Responses of Mouse Liver to Dechlorane Plus Exposure by

Aug 22, 2012 - Dechlorane plus (DP), a chlorinated flame retardant, has been widely detected in different environmental matrices and biota. However, t...
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Responses of Mouse Liver to Dechlorane Plus Exposure by Integrative Transcriptomic and Metabonomic Studies Bing Wu,* Su Liu, Xuechao Guo, Yan Zhang, Xuxiang Zhang, Mei Li, and Shupei Cheng State Key Lab of Pollutant Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, 210093 P.R. China S Supporting Information *

ABSTRACT: Dechlorane plus (DP), a chlorinated flame retardant, has been widely detected in different environmental matrices and biota. However, toxicity data for DP have seldom been reported. In the present study, we investigated hepatic oxidative stress, DNA damage, and transcriptomic and metabonomic responses of male mice administered 500 mg/ kg, 2000 mg/kg, and 5000 mg/kg of DP by gavage for 10 days. The results showed that DP exposure increased the level of superoxide dismutase (SOD) and 8-hydroxy-2-deoxyguanosine (8-OHdG). The microarray-based transcriptomic results demonstrated that DP exposure led to significant alteration of gene expression involved in carbohydrate, lipid, nucleotide, and energy metabolism, as well as signal transduction processes. The NMR-based metabonomic analyses corroborated these results showing changes of metabolites associated with the above altered mechanisms. Our results demonstrate that an oral exposure to DP can induce hepatic oxidative damage and perturbations of metabolism and signal transduction. These observations provide novel insight into toxicological effects and mechanisms of action of DP at the transcriptomic and metabonomic levels.

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INTRODUCTION Dechlorane plus (DP) as a registered trademark is widely used as a chlorinated flame retardant in electrical resistant plastic connectors in wire coatings, televisions and computer monitors, and furniture. DP has been identified as a high volume production chemical by U.S. Environmental Protection Agency (USEPA). Despite its long production history, our understanding of DP in the environment is still limited. It was initially found in the North American Great Lakes Basin in 2006.1 Then, it has been detected in the air,2 water,3 sediments,4 soil,5 and biota (including aquatic and terrestrial wildlife, and human)6−8 in other regions of the world. Although DP levels in the environment are relatively low,9,10 its long-range atmospheric transport has been observed along an oceanic transect from Greenland to Antarctica, indicating a global presence of DP.11 In addition, bioaccumulation of DP has also been found in aquatic and terrestrial wildlife.12,13 Although the persistence and bioaccumulation of DP have been sufficiently demonstrated, toxicity data for DP are not readily available. Most of the existing toxicity data are provided by OxyChem company14 and High Production Volume Information System (HPVIS) of U.S. EPA (http://www.epa. gov/hpvis/). Recently, Crump et al.15 tested the toxicological effects of DP on chicken embryos and found none of mRNA transcripts changed as a result of in vitro or in ovo exposure to DP. Brock et al.16 tested DP toxicity in rats at doses of 0, 750, 1500, and 5000 mg/kg by gavage. After a 28-day oral repeat dose phase and a developmental and reproductive phase, no toxicological effects were observed at three DP exposure doses. © 2012 American Chemical Society

According to the available toxicity data of DP, it has been concluded that the potential toxic effects of DP are low. However, the available toxicity data of DP focused on the in-life parameters or clinical or anatomical pathology, and data gaps exist with regard to “in vivo” toxicity at molecular level and comprehensive hazard classification.9,10 Further research on DP toxicity at molecular levels is required to better understand subtle toxicological effects and mechanisms of action of DP. At present, biomics approaches, such as toxicogenomics and metabonomics approaches, can provide high-throughput tools to characterize the adverse effects of environmental chemicals. The toxicogenomics approaches characterize the gene expression patterns induced by environmental toxicants.17 The metabonomics approaches quantitatively measure the multiparametric metabolic responses to pathophysiological stimuli or genetic modification.18,19 The results of these approaches can help identify useful biological response markers and explore potential molecular mechanism of toxic substances. In recent years, these approaches have been widely applied in environmental toxicology.20 However, there are no reports that apply these approaches to characterize the toxicological effects and mechanisms of action of DP. The objective of this study was to characterize DP toxicity in mice at the molecular level. Liver as the main target organ of Received: Revised: Accepted: Published: 10758

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DP distribution21 was chosen as target organ. Hepatic oxidative stress, DNA damage, and transcriptomic and metabonomic profiles were determined. The toxicological effects and mechanisms of action of DP were systematically analyzed. To our knowledge, this is the first report of systematic analysis of DP toxicity at molecular level.

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Transcriptomic Analysis. Hepatic RNA for each mouse was extracted and cleaned using Trizol reagent protocol (Invitrogen) and Qiagen RNeasy miniRNA cleanup protocol (Qiagen, Valencia), respectively. Then, an equal amount of RNA extraction from three mouse livers in one group was mixed as one sample. Two biological samples for every group were separately analyzed by GeneChip Mouse Exon 1.0 ST array (Affymetrix). Processes for microarray analysis, including RNA extraction, synthesis of double-strand cDNA, labeled transcript and fragment, hybridization, washing, and staining and scanning of hybridization products, were carried out according to manufacturer's protocol of Affymetrix.28 Normalization and comparison analysis of probe values were performed by Expression Console software (Affymetrix). Differentially expressed genes (DEGs) in DP-treated groups were identified by expression as fold change (≥±1.5) and statistical p-value (p < 0.05) of one-way analysis of variance analysis (ANOVA). One-way ANOVA was performed using Partek Genomics Suite software (St. Louis, MO). Biological significances of DEGs were analyzed by gene ontology (GO) analysis, pathway analysis, and cluster analysis. GO analysis of DEGs was performed on the basis of the GO database (http://www.geneontology.org/).29 DEGs were classified to different biological pathways according to Kyoto encyclopedia of genes and genomes (KEGG) pathway database (http://www.genome.ad.jp/kegg/pathway.html)30 and Gene Map Annotator and Pathway Profiler (GenMAPP) database (http://genmapp.org/) using Molecule Annotation System 3.0 (MAS3.0, http://bioinfo.capitalbio.com/mas/). Cluster analysis of DEGs was carried out by Cluster program (http://rana. lbl.gov/EisenSoftware.htm). Results of cluster analysis were visualized and browsed by TreeView program (http:// jtreeview.sourceforge.net/).31 All data obtained from microarray analysis are publicly available at EBI’s ArrayExpress Archive database (Accession Number: E-MEXP-3707). Metabonomic Analysis. Hepatic metabolites were extracted following the procedure described by Lin et al.32 with slight modifications. Briefly, a sample of an individual liver (about 100 mg) was sonicated in 500 μL of ice-cold methanol/ chloroform (v/v = 2:1) for 4 min. Then, 500 μL of ice-cold chloroform/water (v/v = 1:1) was added and mixed. The samples were centrifuged at 10 000 × g for 20 min. The supernatant phase (hydrophilic metabolites) and lower phase (lipophilic metabolites) were separated and lyophilized. The hydrophilic metabolites were reconstituted in 600 μL D2O containing 0.05% trisilylpropionic acid (TSP) and transferred into NMR glass tubes. The lipophilic metabolites were extracted in 600 μL of CDCl 3 containing 1.18 mM tetramethylsilane (TMS) and then vortexed for 20 min, and centrifuged for 15 min at 6000 × g. The supernatants were transferred into NMR glass tubes. The metabolites extracted were analyzed by a Bruker 600 MHz spectrometer (Bruker, Germany). Fourier transformed 1H NMR spectra were manually phased, and the baseline was corrected using MestReC software (MestreC Research, Spain). Each spectrum was segmented into 0.005 ppm bins. Water resonances (5.0−4.5 ppm) and chloroform resonances (7.5−7.0 ppm) were removed for hydrophilic and lipophilic data sets prior to normalization, respectively. A list of metabolites resonances obtained from published literature20,33 was shown in Table S1, Supporting Information. Partial least-squares discriminant analysis (PLS-

MATERIALS AND METHODS

Animal Care and DP Exposure. Five-week-old male mice (Mus musculus, ICR) were purchased from the experimental animal center of Academy of Military Medical Science of China and were housed in stainless-steel cages. Ambient conditions were at 25 ± 3 °C, 50 ± 5% relative humidity, and a 12/12 h light/dark cycle. Following acclimation to laboratory environment for one week, a total of 24 mice were randomly assigned to control and three DP-treated groups. Six mice were applied in every group. For control group, corn oil was given to mice by gavage daily. For three DP-treated groups, mice received a daily dose of DP in corn oil at 500 mg DP/kg bw, 2000 mg DP/kg bw, and 5000 mg DP/kg bw for a period of 10 days, respectively. The DP expsoure doses employed were chosen on the basis of a report of Brock et al.14 and toxicity data provided by OxyChem and U.S. EPA. DP was purchased from Anpon Electrochemical Co., Ltd., in Jiangsu Province of China. After exposure, mice were anaesthetized under isoflurane followed by exsanguination. Livers were removed and immediately frozen. All experimental processes were in accordance with NIH Guide for the Care and Use of Laboratory Animals. Oxidative Stress Analysis. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px)22,23 and level of lipid peroxidation product malonaldehyde (MDA) 24 in mouse livers were determined to identify the oxidative stress of DP. A certain amount of liver was homogenized, and supernatants were used for various estimation. The protein contents of the supernatants were determined using the modified method of Lowry et al.25 SOD, CAT, and GSH-Px activities and MDA level were measured using commercial kits (Nanjing Jiancheng Bioeng. Inst., China), and standardized by protein content. Each experiment was performed in triplicate. 8-Hydroxy-2-deoxyguanosine (8-OHdG) ELISA. The 8OHdG in mouse livers was determined to indicate hepatic oxidative DNA damage using a mouse 8-OHdG enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Bioeng Inst., China). First, genomic DNA of mouse liver was isolated using Genomic DNA Mini Preparation Kit (Beyotime, China). Then, competitive ELISA for 8-OHdG was performed according to manufacturer’s protocol of ELISA kit. Each experiment was performed in triplicate. The average concentration of 8-OHdG per microgram of DNA for each group was calculated. Comet Assay. The level of DNA strand breaks in mouse livers was determined by comet assay using the method described by Sasaki et al.26 After electrophoresis, slides were washed three times with 0.5 M Tris buffer (pH 7.5), and the DNA was stained with ethidium bromide. The stained slides were examined with a fluorescent microscope (BX41, Olympus, Japan). Six slides per group were prepared, and at least 50 cells were analyzed for each slide. Photos were taken with a digital camera (C-5050ZOOM, Olympus, Japan). Images were analyzed by CASP software according to the method of Collins et al.27 10759

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DA) was used to explore the main effects in NMR data sets by SIMCA-P software (Umetric, Sweden). Statistical Analysis. Results were expressed as mean ± standard deviation (SD). Statistical differences of biological parameters between DP-treated groups and control group were evaluated using one-way ANOVA test followed by Tukey’s post hoc test. All analyses were performed by SPSS 15 software (SPSS Inc.). A probability value 0.05). The results were consistent with the toxicity data provided by Oxychem Company, which indicated that oral and inhalation exposure of DP could increase the liver weight of rats. However, body weight and relative organ weight could not effectively reflect the DP toxicity at molecular level, which needed to be further characterized. Oxidative Stress Induced by DP. SOD, CAT, and GSHPx activities and MDA levels in control and DP-treated groups were presented in Figure S1. Compared with the control group, a significant increase in SOD activities in three DP-treated groups was observed. CAT activities increased significantly in mice exposed to 2000 mg/kg DP. MDA showed an apparent increase; however, no significant differences were found. With regards to the GSH-Px, its activities were rather analogous in the control and DP-treated groups. Liver is especially sensitive to oxidative stress since most toxicants are metabolized in this organ. The enzymatic systems, including SOD, CAT, and GSH-Px, are known to inactivate the reactive oxygen species (ROS) formed by environmental contaminants. SOD is in the first line of defense, followed by CAT or GSH-Px.34 In this study, the alterations of SOD and CAT activities indicated that DP induced oxidative stress of mouse livers. It is known that when ROS generation overloads antioxidant defenses, the free radicals can act on macromolecules such as lipid, protein, and nucleic acid, altering their morphology and function. No significant differences in MDA levels in DP-treated groups were found, indicating DP was not causing the lipid peroxidation through the10-day exposure. Hepatic DNA Damage Induced by DP. Quantitative estimates of 8-OHdG in control and three DP-treated groups were performed to identify the DNA adducts formation and oxidative DNA damage (Figure 1). Compared with the control group, the 8-OHdG levels in DP-treated mice showed a significant increase (p < 0.05). Combined with the results of oxidative stress (SOD and CAT activities), it can be deduced that ROS generated by DP exposure overloads antioxidant defenses, and induces oxidative DNA damage of mouse livers.35 Besides the oxidative DNA damage, another type of DNA damage as DNA strand breaks was identified by Comet assay.36 The ratios of olive tail moment and tail length between DPtreated groups and control group were applied to quantify the DNA damage, which were shown in Figure S2. Although these ratios increased in DP-treated groups, no significant changes were found (p > 0.05), indicating that DP exposure could not cause the DNA strand breaks in mouse livers.

Figure 1. Effects of DP exposure on oxidative DNA damage marker 8OHdG in mouse livers.

Transcriptomic Alteration Induced by DP. Hepatic transcriptomic profiles of mice in control and DP-treated groups were analyzed using GeneChip Mouse Gene 1.0 ST Array, which can identify the expression of 28 853 genes. The PLS-DA analysis for all probes showed that the control and DPtreated groups were readily separated, suggesting the DP exposure obviously affected the hepatic transcriptome of mice (Figure.2). A total of 207, 251, and 218 genes were identified to

Figure 2. Partial least-squares discriminant analysis (PLS-DA) of probes identified by microarray analysis. Each symbol is an individual microarray: black, control group; red, 500 mg/kg DP group; blue, 2000 mg/kg DP group; green, 5000 mg/kg DP group.

be DEGs for 500, 2000, and 5000 mg/kg DP-treated groups, respectively. The cluster analysis of DEGs indicated that, with the increase of DP concentration, the differences of expression profiles of DEGs increased (Figure S3). Most DEGs in DP-treated groups changed by ±1.50−1.99 fold (63.1−82.6%) and ±2.00−4.00 fold (13.3−17.9%) (Figure S4). The commonality of DEGs among three DP-treated groups was shown in Figure S5. DEGs synchronously determined in three DP-treated groups were shown in Table S3. The gene ontology analysis shows these DEGs mainly participate in carbohydrate metabolism (GO:0005975), fatty acid elongation (GO:0030497), protein metabolism (GO:0019538), response to chemical stimulus (GO:0042221), superoxide metabolic process (GO:0006801), negative regulation of signal transduction (GO:0009968), and so on. 10760

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The biological significances of DEGs in three DP-treated groups were determined using MAS 3.0 based on KEGG database and GenMAPP database. The heat map of KEGG pathways and GenMAPP pathways related to DEGs was shown in Figure S6. In this study, the KEGG pathways were chosen for intensive analysis. The significantly changed KEGG pathways were identified on the basis of the criteria that have 3 or more DEGs, and a hypergeometric test with p ≤ 0.05. These different KEGG pathways were divided into two groups, i.e., metabolic pathway and biological process pathway. A total of 10 metabolic pathways and 15 biological processes were found (Table S4), which referred to signal transduction, immune system, signaling molecules and interaction, endocrine system, carbohydrate metabolism, lipid metabolism, nucleotide metabolism, and biosynthesis of steroids. Metabonomic Alteration Induced by DP. A total of 56 hepatic metabolites were determined using 1H NMR. PLS-DA analysis for these metabolites showed that control and DPtreated groups were readily separated, suggesting the DP exposure obviously affected hepatic metabonome of mice (Figure 3). The significantly changed metabolites were chosen

Table 1. Altered Metabolites in Mouse Livers in Three DPTreated Groups fold change acetaldehyde NAD/NADP/NADPH phenylalanine uridine uracil glucose-1-phosphate-(glycogen) creatinine creatine carnitine free glycerol choline methionine glutamine pyruvate succinate valine phosphatidyl serine phosphatidyl ethanolamine a

500 mg/kg

2000 mg/kg

5000 mg/kg

1.84 −1.53 −1.05a 1.21a 1.61 1.55 1.52 1.35a −2.0 −1.03a −2.27 1.53 1.62 −2.04 1.62 −1.85 1.58 1.90

2.94 −1.25a 1.50 1.57 2.09 2.07 1.62 1.34a −1.19a −1.67 −2.95 1.96 1.32a −3.12 1.53 −1.51 2.02 1.43a

1.77 −1.50 1.21a 1.50 1.66 1.65 1.47a 1.56 −1.51 −1.35a −2.0 1.94 1.29a −3.22 1.54 −1.12a 1.59 1.57

p > 0.05 or/and fold change 0.05). GSSG is an important endogenous antioxidant providing protection against oxidative stress.44 High increases of PE and PS, and NAD+ breakdown during lipid and carbohydrate metabolism in DP-treated groups, might be associated with the increase of oxidative stress.45 Succinate can be formed by switching of the TCA cycle to the nonenzymatic formation of succinate from aketoglutarate under oxidative stress.46 The increases of creatine and creatinine as key intermediates in energy metabolism were also found to be associated with general cytoprotective effects toward oxidative agents.47 Besides the above-mentioned metabolic pathways, microarray analysis also found significant changes in signal transduction pathways, including MAPK and Jak-STAT signaling pathways. MAPK signaling pathway can couple intracellular responses to the binding of growth factors via cell surface receptors.48 Aberrance of MAPK signaling pathway has been implicated in inflammation and cancers.49 Jak-STAT pathway is a rapid signal transduction pathway used to alter gene

expression. When STAT proteins are activated, they can travel to the nucleus and induce transcription of specific genes.50,51 The heat map of genes involved in both pathways was shown in Figure 5. The fold changes of most genes (such as Spry4, Hsps,

Figure 5. Heat map of differentially expressed genes related to signal transduction processes. Data were mean standardized, and hierarchical clustering was performed. Green represents relative low expression, and yellow represents relative high expression.

Gadd45d, and Piml) were elevated along with the increase of DP concentration. Therefore, the changes of both pathways might be associated with DP concentrations. In this study, the increase of relative liver weight was found in DP-treated groups, indicating DP might induce the cell proliferation of mouse livers. The alterations of signal transduction processes, such as 10762

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(8) Yu, Z. Q.; Ren, G. F.; Ma, S. T.; Li, H. R.; Peng, P. G.; Sheng, G. Y.; Fu, J. M. Determination of dechlorane plus in serum from electronics dismantling workers in south China. Environ. Sci. Technol. 2009, 43 (24), 9453−9457. (9) Sverko, E.; Tomy, G. T.; Reiner, E. J.; Li, Y. F.; McCarry, B. E.; Arnot, J. A.; Law, R. J.; Hites, R. A. Dechlorane plus and related compounds in the environment: A review. Environ. Sci. Technol. 2011, 45 (12), 5088−5098. (10) Xian, Q. M.; Siddique, S.; Li, T.; Feng, Y. L.; Takser, L.; Zhu, J. P. Sources and environmental behavior of dechlorane plus - A review. Environ. Int. 2011, 37 (7), 1273−1284. (11) Moller, A.; Xie, Z. Y.; Sturm, R.; Ebinghaus, R. Large-scale distribution of dechlorane plus in air and seawater from the Arctic to Antarctica. Environ. Sci. Technol. 2010, 44 (23), 8977−8982. (12) Tomy, G. T.; Thomas, C. R.; Zidane, T. M.; Murison, K. E.; Pleskach, K.; Hare, J.; Arsenault, G.; Marvin, C. H.; Sverko, E. Examination of isomer specific bioaccumulation parameters and potential in vivo hepatic metabolites of syn- and anti-dechlorane plus isomers in juvenile rainbow trout (Oncorhynchus mykiss). Environ. Sci. Technol. 2008, 42 (15), 5562−5567. (13) Zhang, X. L.; Luo, X. J.; Liu, H. Y.; Yu, L. H.; Chen, S. J.; Mai, B. X. Bioaccumulation of several brominated flame retardants and dechlorane plus in waterbirds from an e-waste recycling region in south China: Associated with trophic level and diet sources. Environ. Sci. Technol. 2011, 45 (2), 400−405. (14) OxyChem. Occidental Chemical Corporation. Dechlorane Plus Manual; athttp://msds.oxy.com/DWFiles/M41759_NA_EN%232. pdf. (15) Crump, D.; Chiu, S.; Gauthier, L. T.; Hickey, N. J.; Letcher, R. J.; Kennedy, S. W. The effects of dechlorane plus on toxicity and mRNA expression in chicken embryos: A comparison of in vitro and in ovo approaches. Comp. Biochem. Physiol., C: Comp. Pharmacol. 2011, 154 (2), 129−134. (16) Brock, W. J.; Schroeder, R. E.; McKnight, C. A.; VanSteenhouse, J. L.; Nyberg, J. M. Oral repeat dose and reproductive toxicity of the chlorinated flame retardant Dechlorane Plus. Int. J. Toxicol. 2010, 29 (6), 582−593. (17) Lettieri, T. Recent applications of DNA microarray technology to toxicology and ecotoxicology. Environ. Health Perspect. 2006, 114 (1), 4−9. (18) Bino, R. J.; Hall, R. D.; Fiehn, O.; Kopka, J.; Saito, K.; Draper, J.; Nikolau, B. J.; Mendes, P.; Roessner-Tunali, U.; Beale, M. H.; Trethewey, R. N.; Lange, B. M.; Wurtele, E. S.; Sumner, L. W. Potential of metabolomics as a functional genomics tool. Trends Plant Sci. 2004, 9 (9), 418−425. (19) van Ravenzwaay, B.; Cunha, G. C. P.; Leibold, E.; Looser, R.; Mellert, W.; Prokoudine, A.; Walk, T.; Wiemer, J. The use of metabolomics for the discovery of new biomarkers of effect. Toxicol. Lett. 2007, 172 (1−2), 21−28. (20) Ding, L.; Hao, F.; Shi, Z.; Wang, Y.; Zhang, H.; Tang, H.; Dai, J. Systems biological responses to chronic perfluorododecanoic acid exposure by integrated metabonomic and transcriptomic studies. J. Proteome Res. 2009, 8 (6), 2882−91. (21) Zhang, Y.; Wu, J. P.; Luo, X. J.; Wang, J.; Chen, S. J.; Mai, B. X. Tissue distribution of dechlorane plus and its dechlorinated analogs in contaminated fish: High affinity to the brain for anti-DP. Environ. Pollut. 2011, 159 (12), 3647−3652. (22) Oberley, L. W.; Spitz, D. R. Assay of superoxide dismutase activity in tumor tissue. Methods Enzymol. 1984, 105, 457−64. (23) Flohe, L.; Gunzler, W. A. Assays of glutathione peroxidase. Methods Enzymol. 1984, 105, 114−21. (24) Yagi, K. Simple assay for the level of total lipid peroxides in serum or plasma. Methods Mol. Biol. 1998, 108, 101−6. (25) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265−75. (26) Sasaki, Y. F.; Kawaguchi, S.; Kamaya, A.; Ohshita, M.; Kabasawa, K.; Iwama, K.; Taniguchi, K.; Tsuda, S. The comet assay with 8 mouse

MAPK and Jak-STAT signaling pathways, have proven to be correlative with cell proliferation.52,53 In conclusion, DP exposure induced oxidative stress and damage to mouse livers across all concentrations employed in this study. Microarray-based trancriptomic profiles and NMRbased metabonomic profiles showed DP altered hepatic carbohydrate, lipid, nucleotide, and energy metabolism as well as signal transduction processes. Some oxidative stress responses at both transcriptomic and metabonomic levels were identified. These results suggest that DP exposure can cause liver impairment, and oxidative damage might be the potential mechanism of hepatotoxicity. The combination of transcriptomics and metabonomics approaches in this study provided an integrative, systematic view of the DP toxicity at molecular level. Future studies should evaluate the tissue distribution and biochemical responses of DP following longterm, low-level exposure in order to deeply explore its dose− response on toxicological effects and mechanisms of action.

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ASSOCIATED CONTENT

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AUTHOR INFORMATION

S Supporting Information *

Additional tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author

*Phone: 0086-25-89680720; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from the Science Foundation of Nanjing University, Foundation of State Key Laboratory of Pollution Control and Resource Reuse, and National Natural Science Foundation of China (51148003).

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REFERENCES

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dx.doi.org/10.1021/es301804t | Environ. Sci. Technol. 2012, 46, 10758−10764