Article pubs.acs.org/crt
Involvement of Reactive Persulfides in Biological Bismethylmercury Sulfide Formation Yumi Abiko,†,‡ Eiko Yoshida,‡ Isao Ishii,§ Jon M. Fukuto,∥ Takaaki Akaike,⊥ and Yoshito Kumagai*,†,‡ †
Faculty of Medicine, and ‡Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan § Department of Biochemistry, Keio University School of Pharmaceutical Sciences, Tokyo 105-8512, Japan ∥ Department of Chemistry, Sonoma State University, Rohnert Park, California 94928, United States ⊥ Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan S Supporting Information *
ABSTRACT: Bismethylmercury sulfide (MeHg)2S has been found to be a detoxified metabolite of methylmercury (MeHg) that is produced by SH-SY5Y cells and in livers of rats exposed to MeHg. (MeHg)2S could be formed through the interactions between MeHg and sulfur species such as hydrogen sulfide (H2S or HS−), but the origin of its sulfur has not been fully identified. We herein examined the formation of (MeHg)2S through interactions between MeHg and persulfides, polysulfides, and protein preparations. Investigations using HPLC/ atomic absorption spectrophotometry and EI-MS revealed that NaHS and Na2S4 react readily with MeHg to give (MeHg)2S, and similar results were found using GSH persulfide (GSSH) formed endogenously or generated enzymatically in vitro. (MeHg)2S was also formed by incubation of MeHg with liver and heart cytosolic fractions prepared from wild-type mice but not with those from mice lacking cystathionine γ-lyase (CSE) that catalyzes the formation of cysteine persulfide. Consistent with this, (MeHg)2S was detected in a variety of tissues taken from wild-type mice intraperitoneally injected with MeHg in vivo but not in those from MeHg-injected CSE knockout mice. By separating liver fractions by column chromatography, we found numerous proteins that contain persulfides: one of the proteins was identified as being glutathione Stransferase pi 1. These results indicate that the formation of (MeHg)2S can be attributed to interactions between MeHg and endogenous free persulfide species, as well as protein-bound cysteine persulfide.
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INTRODUCTION
ingly, H2S has a pKa of 6.76, indicating that approximately 80% will be in the deprotonated HS− form at physiological pH, which could react with electrophiles such as MeHg to form sulfur adducts. Indeed, we found that MeHg interacts with sodium hydrogen sulfide (NaHS) to produce bismethylmercury sulfide (MeHg)2S in in vitro experiments and that (MeHg)2S is formed in MeHg-treated SH-SY5Y cells and liver extracts of rats intraperitoneally injected with MeHg.12 There are numerous sulfur-containing low-molecular-weight compounds in the body, including cysteine (CysSH), cystine (CysSSCys), GSH, glutathione disulfide (GSSG), methionine (Met), and sulfate ions (SO42−).13 We recently found that some persulfide species in mammalian cells and tissues such as Cys persulfide (CysSSH), Cys polysulfides (CysSSnSH), GSH persulfide (GSSH), and GSH polysulfides (GSSnSH) contain highly reactive nucleophilic sulfur groups and have antioxidant activities.14 CySSH is produced by cystathionine γ-lyase (CSE)
Methylmercury (MeHg) is an electrophile that is present in the environment and is bioconcentrated by tuna (Tunnus) and other marine biota.1 MeHg covalently modifies thiol groups in proteins (a process called S-mercuration), and thus, its negative effect (i.e., toxicity) could be caused, at least in part, by such protein modifications.2−6 However, we previously found that MeHg activates the transcriptional factor Nrf2 through Smercuration of Keap1, a negative regulator of Nrf2. This causes the up-regulation of downstream genes, such as glutamatecysteine ligase (the rate-limiting enzyme for GSH synthesis), facilitating the formation of MeHg−SG adducts and multidrug resistance-associated proteins (MRPs) and allowing the excretion of polar metabolites into the extracellular space.7,8 In vitro and in vivo experiments using Nrf2 knockout (KO) mice have shown that up-regulating glutamate-cysteine ligase and MRPs alleviates the toxicity of MeHg,7,8 suggesting that the S-mercuration of Keap1 contributes to reducing MeHg toxicity. Hydrogen sulfide (H2S) has been proposed to play a role in repressing neuronal and cardiovascular diseases.9−11 Interest© 2015 American Chemical Society
Received: March 12, 2015 Published: April 15, 2015 1301
DOI: 10.1021/acs.chemrestox.5b00101 Chem. Res. Toxicol. 2015, 28, 1301−1306
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Chemical Research in Toxicology and/or cystathionine β-synthase using cystine as a substrate in the presence of vitamin B6. An elongation (polythiolation) reaction can occur, with sulfur interacting with other sulfurcontaining substances to give persulfides and polysulfides such as CysSSnSH, GSSH, and GSSnSH.14 These observations have led us to assume that thiol-containing protein can also react with low-molecular-weight persulfides and polysulfides to give protein-bound persulfide species. In this study, we examined (MeHg)2S production in the reactions between MeHg and various persulfide compounds. Moreover, we attempted to identify, through the formation of (MeHg)2S, a protein that was isolated from mouse liver cytosol that contains persulfide derivatives.
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Blue 3GA column that had been equilibrated with B-I. The column was then washed with the same buffer at a flow rate of 1 mL/min. The proteins that had become bound to the column were eluted using buffer II (B-II; the same as B-I with the addition of 0.5 M potassium chloride) and then buffer III (B-III; the same as B-II with the addition of 10 mM NADH). The proteins eluted by B-I, B-II, and B-III were concentrated using an Ultracell-10K instrument (Millipore, Darmstadt, Germany) to give a final concentration of 15 mg/mL in each. The BIII fraction (containing 10 mg of protein) was applied to a Sephacryl S-100 column (71 cm long, 25 mm i.d.; GE Healthcare, Little Chalfont, UK) that had been equilibrated with 50 mM phosphate buffer (pH 7.4). The flow rate was 1 mL/min. After the concentration step, the proteins in each fraction were quantified using the Bradford assay.17 Preparation of Human Recombinant Glutathione S-Transferase Pi 1 (hGSTP1). E. coli BL21 cells transformed using hGSTP11/pRB269 were kindly donated by Professor Akira Hiratsuka, Tokyo University of Pharmacy and Life Sciences. The E. coli were grown in Luria−Bertani broth at 37 °C until the absorbance of the mixture at 600 nm reached 0.8 absorbance units. The cells were then incubated with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside for 3 h and then harvested by centrifugation. The cells were then suspended in 50 mM Tris−HCl buffer (pH 7.5) containing 4 mg/mL lysozyme and harvested by centrifugation. The pellet was resuspended in the buffer and then incubated on ice for 1 h. The E. coli were lysed by sonication, and then the mixture was centrifuged at 105,000g for 1 h. Human GSTP1-1 was separated from the lysate using a GSH Separose 6B column (75 mm long, 10 mm i.d.; GE Healthcare) following the method described by Baker et al.18 Detection of (MeHg)2S-Forming Reactive Persulfides Generated Endogenously in Tissues. Cytosolic fractions of different mouse tissues (for determining the amount of (MeHg)2S formed by the cytosolic fractions in vitro) were prepared by homogenizing in 4× tissue volume of 50 mM phosphate buffer (pH 7.4), followed by centrifugation at 9,000g for 10 min. MeHg (500 μM) was incubated with each tissue preparation at 25 °C for 1 h. To determine the amount of (MeHg)2S formed in vivo, MeHg (10 mg/kg) dissolved in corn oil was intraperitoneally injected into WT and CSE KO male mice, and sacrificed after 72 h. Cytosolic fractions of various tissues were then prepared as described above. Each sample for (MeHg)2S determination from the in vitro and in vivo tests was mixed with trichloroacetic acid (to give a final concentration of 5% v/v). Each mixture was centrifuged at 9,000g for 5 min, and the supernatant was added to 3× volume of 1 M Tris−HCl buffer (pH 8.5) and then applied to a Sep-Pak C18 cartridge (Waters). The cartridge was washed with water (20 mL), and then the (MeHg)2S was eluted with methanol (1.5 mL). The eluate was evaporated to dryness and redissolved in 10% methanol. A 10 μL aliquot of each sample was subjected to HPLC fractionation followed by AAS analysis as described above.
MATERIALS AND METHODS
Materials. MeHgCl, GSH, NaHS, and sodium tetrasulfide (Na2S4) were obtained from Sigma-Aldrich (San Diego, CA, USA), Wako Pure Chemical Industries (Osaka, Japan), Strem Chemicals (Newburyport, MA, USA), and Alfa Aesar (Ward Hill, MA, USA), respectively. All other reagents were of the highest grade available. GSH polysulfide (GSSSG) was synthesized and purified as described previously.14 The reaction product was identified by LC/MS/MS. Separation was achieved using an Acquity BEH C18 column (50 mm long, 2.1 mm i.d., 1.7 μm particle size; Waters, Milford, MA, USA). The mobile phase was a 98:2 (v/v) mixture of acetonitrile containing 0.1% formic acid and deionized distilled water containing 0.1% v/v formic acid, and the flow rate was 0.3 mL/min. The MS/MS instrument was a Synapt High Definition system (Waters), and it was used with an electrospray source and in positive ion mode. GSSH was generated through the enzymatic reaction between GSH reductase and GSSSG in the presence of NADPH.14 Briefly, GSSSG (0.1 mM) was incubated with 0.1 U of GSH reductase and 0.2 mM NADPH in 20 mM Tris−HCl buffer (pH 7.4) at 25 °C for 5 min. Animals. CSE hemizygous knockout mice were generated and backcrossed for 10−12 generations to a C57BL/6J inbred strain (CLEA Japan, Tokyo, Japan) as previously described.15 CSE hemizygous knockout male and female mice were bred to obtain knockout mice (CSE KO). Mice were housed in an air-conditioned room (temperature 24 ± 1 °C and humidity 55 ± 5%), kept on a 12 h dark/light cycle, and allowed free access to a Certified diet M (Oriental Yeast, Tokyo, Japan) and water. All protocols for animal experiments were approved by the University of Tsukuba Animal Care and Use Committee, and the procedures were performed strictly adhering to the committee’s guidelines for alleviating suffering. Analysis of (MeHg)2S. (MeHg)2S was synthesized as previously described.12 Briefly, MeHg was reacted with NaHS in 50 mM phosphate buffer (pH 7.5), then a 20 μL aliquot of the reaction mixture was applied to an HPLC system. Separation was achieved using a Zorbax Eclipse XDB-C18 column (50 mm long, 2.1 mm i.d., 5 μm particle size; Agilent Technologies, Santa Clara, CA, USA). The mobile phase was a 9:1 (v/v) mixture of 0.1% formic acid and methanol, and the flow rate was 0.5 mL/min. The mercury concentrations in the eluate fractions were determined using an atomic absorption spectrophotometry (AAS) instrument (MA-3000; Nihon Instruments, Osaka, Japan) and the molecular analytes in the eluate fractions were analyzed using an EI-MS instrument (GC/MSQP1100EX; Shimadzu, Kyoto, Japan).12 Incubating MeHg with Various Sulfur-Containing Species. MeHg (5 nmol) was incubated with sulfide, persulfides, or polysulfides in 50 mM phosphate buffer (pH 7.5) for a specified period at 25 °C unless otherwise noted. After the reaction, (MeHg)2S was determined by HPLC/AAS, as described above. Identification of Mouse Liver Proteins That Contain Persulfides. The wild-type (WT) mice were killed, and the liver cytosol was separated using a Cibacron Blue 3GA column (150 mm long, 10 mm i.d.; Sigma-Aldrich) as described previously.16 Briefly, the liver cytosol was dialyzed against buffer I (B-I; 50 mM Tris−HCl buffer (pH 7.2) and 0.25 M sucrose) and then applied to a Cibacron
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RESULTS Interactions between Low-Molecular-Weight SulfurContaining Species and MeHg. (MeHg)2S was produced when MeHg was incubated with NaHS or Na2S4 at 25 °C for 1 h, and its production was NaHS (or Na2S4) amount-dependent (Table 1). For example, the yield of (MeHg)2S was higher than 90% when 5 nmol of MeHg was incubated with 5 nmol of NaHS or Na2S4 (Table 1), demonstrating that MeHg can react well with both HS− and polysulfides. We next examined the reactivity of MeHg with the endogenous persulfide GSSH. As shown in Figure 1A, reacting GSSH (generating from 0.1 mM GSSSG plus GSH reductase) with MeHg (5 mM) at 37 °C for 15 min led to the formation of a reaction product that had a retention time (using the HPLC/AAS system described above) of 12 min, corresponding to that of authentic (MeHg)2S (Figure 1B). (MeHg)2S also formed when MeHg was reacted with GSSSG (data not shown). The formation of (MeHg)2S 1302
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Chemical Research in Toxicology Table 1. (MeHg)2S Formation during the Incubation of MeHg with NaHS or Na2S4 (MeHg)2S formed NaHS added (nmol)
MeHg added (nmol)
predicted (nmol)
observed (nmol)
1.25 2.5 5.0
5.0 5.0 5.0
1.25 2.5 2.5
Na2S4 added (nmol)
MeHg added (nmol)
predicted (nmol)
observed (nmol)
yield (%)
1.25 2.5 5.0
5.0 5.0 5.0
1.25 2.5 2.5
0.1 ± 0.04 1.3 ± 0.11 2.3 ± 0.12
7±4 52 ± 4 93 ± 5
0.2 ± 0.13 1.3 ± 0.05 2.3 ± 0.02 (MeHg)2S formed
yield (%) 15 ± 3 52 ± 5 92 ± 2
Figure 2. Cystathionine γ-lyase (CSE)-dependent formation of (MeHg)2S from MeHg in vitro and in vivo. Red and blue bars indicate MeHg and (MeHg)2S, respectively. (A) Cytosolic fractions of mouse liver and heart were incubated with MeHg at 25 °C for 60 min, and then the proteins were removed by trichloroacetic acid precipitation. Each mixture was then centrifuged (at 9,000g for 5 min), and an aliquot of the reaction mixture was analyzed by HPLC/atomic absorption spectrophotometry (AAS). Mice were intraperitoneally injected with 10 mg/kg MeHg and sacrificed after 72 h, and the cytosolic fractions of brain (B), heart (C), and liver (D) were analyzed by HPLC/AAS.
during the reaction between MeHg and GSSH was confirmed by EI-MS analysis (Figure 1C). Only a small amount of MeHg remained, suggesting that MeHg can be effectively metabolized by GSSH (Figure 1A). This phenomenon may be due to the extensive reaction of GSSH and MeHg, leading to unidentified product(s). These results indicate that MeHg is also capable of interacting with these different persulfide-containing species, resulting in (MeHg)2S formation. CSE Is Required for in Vivo Formation of (MeHg)2S. CSE enzyme catalyzes the formation of persulfide species via initial CysSSH generation, 14 and therefore, (MeHg)2S formation was examined by incubating 5 mM MeHg with liver and heart cytosolic fractions prepared from WT and CSE KO mice. Detectable levels of (MeHg)2S were found in the liver/heart fractions of WT mice but not those of CSE KO mice (Figure 2A). Next, we determined the (MeHg)2S concentrations in the brain, heart, and liver cytosols from WT and CSE KO mice after the mice had been administered MeHg intraperitoneally. As shown in Figure 2B, C, and D, the peaks of (MeHg)2S were only detected in WT tissue fractions, indicating that CSE is essential for (MeHg)2S formation in vivo; persulfides may be required for in vivo formation of (MeHg)2S. Identification of Proteins Containing Persulfides. As shown in Table 2, the amount of (MeHg)2S formed in the mouse liver cytosol when dialysis was applied was 42% of the amount formed when dialysis was not applied, indicating that about 42% of the (MeHg)2S formed was attributable to protein-bound persulfides. To investigate this, we fractioned the dialyzed sample through a Cibacron Blue 3GA column. Many proteins were found that could lead to the formation of
(MeHg)2S, but about 50% of the protein-bound persulfides were found in the B-III fraction, which contained four major bands (25-kDa, 42-kDa, 47-kDa, and 117-kDa) on SDS−PAGE (Table 2 and Figure 3A and B). The subsequent separation of the B-III fraction using Sephacryl S-100 column chromatography revealed that fraction no. 49 contained a single 25-kDa protein on SDS−PAGE (Figure 3C and D). We analyzed the highly purified 25-kDa protein by ultrahigh performance LC/ MS and identified it as GSTP1 (Table S1, Supporting Information). We next examined if GSTP1 contained any persulfide adducts. Incubating human GSTP1 with MeHg indeed produced (MeHg)2S, whereas excluding GSTP1 from the incubated mixture caused negligible amounts of (MeHg)2S to form (Figure 4).
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DISCUSSION We previously found that MeHg reacts with NaHS to produce (MeHg)2S,12 but in the study presented here, we found that MeHg is a potent electrophile so that it can be captured by the
Figure 1. Formation of (MeHg)2S from the mixture of MeHg and GSH persulfide (GSSH). Red and blue bars indicate MeHg and (MeHg)2S, respectively. GSSH was generated from GSSSG (0.05 μmol) incubated with GSH reductase (0.1 U), 0.2 mM NADPH, and 20 mM Tris−HCl buffer (pH 7.4) at 25 °C for 5 min, then the reaction mixture (0.5 mL) was reacted with MeHg (2.5 μmol) at 37 °C for 15 min. Aliquots of the reaction mixture were analyzed by (A) HPLC/atomic absorption spectrophotometry and (C) EI-MS to identify the reaction products. (B) Authentic MeHg and (MeHg)2S. 1303
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Chemical Research in Toxicology Table 2. Protein-Bound Persulfide Species As Determined by (MeHg)2S Formation during the Incubation of MeHg with Protein Fractions of Mouse Liver Cytosol Obtained by Cibacron Blue 3GA Column (Upper) and Sephacryl S-100 Column (Bottom) Chromatographies (MeHg)2S formed fraction mouse liver cytosol dialysis B-I B-II B-III
volume (mL)
protein (mg)
(ng/ fraction)
(ng/mg of protein)
36
4200
7360(100)
35 105 72 60
3990 1266 908 33
3085 (42) 327 224 1598 (MeHg)2S formed
yield (%)
4 2 0.3 0.2 48
Figure 4. HPLC/atomic absorption spectrophotometry (AAS) chromatograms of the mixtures in which glutathione S-transferase pi 1 (GSTP1) reacted with MeHg. Red and blue bars indicate MeHg and (MeHg)2S, respectively. Complete incubation mixture (4 nmol GSTP1, 16 nmol MeHg, and 50 mM Tris−HCl buffer (pH 7.5), A) and the mixture without GSTP1 (B) were incubated at 25 °C for 60 min, and then trichloroacetic acid (to give a final concentration of 5% v/v) was added to the reaction mixtures to remove the proteins. After centrifugation (at 9,000g for 5 min), each supernatant was added to 3× the volume of 1 M Tris−HCl buffer (pH 8.5), and this mixture was applied to a Sep-Pak C18 cartridge. The methanol fraction, containing (MeHg)2S, was evaporated to dryness and then redissolved in 10% methanol. The MeHg and (MeHg)2S was detected using HPLC/AAS.
100 11 7 51
fraction
protein (mg)
(ng/fraction)
(ng/mg of protein)
yield (%)
B-III B-III no. 39 no. 45 no. 49 no. 63 no. 69
15 10 1.5 0.6 0.7 0.04 0.1
1707(100) 1095(64) 126 104 116 107 84
115 110 81 149 124 2822 634
100 12 10 11 10 8
earlier preliminary data that another electrophilic probe for persulfides monobromobimane also produced a remarkable amount of bis-S-bimane adduct in the reaction with a protein fraction of A549 cells.14 In the study presented here, the 25kDa protein containing bound persulfides possibly formed on its particular cysteine residues that we isolated as GSTP1, whereas in the previous proteomics analysis using the Tag− Switch−Tag assay GSTP1 was not identified as having persulfide moieties (Table S1, Supporting Information).14 It has already been established that cystathionine β-synthase, CSE, and 3-mercaptopyruvate sulfurtransferase produce persulfide species such as CysSSH and CysSSnSH.14,19 However, when we determined the persulfide contents of a
reactive persulfide species like Na2S4 and GSSH. This also suggests that the formation of (MeHg)2S is a convenient way of determining the presence of these reacitve persulfides in biological samples (Figure S1, Supporting Information). As shown in Table 2, (MeHg)2S was formed even when a dialyzed sample of mouse liver cytosol was reacted with MeHg, suggesting that MeHg can react with protein-bound persulfide residues. Consistent with this, numerous proteins were able to react with MeHg leading to (MeHg)2S formation (Figure 3A). This supports our previous observations made using the Tag− Switch−Tag assay and is in complete accordance with our
Figure 3. Separation of protein-bound persulfides using Cibacron Blue 3GA and Sephacryl S-100 columns. Dialyzed mouse liver cytosol (3990 mg of protein) was applied to a Cibacron Blue 3GA column, and the proteins were eluted with buffer I, then buffer II, and then buffer III (A and B; see the Materials and Methods section). The fraction (containing 10 mg of protein) that was eluted with buffer III was then applied to a Sephacryl S-100 column, and the proteins were eluted with 50 mM phosphate buffer (pH 7.4) (C and D). The (MeHg)2S formation activity in each fraction of column chromatography was examined (A and C). Protein contents were separated by SDS−PAGE and stained with Coomassie Brilliant Blue (B and D). 1304
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Chemical Research in Toxicology Notes
variety of mouse tissues as evaluated by the amount of (MeHg)2S formed, we found that little (MeHg)2S was formed (1) during the reaction of liver and heart extracts from CSE KO mice with MeHg in vitro (Figure 2) and (2) in tissue preparations from MeHg-injected CSE KO mice. These suggest that CSE-derived reactive persulfide species mainly participates in the formation of (MeHg)2S in vivo. We previously reported that the Keap1/Nrf2 pathway may be crucial to reduce MeHg-mediated toxicity because glutamate-cysteine ligase and MRPs are associated with the formation of MeHg−SG adducts and the excretion of this polar metabolite into the extracellular space.7,8,20 However, our recent observations suggest that the MeHg−SG adduct formed with MeHg is fairly unstable and readily undergoes Stransmercuration by thiol-containing proteins, resulting in the proteins also being modified.21 These results suggest that the Keap1/Nrf2 is not the sole factor repressing MeHg toxicity because exposure of SH-SY5Y cells to the MeHg-SG adduct caused a concentration-dependent cytotoxicity. In contrast, (MeHg)2S itself was not toxic to mice when injected intraperitoneally.12 Taken together, it seems likely that the capture of MeHg by reactive sulfide derivatives formed endogenously to produce (MeHg)2S plays a key role in detoxifying MeHg. While GSH is abundant (>1 mM) in a range of tissues, it has a pKa of 9.12 which indicates that the free anionic GS− is a minority species at physiological pH. Cysteine and GSH hydropersulfides (RSSH) have significantly lower pKa values compared to those of the corresponding thiols, indicating that the anionic species will be at higher concentrations.22 Therefore, reactive cysteine persulfide generation, rather than the Keap1/Nrf2 pathway, may be the primary system for defending against MeHg by scavenging these electrophiles that would otherwise covalently modify proteins through their thiol groups. In summary, (MeHg)2S is produced as a result of MeHg capturing a mobilized sulfur atom from endogenous persulfidecontaining species, such as CysSSH, CysSSnSH, GSSH, and GSSnSH. Since persulfides have high nucleophilicity and antioxidant capability, substantial (MeHg)2S formation is associated with a reduction of nucleophilic/reductive cellular status. Thus, it is proposed that sulfide species protects cells from electrophilic insult and that excessive exposure to electrophilic species may deplete sulfide species, leading to a greater susceptibility to electrophile-dependent toxicity.
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The authors declare no competing financial interest.
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ABBREVIATIONS AAS, atomic absorption spectrophotometry; CSE, cystathionine γ-lyase; GSTP1, glutathione S-transferase pi 1
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ASSOCIATED CONTENT
S Supporting Information *
Detailed LC/MS procedure, a proposed pathway for the formation of (MeHg)2S, and the peptide identification data. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.5b00101.
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REFERENCES
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AUTHOR INFORMATION
Corresponding Author
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[email protected]. Funding
This work was supported by a grant-in-aid (#25220103 to Y.K.) for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. 1305
DOI: 10.1021/acs.chemrestox.5b00101 Chem. Res. Toxicol. 2015, 28, 1301−1306
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DOI: 10.1021/acs.chemrestox.5b00101 Chem. Res. Toxicol. 2015, 28, 1301−1306
