA, the products tended to shift toward higherdegree of oligomers. When NOM was included in the reaction system, cross-coupling between E2 and NOM appeared to occur. Data obtained from E-SCREEN test confirmed that the estrogenicity of E2 can be effectively eliminated following sequential reactions mediated by LiP.

Introduction Endocrine disrupting contaminants have drawn considerable attention in recent years (1-3). Such compounds with hormonal activity disturb the normal biological function of wildlife and thus pose a potential harm to the ecosystem and eventually human health (4-6). Endocrine disruption effects * Corresponding author phone: (770)229-3302; fax: (770)412-4734; e-mail: [email protected]. † Nanjing University. ‡ University of Georgia, Griffin. § Nanjing Agricultural University. | University of Georgia, Athens. 10.1021/es903058k

 2010 American Chemical Society

Published on Web 03/15/2010

on aquatic wildlife have often been linked to some specific chemicals of both synthetic and natural origins, such as 17βestradiol (E2), 17a-ethynyl estradiol (EE2), estrone (E1), bisphenol A (BPA), and nonylphenol (1, 7). E2, a natural female hormone, is of particular concern, because it is carcinogenic (8-12), and its endocrine disrupting potency can be several orders of magnitude greater than others (13). It was reported that fish exposed to E2 at concentrations as low as 0.5 ng/L exhibited changes in biomarkers for estrogenicity (13-17). Studies have revealed a widespread occurrence of endocrine disruptors in natural water bodies, where E2 concentration was as high as 200 ng/L (18, 19). The fact that hormones are capable of eliciting physiological responses at extremely low concentrations presents a unique challenge for the removal of such chemicals to safety levels in water treatment (2, 3). Oxidation by ozone and chlorine was shown to be effective in removing steroidal hormones from water (20-23). However, the efficiency of these processes under field conditions is limited by the low concentrations of the contaminants. Because chemical oxidation is nonspecific, the majority of oxidants and energy are in fact consumed by the nontargeted impurities. In addition, hazardous byproduct can be generated during the oxidation processes (20-23). For instance, mono- and dichlorine substituted E2 can be generated upon chlorination, some of which were found to have greater estrogenicity than the parent compounds (22). Moreover, their sorption on sludge creates additional concerns for solids management (2, 24). As such, in the development of water treatment strategy to address the challenges of endocrine disruptive contaminants, particular attention needs to be given to the following factors: i) the specificity and efficiency of the process; ii) the toxicity and fate of resulting products; and iii) the impact of NOM on the process. Enzymatic reactions have been examined as a potential means for the removal of hormones in water/wastewater (24-30). Horseradish peroxidase (HRP) and laccase are the enzymes that were most frequently studied for this purpose (24-28). Our recent study demonstrated that lignin peroxidase (LiP) can also mediate effective reactions leading to the removal of estrogens (29). LiPs are a class of extracellular enzymes that are produced by certain lignin-degrading fungi, such as Phanerochaete chrysosporium (31, 32). It is known that P. chrysosporium also produces veratryl alcohol (VA) as a secondary metabolite along with LiP (31, 32, 33), which plays a significant role in regulating LiP activity (34). In our previous study (29) the reaction processes of four natural and synthetic estrogens mediated by LiP were systematically investigated, with particular attention to the impact of NOM and VA on the catalytic performance of LiP. It was found that the presence of VA enhanced the catalytic performance of LiP, while such enhancement was mitigated in the presence of NOM. We further elucidated such influences in the context of enzyme/substrate binding mechanisms (29). In the present study, we focused on the characterization of the products resulting from LiP mediated reactions using E2 as a representative hormone. The formation and distribution of various reaction products in the absence and presence of NOM and/ or VA were systematically examined. Their estrogenicity and solubility were evaluated. The information obtained is useful for assessing the feasibility of enzymatic oxidative processes to remove estrogens in water treatment practice.

Experimental Section Materials. 17β-Estradiol (CAS 50-28-2), lignin peroxidase (EC 1.11.1.14), veratryl alcohol (3, 4-dimethoxybenzyl alcohol, VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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96%, CAS 93-03-8), and hydrogen peroxide (30 wt %, CAS 7722-84-1) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade acetonitrile (ACN) and methanol were obtained from Fisher Scientific (Pittsburgh, PA). Citric acid (CAS 77-92-9) and dibasic sodium phosphate (CAS 755879-4) were from J. T. Baker Chemical Co. (Phillipsburg, NJ). Suwannee River Fulvic Acid (SRFA) was obtained from the International Humic Substances Society (IHSS). Human breast cancer estrogen-sensitive MCF-7-BOS cells were kindly provided by Professor Ana M. Soto from Tufts University School of Medicine (Boston, MA). Enzymatic Reactions and Products Characterization. Transformation of E2 mediated by LiP was conducted in 250 mL flasks as batch reactors. Each reactor contained 100 mL of citrate-phosphate buffer solution (CPBS; 10 mM, pH 4.5), containing 10 µM E2, 0.005 U/mL LiP, and 10 µM H2O2, which was incubated on a rotary shaker at 150 rpm. To study the effects of VA and NOM on E2 reaction, reactors having 100 µM VA and/or 5 mg/L (as TOC) SRFA with otherwise composition identical were prepared. Reactors with LiP or H2O2 absent were also prepared to serve as controls. Experiments were performed for each reaction condition in triplicate. After 60 min, the reaction was quenched by adding 1.6 mL of 1 M HCl, and the solution was extracted using C18 solid phase extraction (SPE) (500 mg, J. T. Baker Chemical Co., Phillipsburg, NJ) and analyzed with HPLC. Preliminary tests showed the recovery of SPE process for E2 was 105.7 ((2.61)%. Details of SPE extraction and HPLC analysis are given in Supporting Information I. To further characterize the reaction products, the HPLC effluent was collected at 0.5 min intervals. The fractions were blown dry with nitrogen and reconstituted in 200 µL of methanol. Each of the samples thus obtained were analyzed using mass spectrometry (MS) with negative electron spray ionization (ESI) as described in Supporting Information I. Products Phase Distribution. Experiments were performed to evaluate the separation of soluble and suspended forms of the enzymatic reaction products by both membrane filtration and ultracentrifugation. In the experiment involving membrane filtration, 200 mL of reaction solution comprising 10 µM E2, 0.0065 U/mL LiP, and 15 µM H2O2 in 10 mM CPBS (pH 4.5) was incubated in a rotary shaker at 150 rpm. After 90 min reaction, the solution was filtered through a 0.45-µm pore size syringe filter (cellulose acetate, 25 mm, Sterile, Fisher Scientific, Pittsburgh, PA). The filtrate was collected and extracted using SPE as described above. The filter was eluted with 1 mL of methanol after 3 times rinsing with 5 mL of DI water. Residual E2 and reaction products present in both the filtrate and methanol eluent were analyzed by HPLC. In the experiment involving centrifugation, 1 L of solution comprising 10 µM E2, 0.02 U/mL LiP, and 10 µM H2O2 in 10 mM CPBS (pH 4.5) was prepared. After 120-min incubation, the reaction solution was equally divided into 25 50-mL centrifuge tubes, which were then centrifuged twice at 8000 g for 10 min each time at 4 °C. After centrifugation, 4 mL of top layer solution was carefully withdrawn from each of the 25 centrifuge tubes and combined and then subjected to SPE extraction and HPLC analysis. The remainder of the solution in all tubes was combined and mixed well before measuring out 100 mL for SPE extraction and HPLC analysis. Estrogenic Activity Assay. The estrogenicity of E2 solutions after a different number of enzymatic treatment cycles was assessed. The reactions were performed in glass test tubes, and each reactor contained 2 mL of CPBS (10 mM, pH 4.5) initially comprising 0.1 µM E2. For one set of reactors, 0.01 U/mL LiP and 10 µM H2O2 were added and allowed for 150-min reaction. For the second set of reactors, 0.01 U/mL LiP and 10 µM H2O2 were added at 0 and 75 min, respectively, to accomplish two treatment cycles during the 150-min reaction time. Yet for the third set of reactors, the enzyme 2600

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FIGURE 1. HPLC spectra of E2 reaction solutions having various components: H2O2 + E2 (a), LiP + E2 + H2O2 (b), LiP + E2 + VA + H2O2 (c), LiP + E2 + NOM + H2O2 (d), and LiP + E2 + VA + NOM + H2O2 (e). All samples were incubated for 60 min prior to SPE and HPLC analysis. The compositions of the samples included the following: [E2] ) 10 µM, [LiP] ) 0.005 U/ml, [H2O2] ) 0.01 mM, pH ) 4.5 (10 mM CPBS); [VA] ) 100 µM; [NOM] ) 5 mg/L as TOC. and H2O2 were supplemented at 0, 50, and 100 min to allow for a total of three treatment cycles during the 150-min treatment. After the treatment was completed, 20 µL of 1.12 g/L catalase was added to each reactor to consume excess H2O2 prior to estrogenicity assay. Such a catalase dosage was found to result in complete H2O2 decomposition in our preliminary tests. Blank samples that contained catalase, H2O2, and E2 did not yield discernible change in E2 concentration. Estrogenicity was assayed using an E-Screen test by measuring the proliferation of a MCF-7-BOS cell line. The assay was performed as per a protocol described by Soto et al. (35) with minor changes, and the details are described in Supporting Information I.

Results and Discussion Products. A relatively mild reaction condition (10 µM E2, 10 µM H2O2, and 0.005 U/mL LiP), selected based on our earlier study (29), was used in the experiments so that possible intermediate products that may disappear under stronger reaction conditions can be captured. An hour reaction under such a condition yielded about 39% reduction in E2 concentration. By comparing the chromatograms generated from the control in the absence of LiP and the reaction sample that are displayed in Figure 1a,b, respectively, five peaks likely associated with enzymatic reaction products of E2 can be identified. The same five peaks also appeared in the chromatograms of reaction samples that had VA and/or NOM present (Figure 1c,d,e). The HPLC effluent fractions corresponding to each of the chromatographic peaks present in Figure 1b were further analyzed using negative ESI/MS. In this way, ions (m/z ) M-1) corresponding to deprotonated molecules can be generated. The MS thus determined are shown in Figure S1 in Supporting Information II. Secondary MS spectrometry was attempted, but unfortunately no effective fragmentation

TABLE 1. HPLC Separation and MS Characterization of SPE Extracts of Samples Taken after 60 min of Reaction retention time (min)

peak numbera

molecular ion

molecular weight

possible structure

20.7 23.2 26.1 27.4 31.2 32.5

1 2 3 4 5

269 541 541 541 811 811

270 542 542 542 812 812

estrone E2 dimer I E2 dimer II E2 dimer III E2 trimer I E2 trimer II

a Respectively correspond to the peaks 1-5 shown in Figure 1.

was achieved. Nonetheless, the possible molecular weights (MW) of the products obtained via ESI/MS (Table 1) provide strong indication of possible reaction mechanism. As seen in Table 1, the first three species have a common MW of 542 and the later two have a MW of 812. These molecular masses follow the pattern of nM - 2(n - 1), where n is a natural number (2 or 3 in this case) and M is the molecular weight of E2 (272). Such a pattern has been shown to be indicative of radical-radical coupling reactions (36), i.e. LiP mediated one-electron oxidation of the substrates to generate free radicals, which coupled covalently to each other subsequently. Radical-radical coupling results in covalent bonding of two parent molecules with two hydrogen atoms eliminated, one from each molecule (36). Such a coupling product is still, in most cases, a substrate of the oxidative coupling enzymes and can thus undergo further coupling reactions yielding higher level oligomer products. Molecular weights of the coupling products thus generated have a pattern of nM - 2(n - 1). Such a radical-radical coupling mechanism has been found with the reactions of quite a few EDCs mediated by different peroxidases or laccases (36-39). In addition to these five species generating peaks by PDA (UV) detection, MS analysis of the HPLC effluent fractions other than these peaks revealed a compound with MW of 270 appeared at the retention time of 20.7 min (also included in Table 1). This compound is absent in the control and is presumed to be E1 formed upon oxidation of E2. E1 was not observed in our HPLC/PDA analysis, apparently because its quantity is below the PDA detection limit. Except estrone, no other species having molecular weight smaller than E2 (MW) 272) has been found. This may suggest that E2 transformation was primarily via radical coupling mechanism rather than degradation under LiP catalysis. Treatability of Reaction Products. Residual E2 and the coupling products were analyzed after the reaction solution was filtered through a 0.45-µm pore size membrane. Interestingly, only E2 was detected, while none of the dimer and trimer products were found in the filtrate (Figure 2). However, these coupling products were subsequently eluted out from the membrane residue with methanol. Their relative quantities based on chromatographic peak area were given in Figure 2. This finding suggests that the coupling products can be retained by microfiltration membrane. Such retention could be due to either size exclusion or adsorption. The concentrations of E2 and coupling products in top layer and bulk solution after ultracentrifugation were compared to evaluate the dissolution states of the products. It is evident in Figure 3 that the centrifugation created significant gradients in the concentrations of each dimer and trimer product from the top layer to the bulk of the solution. However, similar concentration gradient was not observed for E2. This finding demonstrates that the coupling products are largely present in water as undissolved colloids. Therefore, their retention by membrane was more likely

FIGURE 2. The HPLC responses of E2 and products present in filtrate and methanol eluent in a reaction solution that was filtered by a 0.45-µm pore size membrane filter followed by methanol elution. The reaction solution comprised 10 µM E2, 0.0065 U/mL LiP, and 15 µM H2O2 in 10 mM CPBS buffer (pH 4.5). The reaction time was 90 min. Error bars represent standard deviation of three replicates. The left ordinate is for E2, while the right one is for all the dimer and trimer products.

FIGURE 3. The HPLC responses of E2 in a blank control sample and E2 coupling products in a reaction sample after centrifugation. The black and red bars respectively indicate the concentrations in the top layer and the bulk solution. The reaction solution comprised 10 µM E2, 0.02 U/mL LiP, and 15 µM H2O2 in 10 mM CPBS buffer (pH 4.5). The reaction time was 120 min. Error bars represent standard deviation of three replicates. attributed to size exclusion of colloidal aggregates. The results of filtration and centrifugation experiments together suggest that LiP-mediated E2 reactions yielded coupling products with solubility significantly lower than E2. The much reduced solubility enables the products to form aggregates that are essentially colloids and can be readily filterable and settable. The insolubility of the coupling products is apparently attributable to their increased molecular sizes and the fact that the phenolic group in E2 may be converted into more hydrophobic ether moieties upon coupling (40). This finding is of great practical significance because it supports the notion that catalyzed oxidative coupling reactions may be combined with microfiltration and/or coagulation to provide a novel scheme to effectively remove E2 and other micropollutants of similar nature from water. It also implies that such reactions VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Dose-response curves for E2 (a) and the estrogenicity change after sequenced reaction treatments (b). The reaction system initially contained a 1 × 10-7 M E2. Ten µM H2O2 and 0.01 U/mL of LiP were added at each treatment step. Error bars represent standard deviations (n ) 3). occurring in the natural soil/water systems would convert micropollutants into products having greatly reduced mobility. Estrogenicity. We employed MCF-7-BOS cell line to examine estrogenicity of E2 solution after enzymatic treatment. Figure 4a shows a dose-response curve for E2 standards ranging from 1 × 10-7 to 1 × 10-13 M. It is demonstrated that the estrogenic response was not detectable when E2 concentration dropped below 1 × 10-12 M. Figure 4b shows the estrogenicity of the solutions initially containing 1 × 10-7 M E2 followed by sequential LiP treatments as described in the Experiment Section. It can be seen that the estrogenicity was nearly completely removed after three treatment cycles. This suggests that E2 remaining in solution was below 1 × 10-12 M and that the coupling products were not estrogenic, indicative of the great efficiency of the LiP reaction in E2 and estrogenicity removal. Influence of VA and NOM on Product Formation. As described in the Experimental Section, the influence of VA (100 µM) and/or NOM (5 mg/L as TOC) on LiP-mediated E2 reaction was probed under a relatively mild reaction condition (10 µM E2, 10 µM H2O2, and 0.005 U/mL LiP). Based on our HPLC/PDA analysis described in Supporting Information I, the removal rate of E2 increased from 39% to 90% in the presence of VA and decreased to 24% in the presence of NOM, while 75% removal was achieved in the presence of both VA and NOM. The results are consistent with our earlier study (29) wherein the presence of VA enhanced the removal of E2, which was attributed to the ability of this compound to protect LiP from inactivation and to enhance LiP activity. It was concluded in that study (29) NOM competed with VA for the active site located on LiP surface; therefore, the

FIGURE 6. Comparison of the relative yield of each product after 60 min of E2 reaction in LiP-mediated systems with and without the presence of NOM, including a reaction system that did not contain VA or NOM (E2); a system containing NOM at 5 mg TOC/L (E2 + NOM); a system containing 100 µM VA (E2 + VA); and a system containing both 5 mg TOC/L NOM and 100 µM VA (E2 + NOM + VA). Experimental conditions: [E2] ) 10 µM; [LiP] ) 0.005 U/mL, [H2O2] ) 10 µM, pH ) 4.5 (10 mM CPBS). Error bars represent standard deviations (n ) 3). enhancement effect of VA on LiP performance was mitigated in the presence of NOM. All of the five major products were detected in the reaction systems having VA and/or NOM present (Figure 1c,d,e). In addition, a product with m/z value of 539 was detected in the HPLC effluent fraction at retention time between 24 and 25 min. This compound was only formed in the reaction systems having VA present. According to its MW (540), this product likely resulted from the cross-coupling between E2 and E1 (272-1 + 270-1)540). Although the absolute concentrations of the products cannot be quantified without standards available, their relative yields and distribution can be determined and

FIGURE 5. HPLC peak area of the products generated after 60 min of reaction in LiP-mediated reaction systems variously containing VA and/or NOM, including a reaction system that did not contain VA or NOM (E2); a system containing NOM at 5 mg TOC/L (E2 + NOM); a system containing 100 µM VA (E2 + VA); and a system containing both 5 mg TOC/L NOM and 100 µM VA (E2 + NOM + VA). Experimental conditions: [E2] ) 10 µM; [LiP] ) 0.005 U/mL, [H2O2] ) 10 µM, pH ) 4.5 (10 mM CPBS). Error bars represent standard deviations (n ) 3). 2602

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compared according to the peak area. The data are presented in Figure 5. It is demonstrated that the presence of VA increased the yield of each of the coupling products, which is attributable to the ability of VA to enhance the catalytic performance of LiP (29, 41, 42). Data presented in Figure 5 also demonstrate that the ratio of trimer to dimer products tends to increase in the presence of VA, regardless of the absence or presence of NOM. This result is verified by the data shown in Figure S2 in Supporting Information II. The ratio of the peak intensity of E2 dimer to trimer changed from 100/34 to 100/42 when VA was included in the system. Therefore, VA not only increased the transformation of the reactant but also increased the proportion of products with higher degree of polymerization. The HPLC peak area of each product was further normalized by the removal ratio of E2 in the reaction system to reflect the yield of each product relative to the removed E2. As shown in Figure 6, the presence of NOM tends to decrease the relative yield of most products regardless of the presence or absence of VA. We hypothesize that crosscoupling between NOM and E2 may have occurred. NOM molecules contain phenolic functionalities, and thus they are active substrates of LiP. Upon oxidation, free radicals can be formed from NOM moieties which may nonselectively couple with E2 radicals to form cross-coupling species. As a result, the yields of E2 self-coupling were reduced, but E2 moieties were incorporated into NOM macromolecules. The reactivity of NOM and its potential to bind micropollutants in catalyzed oxidative coupling systems have been demonstrated in a few earlier studies (40, 43-47).

Acknowledgments S.G. thanks the support from Key Projects of the National Natural Science Foundation of China (20737001 and 20677024). L.M. thanks the China Scholarship Council for supporting him to participate in this study as a visiting student at UGA. The study was supported in part by the U.S. EPA STAR grant G6M10518 and HATCH fund. The content of the paper does not necessarily represent the views of the funding agencies. We are grateful to Professor Ana M. Soto in Tufts University School of Medicine for kindly providing the human breast cancer MCF-7-BOS cells. We thank Vijayalakshmi Mantripragada for her technical assistance with the ESCREEN test.

Supporting Information Available Additional description of certain experimental procedure (I) and selected MS spectra (II). This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Jobling, S.; Nolan, M.; Tyler, C. R.; Brighty, G.; Sumpter, J. P. Widespread sexual disruption in wild fish. Environ. Sci. Technol. 1998, 32, 2498–2506. (2) Johnson, A. C.; Sumpter, J. P. Removal of endocrine-disrupting chemicals in activated sludge treatment works. Environ. Sci. Technol. 2001, 35, 4697–4703. (3) Huang, Q.; Weber, W. J., Jr. Transformation and removal of bisphenol A from aqueous phase via peroxidase-mediated oxidative coupling reactions: efficiency, products, and pathways. Environ. Sci. Technol. 2005, 39, 6029–6036. (4) Kidd, K. A.; Blanchfield, P. J.; Mills, K. H.; Palace, V. P.; Evans, R. E.; Lazorchak, J. M.; Flick, R. W. Collapse of fish population after exposure to a synthetic estrogen. Proc. Natl. Acad. Sci. 2007, 104, 8897–8901. (5) Michael, J. Are problems with male reproductive health caused by endocrine disruption. Occup. Med. Educ. 2001, 58, 281–288. (6) Welshons, W. V.; Thayer, K. A.; Judy, B. M.; Taylor, J. A.; Curran, E. M.; vom Saal, F. S. Large effects from small exposures. I. mechanisms for endocrine-disrupting chemicals with estrogenic activity. Environ. Health Perspect. 2003, 111, 994–1006.

(7) Miyamoto, J.; Klein, W. Environmental exposure, specials differences and risk assessment. Pure Appl. Chem. 1998, 70, 1829–1845. (8) Yager, J. D.; Liehr, J. G. Molecular mechanisms of estrogen Carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 1996, 36, 203– 232. (9) Bisagno, V.; Bowman, R. E.; Luine, V. E. Functional aspects of estrogen neuroprotection. Endocrine 2003, 21, 33–41. (10) Kajta, M.; Beyer, C. Cellular strategies of estrogen-mediated neuroprotection during brain development. Endocrine 2003, 21, 3–9. (11) Bolton, J. L.; Yu, L.; Thatcher, G. R. Quinoids formed from estrogens and antiestrogens. Methods Enzymol. 2004, 378, 110– 123. (12) Pezzella, A.; Lista, L.; Napolitano, A.; d’Ischia, M. Oxidative coupling of 17β-estrodiol: inventory of oligomer products and configuration assignment of atropoisomeric C4-linked biphenyltype dimers and trimers. J. Org. Chem. 2004, 69, 5652–5659. (13) Gaido, K. W.; Leonard, L. S.; Lovell, S.; Gould, J. C.; Babai, D.; Portier, C. J.; Mcdonnell, D. P. Evaluation of chemicals with endocrine modulating activity in a yeast-based steroid hormone receptor gene transcription assay. Toxicol. Appl. Pharmacol. 1997, 143, 205–212. (14) Desbrow, C.; Routledge, E. J.; Brighty, G. C.; Sumpter, J. P.; Waldock, M. Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol. 1998, 32, 1549–1558. (15) Kramer, V. J.; Miles-Richardson, S.; Pierens, S. L.; Giesy, J. P. Reproductive impairment and induction of alkaline-labile phosphate, a biomarker of estrogen exposure, in fathead minnows (Pimephales promelas) exposed to waterborne 17βestradiol. Aquat. Toxicol. 1998, 40, 335–360. (16) Routledge, E. J.; Sheahan, D.; Desbrow, C.; Brighty, G. C.; Waldock, M.; Sumpter, J. P. Identification of estrogenic chemicals in STW effluent. 2. in vivo responses in trout and roach. Environ. Sci. Technol. 1998, 32, 1559–1565. (17) Zhao, Y.; Hu, J.; Jin, W. Transformation of oxidation products and reduction of estrogenic activity of 17β-estradiol by a heterogeneous photo-fenton reaction. Environ. Sci. Technol. 2008, 42, 5277–5284. (18) Johnson, J. Q.; Belfroid, A.; Di Corcia, A. C. Estimating steroid oestrogen inputs into activated sludge treatment works and observations on their removal from the effluent. Sci. Total Environ. 2000, 256, 163–173. (19) Kolpin, D. W.; Furlong, E. T.; Meyer, M. T.; Thurman, E. M.; Zaugg, S. D.; Barber, L. B.; Buxton, H. T. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999-2000: A national reconnaissance. Environ. Sci. Technol. 2002, 36, 1202–1211. (20) Bila, D.; Montalva˜o, A. F.; Azevedo, D; de, A.; Dezotti, M. Estrogenic activity removal of 17 β-estradiol by ozonation and identification of by-products. Chemosphere 2007, 69, 736–746. (21) Nakamura, H.; Shiozawa, T.; Terao, Y.; Shiraishi, F.; Fukazawa, H. By-products produced by the reaction of estrogens with hypochlorous acid and their estrogen activities. J. Health Sci. 2006, 52, 124–131. (22) Hu, J.; Chen, S.; Aizawa, T.; Terao, Y.; Kunikane, S. Products of aqueous chlorination of 17β-estradiol and their estrogenic activities. Environ. Sci. Technol. 2003, 37, 5665–5670. (23) Huber, M. M.; Ternes, T. A.; Gunten, U. V. Removal of estrogenic activity and formation of oxidation products during ozonation of 17R-ethinylestradiol. Environ. Sci. Technol. 2004, 38, 5177–5186. (24) Auriol, M.; Filali-Meknassi, Y.; Adams, C. D.; Tyagi, R. D. Natural and synthetic hormones removal using the horseradish peroxidase enzyme: temperature and pH effects. Water Res. 2006, 40, 2847–2856. (25) Klibanov, A. M.; Tu, T. M.; Scott, K. P. Peroxidase-catalyzed removal of phenols from coal-conversion waste waters. Science 1983, 221, 259–261. (26) Yu, J.; Taylor, K. E.; Zou, H.; Biswas, N.; Bewtra, J. K. Phenol conversion and dimeric intermediates in horseradish peroxidase-catalyzed phenol removal from water. Environ. Sci. Technol. 1994, 28, 2154–2160. (27) Weber, W. J., Jr.; Huang, Q. Inclusion of persistent organic pollutants in humification processes: direct chemical incorporation of phenanthrene via oxidative coupling. Environ. Sci. Technol. 2003, 37, 4221–4227. (28) Karam, J.; Nicell, J. A. Potential applications of enzymes in waste treatment. J. Chem. Technol. Biotechnol. 1997, 69, 141–153. (29) Mao, L.; Huang, Q.; Lu, J.; Gao, S. Ligninase-mediated removal of natural and synthetic estrogens from water: I. Reaction behaviors. Environ. Sci. Technol. 2009, 43, 374–379. VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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(30) Suzuki, Kestrogenic activities of 17 beta-estradiol and ethinylestradiol by ligninolytic enzymes.;Hirai, H.; Murata, H.; Nishida, T. Removal of from white rot fungi. Water Res. 2003, 37 (8), 1972–1975. (31) Tien, M.; Kirk, T. K. Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 1988, 161, 238–249. (32) Bumpus, J. A.; Tien, M.; Wright, D.; Aust, S. D. Oxidation of persistent environmental pollutants by a white rot fungus. Science 1985, 228, 1434–1436. (33) Harvey, P. J.; Schoemaker, H. E.; Palmer, J. M. Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium. FEBS Lett. 1986, 195, 242–246. (34) Wariishi, H.; Gold, M. H. Lignin peroxidase compound II and III: Merchanism of formation and decomposition. J. Biol. Chem. 1990, 265, 2070–2077. (35) Soto, A. M.; Sonnenschein, C.; Chung, K. L.; Fernandez, M. F.; Olea, N.; Olea Serrano, F. The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health Perspect. 1995, 103, 113–122. (36) Lu, J.; Huang, Q.; Mao, L. Removal of acetaminophen using enzyme-mediated oxidative coupling process: I. reaction rates and pathways. Environ. Sci. Technol. 2009, 43, 7062–7067. (37) Huang, Q.; Weber, W. J. Transformation and Removal of Bisphenol A from Aqueous Phase via Peroxidase-Mediated Oxidative Coupling Reactions: Efficacy, Products, and Pathways. Environ. Sci. Technol. 2005, 39 (16), 6029–6036. (38) Auriol, M.; Filali-Meknassi, Y.; Tyagi, R. D.; Adams, C. D. Laccase-catalyzed conversion of natural and synthetic hormones from a municipal wastewater. Water Res. 2007, 41 (15), 3281–3288.

2604

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 7, 2010

(39) Yu, J.; Taylor, K. E.; Zou, H. X.; Biswas, N.; Bewtra, J. K. Phenolic conversion and dimeric intermediates in horserahish peroxidase-catalyzed phenol removal from water. Environ. Sci. Technol. 1994, 28 (12), 2154–2160. (40) Weber, W. J., Jr.; Huang, Q.; Pinto, R. A. Reduction of disinfection byproduct formation by molecular reconfiguration of the fulvic constituents of natural background organic matter. Environ. Sci. Technol. 2005, 39, 6446–6452. (41) Khindaria, A.; Nie, G.; Aust, S. D. Detection and characterization of the lignin peroxidase compound II - veratryl alcohol cation radical complex. Biochemistry 1997, 36, 14181–14185. (42) Khindaria, A.; Yamazaki, I.; Aust, S. D. Veratryl alcohol oxidation by lignin peroxidase. Biochemistry 1995, 34, 16860–16869. (43) Lu, J.; Huang, Q. Removal of acetaminophen using enzymemediated oxidative coupling processes: II. Cross-coupling with natural organic matter. Environ. Sci. Technol. 2009, 43 (18), 7068–7073. (44) Cozzolino, A.; Piccolo, A. Polymerization of dissolved humic substances catalyzed by peroxidase. Effects of pH and humic composition. Org. Geochem. 2002, 33, 281–294. (45) Piccolo, A.; Cozzolino, A.; Conte, P.; Spaccini, R. Polymerization of humic substances by an enzyme-catalyzed oxidative coupling. Naturwissenschaften 2000, 87, 391–394. (46) Conte, P.; Piccolo, A. Conformation arrangement of dissolved humic substances. Influence of solution composition on association of humic molecules. Environ. Sci. Technol. 1999, 33, 1682–1690. (47) Bialk, H. M.; Pedersen, J. A. NMR investigation of enzymatic coupling of sulfonamide antimicrobials with humic substances. Environ. Sci. Technol. 2008, 42 (1), 106–112.

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