The Metabolic Mechanism of Aryl Phosphorus Flame Retardants by

Subscriber access provided by Université de Strasbourg - Service Commun de la Documentation

Ecotoxicology and Human Environmental Health

The Metabolic Mechanism of Aryl Phosphorus Flame Retardants by Cytochromes P450: A Combined Experimental and Computational Study on Triphenyl Phosphate Quan Zhang, Shujing Ji, Lihong Chai, Fangxing Yang, Meirong Zhao, Weiping Liu, Gerrit Schüürmann, and Li Ji Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03965 • Publication Date (Web): 13 Nov 2018 Downloaded from http://pubs.acs.org on November 14, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36

Environmental Science & Technology

1

The Metabolic Mechanism of Aryl Phosphorus Flame Retardants by

2

Cytochromes P450: A Combined Experimental and Computational

3

Study on Triphenyl Phosphate

4

Quan Zhang1, Shujing Ji2, Lihong Chai2, Fangxing Yang2, Meirong Zhao1, Weiping Liu2, Gerrit

5

Schüürmann3,4, and Li Ji*,2

6 7 8

1 College

2 College

9 10

13

of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058, China

3 UFZ

11 12

of Environment, Zhejiang University of Technology, Hangzhou 310032, China

Department of Ecological Chemistry, Helmholtz Centre for Environmental Research, Permoserstrasse 15, 04318 Leipzig, Germany

4

Institute for Organic Chemistry, Technical University Bergakademie Freiberg, Leipziger Strasse 29, 09596 Freiberg, Germany

14 15 16 17 18

ACS Paragon Plus Environment

1


Page 2 of 36

19

Abstract

20

Understanding the metabolic mechanisms is critical and remains a difficult task in risk assessment

21

of emerging pollutants. Triphenyl phosphate (TPHP), one of the widely-used aryl phosphorus

22

flame retardants (aryl-PFRs), has been frequently detected in the environment, whose major

23

metabolite was deemed as diphenyl phosphate (DPHP). However, knowledge of the mechanism

24

for TPHP leading to DPHP and other metabolites is lacking. Our in vitro study shows TPHP is

25

metabolized into its diester metabolite DPHP, and mono-, di-hydroxylated metabolites by

26

cytochromes P450 (CYP) in human liver microsomes, while CYP1A2 and CYP2E1 isoforms are

27

mainly involved in such process. Molecular docking gives the conformation for TPHP binding

28

with the active species Compound I (an iron IV-oxo heme cation radical) in specific CYP isoforms,

29

showing the aromatic ring of TPHP is likely to undergo metabolism. Quantum chemical

30

calculations have shown the dominant reaction channel is O-addition of Compound I onto the

31

aromatic ring of TPHP, followed by hydrogen-shuttle mechanism leading to ortho-hydroxy-TPHP

32

as the main mono-hydroxylated metabolite; the subsequent H-abstraction/OH-rebound reaction

33

acting on ortho-hydroxy-TPHP yields the meta- and ipso-position quinol intermediates, while the

34

former of which can be metabolized into di-hydroxy-TPHP by fast protonation, and the latter

35

species needs to go through type I ipso-substitution and fast protonation to be evolved into DPHP.

36

We envision the identified mechanisms may give inspiration for studying the metabolism of

37

several other aryl-PFRs by CYP.

38 39 40 41


2

Page 3 of 36

42


1. Introduction

43

In response to the phase-out of polybrominated diphenyl ethers (PBDEs) as flame retardants

44

in the mid-2000s,1 there is growing interest in development for appropriate alternatives of PBDEs,

45

such as phosphorus flame retardants (PFRs).2 The production of PFRs has exploded in recent years,

46

e.g. the output of PFRs reached 100,000 tons in China in 2011 and increased by 15% per year,

47

leading to substantial PFRs releasing into the environment.3 The rapid increase of PFRs in the

48

environment takes more and more attention on their effects concerning with ecotoxicology and

49

human health. PFRs have been shown to potentially cause adverse reproductive, endocrine and

50

systemic effects in animals owing to long-term exposure.2 For instance, tris(2-chloroethyl)

51

phosphate (TCEP) and tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) are suspected

52

carcinogens,4 and triphenyl phosphate (TPHP) may cause hepatotoxicity and reduced fertility,5-7

53

which showed the strongest estrogenic activity among nine PFRs in our previous in vitro study.8

54

Biotransformation, especially by phase I metabolizing enzymes cytochromes P450 (CYP),

55

may produce metabolites with substantially altered toxicological and physicochemical profiles.9

56

Several studies have shown PFRs can be easily metabolized in humans,10 while early studies

57

revealed some PFR metabolites, e.g. the metabolites of TDCIPP, were mutagenic in vitro.11 The

58

diesters resulting from O-dealkylation metabolism were characterized as the primary metabolites

59

of PFRs in urine samples of humans through several biomonitoring studies,10 such as diphenyl

60

phosphate (DPHP), bis(2-chloroethyl) phosphate (BCEP) and bis(1,3-dichloro-2-propyl)

61

phosphate (BDCIPP), metabolized from TPHP, TCEP and TDCIPP, respectively, as shown in

62

Table S1 in the Supporting Information (SI). Previous in vitro studies showed TPHP as one of

63

typical aryl-PFRs could be decomposed into DPHP by both cytochromes P450 (CYP) and

64

arylesterase in microsomes,12,13 while the formation of diester metabolites only involved CYP


3


Page 4 of 36

65

rather than hydrolase for alky-PFRs, such as TCEP and TDCIPP.13 In addition to diester metabolite

66

formation, it was reported that the C-hydroxylation pathway was another primary metabolic

67

pathway for TPHP exposed to chicken embryonic hepatocytes,6,14 as well as tris(2-butoxyethyl)

68

phosphate (TBOEP) and tris(n-butyl) phosphate (TNBP) in fish liver microsomes;15 especially,

69

some new evidences suggested that the C-hydroxylation metabolites of diverse aryl- and alky-

70

PFRs produced by CYP in human liver microsomes were significant metabolites as well.13

71

Therefore, different pathways such as O-dealkylation and C-hydroxylation may compete for the

72

biotransformation of PFRs in human liver microsomes involving CYP as the major enzyme system,

73

and understanding the mechanisms of relevant pathways is undoubtedly essential to access the fate

74

and toxicity of PFRs.

75

The enzyme-mediated metabolic mechanism can be addressed through two procedures: 1)

76

how does the molecule enter into the active site of CYP (structure-based); 2) then how does CYP

77

activate the molecule resulting in bond breaking or forming to undergo metabolism (reactivity-

78

based).9 CYP comprise a superfamily of enzymes, although it was shown CYP3A4 and CYP1A

79

were the primary CYP isoforms for metabolizing TBOEP and TNBP in fish liver microsomes,15

80

the specific CYP isoforms involved in metabolism of PFRs in human liver microsomes have not

81

been identified yet. Chemical inhibitors have been frequently applied to define the catalytic

82

specificity of CYPs. The major advantage of using selective inhibitors of individual CYPs is that

83

the fractional inhibition of a reaction in microsomes indicates the extent to which particular CYP

84

isoform is responsible for a reaction.16 The experimental identification of specific CYP isoform

85

responsible for metabolizing PFRs will provide the feasibility for using a structure-based method

86

such as molecular docking to determine the proximity of ligand atoms in a pose to the active

87

species of CYP.9


4

Page 5 of 36


88

The active species of CYP, an iron(IV)-oxo heme cation radical, called Compound I (Cpd I),

89

as shown in Figure 1. Due to the transient nature of Cpd I,17,18 many details of its catalytic

90

mechanism are not accessible with experimental means, although many advanced analytical

91

methods comprise the workhorse for detecting metabolites.19 Nevertheless, quantum chemical

92

calculation as the reactivity-based method, able to simulate the electronic structure of the reaction

93

system, allow rationalization of the reaction mechanisms relevant to diverse experimentally

94

detected metabolites at the molecular level.20-27 This method has already been used to reveal

95

several long-term unsolved mechanisms of CYP-mediated metabolic reactions, such as

96

hydroxylation of C−H bonds,28-30 epoxidation of C=C bonds,31,32 oxidation of aromatic rings,33-35

97

and oxidation of heteroatoms.36-40 Especially, in recent years, the potential of quantum chemical

98

calculation applied in environmental chemistry has attracted wide concern, which has been already

99

performed to provide the molecular-level insight into CYP-mediated metabolic process of some

100

widely-concerned emerging pollutants, such as 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47),41,42

101

atrazine,43 BPA and its analogues.44

102

103 104

Figure 1. Cpd I of CYP (extract of the active site of CYP1A2 as taken from the 2HI4 PDB file

105

visualized by VMD program45)

106

Therefore, there is enormous potential for synergy in experiments closely integrated with

107

computations, which may allow the analysis and prediction of metabolism to make a leap forward.


5


Page 6 of 36

108

The main purpose of this work is to reveal the molecular mechanism of the CYP-mediated

109

metabolism of TPHP in human liver microsomes in vitro, as TPHP is one of the prominent aryl-

110

PFRs detected in environment. Firstly, the advanced analysis methods were performed to identify

111

the metabolites of TPHP incubated in human liver microsomes, and the specific inhibitors of CYP

112

isoforms were used to figure out the individual CYP isoforms responsible for the metabolism of

113

TPHP. Secondly, TPHP was docked into the specific CYP isoforms responsible for TPHP

114

metabolism to know the potential reactive moiety from the optimal binding mode of TPHP with

115

the active species of CYP, and the quantum chemical calculations were done to obtain the

116

fundamental electronic drivers that govern diverse reaction pathways relevant for TPHP

117

metabolized by CYP. Although the multiple in vitro metabolic pathways of TPHP have been

118

reported across different species,6,13-15 to the best of our knowledge, this is the first combined

119

experimental and computational study to report new reaction mechanisms which can rationalize

120

the complex metabolic pathways of aryl-PFR by CYP in human liver microsomes at the molecular

121

level, especially to reveal the long-term unsolved mechanism of O-dealkylation with resistant C-

122

O bond scission for formation of the frequently detected diester metabolite DPHP.

123 124

2. Materials and Methods

125

2.1 Experimental Section

126

2.1.1 Chemicals and Reagents

127

Pooled human liver microsomes (50 donors, mixed gender) were purchased from BD

128

Biosciences (San Jose, CA). β-nicotinamide adenine dinucleotide 2’-phosphate sodium salt

129

hydrate (NADP), glucose-6-phosphate Dehydrogenase (G-6-P-DH), d-glucose 6-phosphate

130

solution, pilocarpine, ticlopidine, quinindium, sodium diethyldithiocarbamate, and ketoconazole


6

Page 7 of 36


131

were obtained from Sigma-Aldrich (Bornem, Belgium). Sulfaphenazole was offered by Dr.

132

Ehrenstorfer GmbH (Augsburg, Germany). TPHP was purchased from J&K Chemical (Beijing,

133

China), and HPLC-grade acetonitrile was obtained from TEDIA (Tedia Company, USA).

134 135

2.1.2 in Vitro Metabolism Assay

136

To identify the main metabolites of TPHP, the preparation of the biotransformation system

137

of human liver microsomes referenced the general procedures in previous publications.13,46 The

138

final volume of the incubation system was 750 μl, which contained 50 mM TRIS buffer (pH=7.4,

139

37 °C), 0.5 mg/ml (final protein concentration) human liver microsomes, 4 μM TPHP. After 6 min

140

pre-incubation, then the reaction was initiated by adding NADPH regeneration system (1.3 mM

141

NADP, 3.3 mM G-6-P,1 U/ml G-6-P-DH, 3.3 mM MgCl2), and the final concentration of organic

142

solvent did not exceed 0.2%.13 To monitor the metabolic process of TPHP, the incubation times

143

are 0, 8, 16, 24, 32, 40, 48, 56 min respectively. Reaction was terminated by 750 μl ice-cold

144

acetonitrile as well as sample was centrifuged for 8 min at 14000 xg. Then the solution was

145

extracted with ethyl acetate and evaporated by N2. Before sample injection, it was filtered through

146

a disc filter (0.2 μm, Waters, USA). The control experiments, such as enzyme negative control

147

without human liver microsomes, negative control using heat-deactivated microsomes (heating at

148

100 degree for 5 min), NADPH negative control, and blank control without TPHP, were performed

149

to ensure the reliability of the experimental results. All incubations were performed in at least

150

triplicate.

151 152

2.1.3 CYP Enzymes Inhibition Assay

153

To clarify the contribution of human CYP isoforms to the metabolism of TPHP, the effect of

154

specific inhibitors on the ration of TPHP metabolism were performed. The substrate concentration

155

is 4μM. We determined the inhibitory effect of specific inhibitors α-naphthoflavone (α-NF 1 μΜ


7


Page 8 of 36

156

the specific inhibitor for CYP1A2), pilocarpine (PIL 1 μΜ,the specific inhibitor for CYP2A6),

157

sulfaphenazole (SUL 10 μΜ, the specific inhibitor for CYP2C9), ticlopidine (TIC 10 μΜ, the

158

specific inhibitor for CYP2C19), quinidine (QUI 1 μΜ, the specific inhibitor for CYP2D6),

159

ketoconazole (KET,1 μΜ the specific inhibitor for CYP3A4), sodium diethyldithiocarbamate (DIE

160

50 μΜ, the specific inhibitor for CYP2E1).47 All concentrations were selected on the basis of

161

previously reported values to insure enough inhibitory selectivity and maximal inhibitory

162

efficiency. Except the mechanism-based inhibitors diethyldithiocarbamate and ticlopidine

163

preincubated with human liver microsomes for 10 min, other inhibitors were for 6 min, all other

164

conditions were prepared as described above. All incubations were performed in at least triplicate.

165 166

2.1.4 Metabolites Analysis

167

The major metabolites of TPHP were identified by liquid chromatography-mass

168

spectrometry/mass spectrometry (LC-MS/MS) using a Waters Xevo TQ-S (Waters, USA) with a

169

Waters BEH C18 column (2.1 mm × 50 mm, 1.7 μm). The chromatographic conditions were

170

referred to previous report.48 In addition, triple TOF 5600 (AB Sciex, Framingham, USA) was

171

applied for qualitative measurement of the major metabolites. The following chromatographic

172

parameters were used: A 5 μl sample was injected into a Waters BEH C18 column (2.1 mm × 50

173

mm, 1.7 μm). The flow rate was 0.3 mL/min and the composition were 0.1% formic acid in

174

acetonitrile (A) and water (B). Gradient elution was as follows: 0 min 95% B, 0.5 min 95% B, 10

175

min 5% B, 10.5 min 95% B, 13 min 95% B. MS/MS was operated in the negative and positive

176

electrospray mode with a mass range from m/z 100 to 500 amu for detection of metabolites of

177

TPHP. The following MS parameters were used: source temperature 550 °C, desolvation 50 L/h,

178

auxiliary gas atomization 50 L/h, the atomization gas 50 L/h, cutain gas 30 L/h. Capillary voltage

179

and declustering potential voltage were set to 5.5 kV and 100 V, respectively. Besides, the


8

Page 9 of 36

180


Collision energy was 35 eV.

181

The metabolites ionized to (M + H)+ or (M+H)- species were affirmed by MS data analysis

182

software (Peakview 1.2). Statistical analysis was performed using ORIGIN 9.1 (OriginLab,

183

Northampton, MA). One-Way ANOVA was used to assess the significant difference between

184

control and inhibitory group. Data was expressed as mean ± standard deviations (SD), and

185

differences of means were considered statistically significant when p< 0.05.

186 187

2.2 Computational Section

188

2.2.1 Molecular Docking

189

Molecular docking was carried out in the Discovery Studio 2.5 (DS 2.5; Accelrys, San Diego,

190

USA). The crystal structures of CYP were obtained from the Protein Data Bank

191

(http://www.rcsb.org): CYP1A2 (PDB ID: 2HI4), CYP2E1 (PDB ID: 3LC4), CYP2D6 (PDB ID:

192

4WNV), CYP2C9 (PDB ID: 4NZ2), CYP2A6 (PDB ID: 1Z10), and CYP2C19 (PDB ID: 4GQS).

193

Hydrogen atoms were added to the initial CYP enzymes, especially the charged residues (Arg,

194

Asp, Lys, Glu) were neutralized to cause an overall charge of zero for each system. The 3D

195

structure of each CYP on resting state was applied with the force field of CHARMM, after the

196

crystallographic water molecules removed from each CYP structure. Together, the 3D structure of

197

TPHP was sketched and energy minimized using the protocol of minimization. Then, molecular

198

docking was performed to simulate the binding of TPHP to CYP isoforms using CDOCKER. In

199

CDOCKER, 10 confirmations of TPHP for each CYP isoform were generated with the default

200

parameters. As shown in Table S5 in the SI, the -CDOCKER_INTERACTION_ENERGY of the

201

first-ranked TPHP confirmation was applied to reflect the interaction between TPHP and each

202

CYP isoform.

203 204

2.2.2 Quantum Chemical Calculations


9


Page 10 of 36

205

The same as previous studies,37,44 Cpd I of CYP active site was modeled as the six-

206

coordinated triradicaloid oxoferryl complex Fe4+O2−(C20N4H12)−1(SH)−1. The reactions were only

207

reported in the doublet-spin state in this work, since the former calculations on Cpd I oxidation of

208

benzene systems showed that the barriers of rate-determining steps for Cpd I in the quartet-spin

209

state were remarkably higher than in the doublet-spin state by about 3.0 kcal/mol,33 and our

210

calculations in this work also ruled out the unfavorable quartet-spin pathway for both the mono-

211

and di-hydroxylation of TPHP as discussed in details in Figure S2 in the SI. All of the calculations

212

for geometry optimizations and frequency analyses were carried out using the unrestricted hybrid

213

B3LYP (UB3LYP) density functional49,50 in combination with the LANL2DZ51 basis set on iron

214

and 6–31G** basis set on other atoms (denoted BSI). The B3LYP exchange-correlation functional

215

was chosen since it had been shown to accurately reproduce experimentally measured kinetic

216

isotope effects for CYP-catalyzed reactions,52 and electron paramagnetic resonance parameters for

217

penta-coordinated heme in CYP enzyme,53 yield geometries in good agreement with experimental

218

crystal structures,54 and have qualitatively accurate relative energies vs benchmark CASSCF

219

calculations.55 The computed vibrational frequencies were further used to quantify the zero-point

220

energy correction (ZPE) and thermal and entropic corrections to the free energy at 298.15 K and

221

101.325 kPa. All ground states were confirmed by the presence of only real frequencies, whereas

222

the transition states had one imaginary frequency. More accurate energies were obtained using

223

single-point calculations with the SDD basis set56 on iron and the 6–311++G** basis set for all

224

other atoms (denoted BSII). Bulk polarity effects were evaluated with the PCM continuum-

225

solvation model57 (ε = 5.6, chlorobenzene) at the UB3LYP/BSI level of theory. Dispersion

226

interactions were included by performing single-point energy calculations with the UB3LYP-

227

D3/BSI level of theory because B3LYP lacks such interactions.58


10

Page 11 of 36


228

We also did full geometry optimizations for transition states at the UBLYP-D3/BSI level of

229

theory to test the reliability of the above approach, the results of which gave only minor geometric

230

differences to those obtained with UB3LYP/BSI optimizations (detailed comparisons see Table

231

S6 in the SI). Moreover, the quantum cluster approach was performed to verify the reaction

232

mechanisms through treating the active site of CYP through comprising important ambient amino

233

acids fully quantum mechanically,21 which are described in details in the SI. Based on our

234

experiments in this work, TPHP was mainly catalyzed by CYP1A2 and CYP2E6, and thus we

235

used CYP1A2 protein structure (PDB code: 2HI4) to yield a large cluster model (Figure S3 in the

236

SI); the results showed that the large cluster model was in good agreement with the small model

237

on the mechanisms, especially the geometric constraints near the active site of CYP1A2 did not

238

restrict the preferred pathways obtained from the small model (Figure S4 in the SI). Furthermore,

239

it was shown that the prediction model based on reactivity gives good results for prediction of

240

metabolism mediated by CYP isoforms 1A2 and 2E1, indicating that the reactivity is the most

241

important factor for determining the sites of metabolism and structures of metabolites for certain

242

moiety of substrates accessible to Cpd I.59,60 We thus performed quantum chemical calculations in

243

this work based on the small Cpd I model of CYP.

244

Therefore, the relative free energies of the CYP oxidation reactions shown below were

245

estimated by combining B3LYP/BSII single-point energies with PCM solvation and dispersion

246

corrections, as well as Gibbs free energy corrections from optimizations at the BSI level, unless

247

pointed out specifically. All quantum chemical calculations were performed with the Gaussian 09

248

D.01 program.61

249 250

3. Results and Discussions

251

3.1 TPHP Metabolism in Human Liver Microsomes


11


Page 12 of 36

252

As shown in Figure S1 in the SI, the initial concentration of TPHP was 4.00 μM, while such

253

concentration fell to 2.55 μM after 15 min of incubation. By the incubation period of 60 min, only

254

0.66μM of TPHP was detected, thus the half-life for the metabolism is 23.1 min. In the human

255

liver microsomes incubations, we observed a number of hydroxylated metabolites in the presence

256

of NADPH. Figure 2 shows the identified metabolites of TPHP formed in human liver microsomes

257

through LC-MS/MS method, which are a mono-hydroxylated metabolite, a di-hydroxylated

258

metabolite, and a diester metabolite (DPHP), although some previous in vitro studies reported

259

DPHP as the only metabolite of TPHP in human liver microsomes.12,62 In the meanwhile, no

260

metabolites were detected in negative control using heat-deactivated microsomes, which

261

demonstrates that enzyme-catalyzed metabolism is the dominant pathway as opposed to

262

nonenzymatic hydrolysis within relatively short-time scales of enzyme-catalysis. More

263

importantly, there are no metabolites detected in NADPH negative control samples, while addition

264

of NADPH is the necessary condition to activate CYP in human liver microsomes, which points

265

out the prominent role of CYP in catalytic production of mono-hydroxylated, di-hydroxylated, and

266

diester metabolites simultaneously during the metabolism of TPHP in human liver microsomes.

267

Note previous findings suggested the formation of diester metabolite DPHP from TPHP in human

268

liver microsomes depended on both the contribution from CYP and hydrolase,12,13 herein the

269

catalytic function of CYP for production of diester metabolite has been further strengthened.

270

However, there are two mechanistic puzzles concerning about CYP reactivity in experiments: 1)

271

it is difficult to identify the specific conformations of metabolites regarding the position of

272

hydroxylation derived by mass spectrometry, and thus difficult to quantitatively determine the

273

proportions of different conformations of metabolites; 2) CYP has already been proved to play an

274

important role in DPHP formation, but the C-O bond attaching to the aromatic ring of TPHP is


12

Page 13 of 36


275

generally resistant, yet the reaction mechanism for CYP eliminating the associated C-O bond

276

cleavage of TPHP with formation of DPHP has not been resolved. OH

O O P O O

mono-hydroxylated metabolite

O

O

OH

O

P

P

O

O

O

O

triphenyl phosphate (TPHP)

diphenyl phosphate (DPHP)

OH

OH

O O P O O

277

di-hydroxylated metabolite

278

Figure 2. The identified main metabolites of TPHP catalyzed by CYP in human liver microsomes

279

through LS-MS/MS method

280 281

3.2 Inhibition of TPHP Metabolism by Specific CYP Inhibitors

282

For inhibition studies, we investigated the in vitro metabolism of TPHP by human CYP

283

involving mostly common isoforms such as CYP1A2, CYP2E1, CYP2A6, CYP2C9, CYP2D6,

284

CYP2C19, and CYP3A4 using their inhibitors. The TPHP clearance ratios with different inhibitors

285

is shown in Figure 3, which demonstrates that α-NF (specific inhibitor for CYP1A2) and DIE

286

(specific inhibitor for CYP2E1) exerted the strongest inhibitory effect on the metabolism rate of

287

TPHP (up to 30% and 40% of control activity), while PIL (specific inhibitor for CYP2A6), SUL

288

(specific inhibitor for CYP2C9), and QUI (specific inhibitor for CYP2D6) inhibited the reaction


13


Page 14 of 36

289

up to 60% or so. The TIC (specific inhibitor for CYP2C19) and KET (specific inhibitor for

290

CYP3A4) exhibited no inhibitory effect or only marginally inhibited the metabolism of TPHP.

291

Therefore, we find that CYP1A2 and CYP2E1 play important roles in the TPHP metabolism in

292

human liver microsomes, especially CYP1A2 seems the most relevant CYP isoforms involving in

293

the metabolism of TPHP. Many previous studies have shown that CYP1A2 exhibited much higher

294

activities for phenacetin and alkoxyresorufin O-dealkylation as well, and especially O-dealkylation

295

of phenacetin is a well-known marker reaction of CYP1A2 activity.63

296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315

Figure 3. The TPHP clearance ratios with different inhibitors referenced with the control

316

experiment in human liver microsomes (* Statistical significance of p < 0.05, ** Statistical significance of

317

p < 0.01, *** Statistical significance of p < 0.001)

318 319

3.3 Molecular Docking

320

To further account for above results of experimental inhibition, the frequently used structure-

321

based method in studying molecular metabolism, i.e. molecular docking, has been conducted to

322

obtain the optimal orientation of TPHP in specific CYP isoforms. The crucial cofactor of CYP is


14

Page 15 of 36


323

a heme group, it is clear that the substrate needs to get close enough to the reactive center to

324

undergo a reaction. A very simple rule of thumb that may be applied is the “6Å rule,” which states

325

that the site of metabolism is likely to be within 6Å of the heme iron.9 Table S5 in the SI shows

326

the interaction energy and geometry results for TPHP docking into the CYP pocket. All distances

327

between TPHP and the heme Fe of each CYP (4.319 Å for CYP1A2, 4.758 Å for CYP2E1, 5.286

328

Å for CYP2D6, 5.8227 Å for CYP2C9, 3.059 Å for CYP2A6, and 5.463 Å for CYP2C19) are less

329

than 6 Å. However, it is evident that CYP1A2 and CYP2E1 are more efficient catalysts toward

330

TPHP, with shorter distances between TPHP and the heme Fe. Although there is the shortest

331

distance

332

CDOCKER_INTERACTION_ENERGY for CYP2A6 (-32.577 kcal/mol) indicates the large

333

instability between TPHP and the CYP2A6. As shown in Figure 4, one aromatic ring of TPHP is

334

very close to the heme iron for both CYP1A2 and CYP2E1, which is the most likely reactive moiety

335

of metabolism. Note that the ortho-, meta- and para-carbon of the aromatic ring of TPHP in

336

CYP1A2 and CYP2E1 protein pockets are all within 6Å of the heme iron, thus all these three

337

positions are likely to be the sites of metabolism (SoMs), however, the ortho-carbon outside 6Å

338

of the heme iron rather than the para- and meta-carbon of the aromatic ring is not the feasible SoM

339

for TPHP in the protein pockets of other CYP isoforms. Such a prediction based on structure-

340

based principle can provide the first general suggestion as to which SoMs of the molecular is likely

341

to be accessible for a reaction to occur, whereas the reactivity of this moiety determines which

342

products can be formed afterwards.

between

TPHP

and

the

heme

Fe

of

CYP2A6,

the

low

value

of

-

343 344 345 346 347 348 349


15


Page 16 of 36

350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373

Figure 4. The interaction between TPHP and the heme in CYP1A2 (A) and CYP2E1 (B) through

374

molecular docking along with the shortest distance (Å) between TPHP and the heme Fe

(A)

(B)

375 376

3.4 Quantum Chemical Calculations

377

Mono-Hydroxylation of TPHP by CYP. Previous studies ruled out the H-abstraction/OH-

378

rebound mechanism as viable alternative for benzene hydroxylation due to the extremely high H-

379

abstraction barriers,33 which was verified in this work again that the H-abstraction from the para-

380

carbon of the aromatic ring of TPHP by CYP was nearly 30 kcal/mol, thus we only focused on the

381

mechanisms of O-addition on the aromatic ring in this work. Figure 5 shows the potential energy

382

profile for the hydroxylation pathway of the aromatic ring of TPHP by CYP (aromatic ring is the

383

moiety nearest to Cpd I in CYP pocket as revealed by molecular docking). The initial and rate-

384

determining step involves electrophilic addition of the oxo group of Cpd I onto the unsubstituted


16

Page 17 of 36


385

aromatic ring of TPHP via C−O bond-forming transition states (TSO), leading to an σ-complex

386

intermediate. As stated above, all the ortho-, meta- and para-carbon of the aromatic ring of TPHP

387

in CYP1A2 and CYP2E1 pockets are likely to be the SoMs. The reaction barriers for O-addition

388

at the ortho-position (TSOo: 16.0 kcal/mol) are lower than such values for O-addition at meta-

389

position (TSOm: 20.2 kcal/mol) and para-position (TSOp: 20.4 kcal/mol). Introduction of these

390

values into the Eyring equation (see details in the SI) yielded a preponderance of O-addition at the

391

ortho-position over the para- and orthro-position by roughly a factor of 1700:1. The much more

392

favorable O-addition intrinsic reactivity on the ortho-carbon than para-carbon further explains

393

why CYP1A2 and CYP2E1 play important roles in the TPHP metabolism in human liver

394

microsomes, that their active site are accessible to the most reactive ortho-carbon. As shown in

395

Figure 5, the aromatic ring of TPHP is almost perpendicular to the porphyrin ring (side-on) for

396

TSOo. Furthermore, the O-addition at the meta-position and para-position has reaction energies of

397

-5.8 and -5.7 kcal/mol respectively, whereas such exothermic reaction at the ortho-position is

398

energetically more favorable by ca. 7 kcal/mol. Thus, the major mono-hydroxylation metabolite of

399

TPHP by human CYP1A2 and CYP2E1 originates from oxidation of ortho-position of the aromatic

400

ring of TPHP through both the kinetic and thermodynamic analysis, although the obtained energy

401

difference would translate into a still lower fraction of the products from O-addition at the para-

402

and meta-positions.

403

As can be seen in Figure 5, starting from σ-complex intermediate with O-addition at the

404

ortho-position (IMOo), one of the nitrogen atoms of the porphyrin ring of Cpd I abstracts the ortho

405

hydrogen from the tetrahedral carbon of the σ-complex with formation of the protonated porphyrin

406

complex (IMpp), which is a favorable exothermic reaction with reaction energy of -27.6 kcal/mol.

407

This hydrogen-transfer process is essentially barrierless, which should have a very small barrier


17


Page 18 of 36

408

that could not be localized precisely due to the extreme flatness of the surface. An interesting

409

feature of IMPP is the proximity of the hydrogen end of the N-H bond to the oxygen atom of the

410

phenoxyl moiety with O···H distance of only 1.793 Å. As such, with significant reaction energy

411

of -14.2 kcal/mol, the protonated porphyrin species can shuttle the hydrogen back to the oxygen

412

atom through an essentially barrierless manner with production of the complex POH with the major

413

mono-hydroxylation metabolite ortho-hydroxy-TPHP and the ferric iron porphyrin.

414

Note it was reported that TPHP was mainly metabolized into the para-hydroxy-TPHP using

415

a chicken embryonic hepatocyte assay;14 although the specific protein structure of the CYP

416

involved in such metabolism is unknown yet, the main reason for no ortho-hydroxy-TPHP

417

formation in this chicken embryonic hepatocyte assay can be deduced that the ortho-carbon of the

418

aromatic ring of TPHP is outside 6Å of the heme iron in the relevant CYP pocket leading to this

419

site unlikely to be accessible for a reaction to occur, while the para-carbon of the aromatic ring is

420

generally more accessible to the active site in most CYP isoforms.

421 422 423 424 425 426 427 428 429 430


18

Page 19 of 36


431 432

Figure 5. Free energy profile for mono-hydroxylation of TPHP catalyzed by Cpd I of CYP, along

433

with the optimized geometries (geometrical parameters: lengths in Å) of the key reaction species

434

in the doublet spin state. Free energies (kcal/mol) are relative to the reactant complex (Cpd I

435

+TPHP) at the B3LYP/BSII//BSI level including solvation (ε=5.6) and dispersion corrections. For

436

transition state, the imaginary frequency is shown.

437 438

Di-hydroxylation of TPHP by CYP. Figure 6 shows the potential energy profile for the di-

439

hydroxylation pathway of TPHP by CYP, i.e. hydroxylation of ortho-hydroxy-TPHP (major

440

mono-hydroxylation metabolite) by CYP. The reaction starts from the reactant complex (RC), with

441

the phenolic group of ortho-hydroxy-TPHP interacting with the Cpd I iron-oxo moiety through H-

442

bonding. Then, RC can pass through hydrogen abstraction transition states TSH with reaction

443

barrier of just 1.3 kcal/mol, producing an intermediate complex (IMH) with the iron-hydroxo

444

species and the phenoxy radical, which is an exothermic process with reaction energy of -3.9

445

kcal/mol. This hydrogen abstraction transition state is confirmed by nearly linear O…H…O angles

446

(179.0 degree) and high imaginary frequency (i1463 cm-1). The other possible route is the addition

447

of the oxo group of Cpd I onto the aromatic ring of ortho-hydroxy-TPHP. The obtained barriers

448

for O-addition at all unsubstituted positions of aromatic ring of ortho-hydroxy-TPHP are between

449

24.2 and 28.9 kcal/mol. Thus, it is significant that the H-abstraction pathway for ortho-hydroxy-

450

TPHP is much more favorable, whereas the O-addition pathway is ruled out, which is similar to

451

previously revealed metabolic mechanisms of paracetamol and BPA by CYP.39,44

452

Formation of IMH is followed by rebound of the OH radical from iron onto the phenoxy

453

radicals. This occurs via formation of covalent bonds at the ipso-, ortho-, meta-, para-carbon of


19


Page 20 of 36

454

the aromatic ring of ortho-hydroxy-TPHP to produce respective addition quinol intermediates

455

IMipso, IMortho, IMmeta1, IMmeta2 or IMpara. As shown in Figure 6, the essentially barrierless rebound

456

reactions at the ipso-, meta1- and meta2-carbon are exothermic, with reaction energies of -28.0, -

457

23.1 and -27.1 kcal/mol, respectively, while the rebound reactions at the ortho- and para-carbon

458

are endothermic (+2.6 and +9.3 kcal/mol). Therefore, OH radical rebounds under thermodynamic

459

control, and the exothermic reaction energy difference between formation of IMipso, IMmeta1 and

460

IMmeta2, favors IMipso and IMmeta2 formation but also produces minor fraction of IMmeta1.

461 462 463 464 465 466 467 468 469 470 471 472 473

Figure 6. Free energy profile for di-hydroxylation of TPHP (hydroxylation of ortho-hydroxy-

474

TPHP) catalyzed by Cpd I of CYP, along with the optimized geometries (geometrical parameters:

475

lengths in Å, angles in degrees) of the key reaction species in the doublet spin state. Free energies

476

(kcal/mol) are relative to the reactant complex RC at the B3LYP/BSII//BSI level including


20

Page 21 of 36


477

solvation (ε=5.6) and dispersion corrections. For transition states, the imaginary frequencies are

478

shown.

479

It is evident that IMmeta1 and IMmeta2 can further evolve into di-hydroxy-TPHP by fast

480

protonation after they enter into the nonenzymatic environment. However, considering the fate of

481

IMipso, the diester metabolite formed upon C-O bond scission, i.e. DPHP, has been detected in the

482

experiments of CYP-catalyzed metabolism of TPHP, showing ipso-position metabolism of ortho-

483

hydroxy-TPHP will further proceed from its quinol form (IMipso). The quinol intermediate

484

decomposition is an ipso-substitution reaction, which can be categorized into two types depending

485

on the eliminating group, i.e. type I ipso-substitution means that the substituent eliminates as an

486

anion with formation of a quinone, whereas in type II ipso-substitution the eliminating group is a

487

cation with formation of a hydroquinone.64 As shown in Table 1, the heterolytic decomposition of

488

IMipso may proceed charge-neutrally or after protonation or deprotonation in the aqueous

489

environment. All geometries for the decomposition reactions of various ipso-addition quinol

490

intermediates were optimized at the B3LYP/6-311++G** level in water solution (ε = 78.4) with

491

PCM. The computations suggest that the most feasible pathway is the decomposition of IMipso

492

after deprotonation producing a diester anion and a quinone (type I ipso-substitution) with

493

exothermic reaction of -10.4 kcal/mol. Such diester anion can readily evolve into DPHP by fast

494

protonation in a nonenzymatic environment. Furthermore, the decomposition reactions of quinol

495

of para-hydroxy-TPHP was also investigated, as shown in Table S7 in the SI, the most feasible

496

decomposition pathway is type I ipso-substitution leading to a diester anion as well, so the type I

497

ipso-substitution seems a common characteristic for DPHP formation regardless of any mono-

498

hydroxy-TPHP as the precursor. Thus, this study provides a complete mechanistic picture for

499

DPHP formation from TPHP by CYP, which has been a long-term puzzled mechanistic problem.


21


Page 22 of 36

500 501

Table 1. Computed Aqueous-Phase Free Energies (ΔG) (kcal/mol) for the Decomposition

502

Reactions of Quinol of Ortho-Hydroxy-TPHPa Condition

Type I

Type II

P

O

Type I

Type II

+

O

O

P

O

O-

OH O-

O

OH

+

O

O

P

O

O

O

O-

+

P

O

O+

O

O

O

OH OH

O

O

OH OH+

+

O O

67.2

O P

O-

O

P

6.1

O

O

O

O

Protonation

OH

P

O

Deprotonation

O

O

O

O O

O

Neutralization

OH+

O

O

Neutralization

ΔG (kcal/mol)

Elimination Type

-10.4

O P

O+

20.7

503

a The

504

the B3LYP-PCM/6-311++G** level of theory with water solution and free energy corrections.

reported reaction free energies are described by the energies from geometry optimizations at

505 506

4. Environmental Implications

507

Emerging pollutants are subject to biotransformation processes, which may produce

508

metabolites that can differ in their environmental behavior and toxicological profile.65 The

509

development of methods for analyzing and predicting the metabolites of emerging pollutants have

510

become a thriving field of research during past few years, however, identification of such

511

metabolites is challenging, due to their often trace amounts, requiring highly selective and sensitive

512

analytic techniques.66 Thus, understanding the metabolic mechanisms of emerging pollutants to

513

develop corresponding mechanism-based methods for prediction of metabolites will aid in the


22

Page 23 of 36


514

metabolites-oriented analysis in environmental risk assessment. CYP are responsible for most of

515

the phase-I biotransformation,47 however, the vital roles of CYP in biotransformation of substantial

516

emerging pollutants have not been clear yet.

517

The major role of CYP in oxidation of aryl-PFRs has been in doubt, since considering the

518

reactivity of oxidative enzymes in human liver microsomes such as CYP, the formation of the

519

major diester metabolites with resistant C-O bond scission by CYP seems uncommon, thus the

520

hydrolytic enzymes have been naturally considered to be mainly involved in such process. Our

521

experimental study suggests that CYP-mediated oxidation is one of the primary pathways for

522

metabolism of TPHP in human liver microsomes, leading to diester, mono- and di-hydroxylated

523

metabolites together, and CYP1A2 and CYP2E1 isoforms are mainly involved in such metabolic

524

process. It is a common sense that CYP in human liver microsomes can produce the hydroxylated

525

products from oxidation of aromatic ring, while other oxidative enzymes in microsomes involved

526

in such hydroxylation reactions are seldom reported.19,47 It is worth noting that the importance of

527

the hydroxylation pathway was emphasized previously in the CYP-mediated metabolism of TPHP

528

in in vitro assay studies with chicken embryonic hepatocytes,14 while the following studies showed

529

the reduced metabolic depletion of the alkyl-substituted TPHPs relative to TPHP in vitro

530

metabolism in polar bear and ringed seal liver microsomes might originate from that the alkyl

531

substituents on TPHP aryl groups hindered the hydroxylation pathways.67 The computations in

532

this study shows the meta-position quinol intermediate (precursor of di-hydroxy-TPHP) and ipso-

533

position quinol intermediate (precursor of DPHP) can be simultaneously formed through the same

534

reaction mode by CYP from the same parent compound (ortho-hydroxy-TPHP); especially the

535

formation of the latter species is even more thermodynamically favorable (See Figure 6). Thus,

536

this work has carried out the strategy of computations regurgitation-feeding experiments, that the


23


Page 24 of 36

537

computations give powerful support on experiments that CYP in human liver microsomes can

538

really eliminate the C-O bond cleavage of TPHP through ipso-substitution mechanism.

539

Although there have been great efforts to study the fate, occurrence and toxicity of PFRs in

540

the environment,2 there are only few existed studies on their biotransformation mechanisms and

541

toxicological effects of metabolites currently.6,13-15,67,68 The identified metabolic mechanisms of

542

TPHP in this work can be potentially extended to the metabolism of several other aryl-PFRs by

543

CYP, which will enable the screening of metabolites for toxicity assay and biomonitoring. This

544

study further enhances the importance of ipso-substitution mechanism, which was reported to play

545

an important role in biotransformation of many emerging pollutants, such as bisphenol analogues,

546

alkylphenols, and sulfonamide antibiotics.44,69-73 The close integration of experiments with

547

computations herein gives an example that both methods together can lead to synergistic effects,

548

which will be helpful to reveal some unreported and uncommon metabolic mechanisms of

549

substantial emerging pollutants.

550 551

ASSOCIATED CONTENT

552

Supporting Information. The structures of parent PFRs and their diester metabolites reported

553

in the references; detailed experimental data; detailed molecular docking results; free energy

554

profiles for mono- and di-hydroxylation of TPHP catalyzed by CYP in both quartet and doublet

555

spin states; geometrical comparison between B3LYP/BSI and B3LYP-D3 optimized transition

556

states; details for quantum chemical cluster calculations; detailed description of Eyring equation;

557

computed aqueous-phase free energies for the decomposition reactions of quinol of para-hydroxy-

558

TPHP; Mulliken spin densities; energies for all molecular species and Cartesian coordinates of all


24

Page 25 of 36


559

structures from quantum chemical calculations. This material is available free of charge via the

560

Internet at http://pubs.acs.org.

561

Corresponding Author

562

*(L.J.) E-mail: [email protected]

563

Notes

564

The authors declare no competing financial interest.

565 566

ACKNOWLEDGMENT

567

This work was supported by the National Natural Science Foundation of China (NO. 21337005

568

and 21677125). The China National Supercomputing Center in Shenzhen is acknowledged for

569

providing the computing resources.

570 571

REFERENCES

572

(1) Stapleton, H. M.; Sharma, S.; Getzinger, G.; Ferguson, P. L.; Gabriel, M.; Webster, T. F.;

573

Blum, A. Novel and high volume use flame retardants in us couches reflective of the 2005 pentabde

574

phase out. Environ. Sci. Technol. 2012, 46, (24), 13432-13439.

575 576

(2) van der Veen, I.; de Boer, J. Phosphorus flame retardants: Properties, production, environmental occurrence, toxicity and analysis. Chemosphere 2012, 88, (10), 1119-1153.

577

(3) Wang, R. M.; Tang, J. H.; Xie, Z. Y.; Mi, W. Y.; Chen, Y. J.; Wolschke, H.; Tian, C. G.;

578

Pan, X. H.; Luo, Y. M.; Ebinghaus, R. Occurrence and spatial distribution of organophosphate


25


Page 26 of 36

579

ester flame retardants and plasticizers in 40 rivers draining into the bohai sea, north china. Environ.

580

Pollut. 2015, 198, 172-178.

581 582

(4) California office of environmental health hazard assessment proposition 65. Http://oehha.Ca.Gov/prop65.Html (accessed sept 30, 2014).

583

(5) Meeker, J. D.; Stapleton, H. M. House dust concentrations of organophosphate flame

584

retardants in relation to hormone levels and semen quality parameters. Environ. Health Perspect.

585

2010, 118, (3), 318-323.

586

(6) Su, G.; Crump, D.; Letcher, R. J.; Kennedy, S. W. Rapid in vitro metabolism of the flame

587

retardant triphenyl phosphate and effects on cytotoxicity and mrna expression in chicken

588

embryonic hepatocytes. Environ. Sci. Technol. 2014, 48, (22), 13511-13519.

589

(7) Van den Eede, N.; Cuykx, M.; Rodrigues, R. M.; Laukens, K.; Neels, H.; Covaci, A.;

590

Vanhaecke, T. Metabolomics analysis of the toxicity pathways of triphenyl phosphate in heparg

591

cells and comparison to oxidative stress mechanisms caused by acetaminophen. Toxicol. In Vitro

592

2015, 29, (8), 2045-2054.

593

(8) Zhang, Q.; Lu, M. Y.; Dong, X. W.; Wang, C.; Zhang, C. L.; Liu, W. P.; Zhao, M. R. Potential

594

estrogenic effects of phosphorus-containing flame retardants. Environ. Sci. Technol. 2014, 48,

595

(12), 6995-7001.

596

(9) Kirchmair, J.; Goller, A. H.; Lang, D.; Kunze, J.; Testa, B.; Wilson, I. D.; Glen, R. C.;

597

Schneider, G. Predicting drug metabolism: Experiment and/or computation? Nat. Rev. Drug

598

Discov. 2015, 14, (6), 387-404.


26

Page 27 of 36


599

(10) Dodson, R. E.; Van den Eede, N.; Covaci, A.; Perovich, L. J.; Brody, J. G.; Rudel, R. A.

600

Urinary biomonitoring of phosphate flame retardants: Levels in california adults and

601

recommendations for future studies. Environ. Sci. Technol. 2014, 48, (23), 13625-13633.

602 603 604 605

(11) Gold, M. D.; Blum, A.; Ames, B. N. Another flame retardant, tris-(1,3-dichloro-2-propyl)phosphate, and its expected metabolites are mutagens. Science 1978, 200, (4343), 785-787. (12) Sasaki, K.; Suzuki, T.; Takeda, M.; Uchiyama, M. Metabolism of phosphoric acid triesters by rat liver homogenate. Bul. Environ. Contam. Toxicol. 1984, 33, (3), 281-288.

606

(13) Van den Eede, N.; Maho, W.; Erratico, C.; Neels, H.; Covaci, A. First insights in the

607

metabolism of phosphate flame retardants and plasticizers using human liver fractions. Toxicol.

608

Lett. 2013, 223, (1), 9-15.

609

(14) Su, G. Y.; Letcher, R. J.; Crump, D.; Gooden, D. M.; Stapleton, H. M. In vitro metabolism

610

of the flame retardant triphenyl phosphate in chicken embryonic hepatocytes and the importance

611

of the hydroxylation pathway. Environ. Sci. Technol. Lett. 2015, 2, (4), 100-104.

612

(15) Hou, R.; Huang, C.; Rao, K. F.; Xu, Y. P.; Wang, Z. J. Characterized in vitro metabolism

613

kinetics of alkyl organophosphate esters in fish liver and intestinal microsomes. Environ. Sci.

614

Technol. 2018, 52, (5), 3202-3210.

615 616 617 618

(16) Halpert, J. R.; Guengerich, F. P.; Bend, J. R.; Correia, M. A. Selective inhibitors of cytochromes p450. Toxicol. Appl. Pharmacol. 1994, 125, (2), 163-175. (17) Rittle, J.; Green, M. T. Cytochrome p450 compound i: Capture, characterization, and c-h bond activation kinetics. Science 2010, 330, (6006), 933-937.


27


619 620

Page 28 of 36

(18) Guengerich, F. P. Common and uncommon cytochrome p450 reactions related to metabolism and chemical toxicity. Chem. Res. Toxicol. 2001, 14, (6), 611-650.

621

(19) Drug metabolism prediction. In Kirchmair, J., Ed. John Wiley & Sons: Weinheim, 2014.

622

(20) Shaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. P450 enzymes: Their

623

structure, reactivity, and selectivity-modeled by qm/mm calculations. Chem. Rev. 2010, 110, (2),

624

949-1017.

625 626 627 628

(21) Himo, F. Recent trends in quantum chemical modeling of enzymatic reactions. J. Am. Chem. Soc. 2017, 139, (20), 6780-6786. (22) Paneth, P. Chlorine kinetic isotope effects on enzymatic dehalogenations. Acc. Chem. Res. 2003, 36, (2), 120-126.

629

(23) Li, Y.; Shi, X.; Zhang, Q.; Hu, J.; Chen, J.; Wang, W. Computational evidence for the

630

detoxifying mechanism of epsilon class glutathione transferase toward the insecticide ddt. Environ.

631

Sci. Technol. 2014, 48, (9), 5008-5016.

632 633

(24) Sadowsky, D.; McNeill, K.; Cramer, C. J. Dehalogenation of aromatics by nucleophilic aromatic substitution. Environ. Sci. Technol. 2014, 48, (18), 10904-10911.

634

(25) Pati, S. G.; Kohler, H. P.; Pabis, A.; Paneth, P.; Parales, R. E.; Hofstetter, T. B. Substrate

635

and enzyme specificity of the kinetic isotope effects associated with the dioxygenation of

636

nitroaromatic contaminants. Environ. Sci. Technol. 2016, 50, (13), 6708-6716.

637

(26) Wijker, R. S.; Adamczyk, P.; Bolotin, J.; Paneth, P.; Hofstetter, T. B. Isotopic analysis of

638

oxidative pollutant degradation pathways exhibiting large h isotope fractionation. Environ. Sci.

639

Technol. 2013, 47, (23), 13459-13468.


28

Page 29 of 36

640 641


(27) Shaik, S.; Kumar, D.; de Visser, S. P.; Altun, A.; Thiel, W. Theoretical perspective on the structure and mechanism of cytochrome p450 enzymes. Chem. Rev. 2005, 105, (6), 2279-2328.

642

(28) Ogliaro, F.; Harris, N.; Cohen, S.; Filatov, M.; de Visser, S. P.; Shaik, S. A model "rebound"

643

mechanism of hydroxylation by cytochrome p450: Stepwise and effectively concerted pathways,

644

and their reactivity patterns. J. Am. Chem. Soc. 2000, 122, (37), 8977-8989.

645

(29) Ji, L.; Schuurmann, G. Computational evidence for alpha-nitrosamino radical as initial

646

metabolite for both the p450 dealkylation and denitrosation of carcinogenic nitrosamines. J. Phys.

647

Chem. B 2012, 116, (2), 903-912.

648

(30) Ji, L.; Faponle, A. S.; Quesne, M. G.; Sainna, M. A.; Zhang, J.; Franke, A.; Kumar, D.; van

649

Eldik, R.; Liu, W. P.; de Visser, S. P. Drug metabolism by cytochrome p450 enzymes: What

650

distinguishes the pathways leading to substrate hydroxylation over desaturation? Chem. Eur. J.

651

2015, 21, (25), 9083-9092.

652

(31) Kumar, D.; Karamzadeh, B.; Sastry, G. N.; de Visser, S. P. What factors influence the rate

653

constant of substrate epoxidation by compound i of cytochrome p450 and analogous iron(iv)-oxo

654

oxidants? J. Am. Chem. Soc. 2010, 132, (22), 7656-7667.

655

(32) Zhang, J.; Ji, L.; Liu, W. In silico prediction of cytochrome p450-mediated

656

biotransformations of xenobiotics: A case study of epoxidation. Chem. Res. Toxicol. 2015, 28, (8),

657

1522-1531.

658

(33) de Visser, S. P.; Shaik, S. A proton-shuttle mechanism mediated by the porphyrin in

659

benzene hydroxylation by cytochrome p450 enzymes. J. Am. Chem. Soc. 2003, 125, (24), 7413-

660

7424.


29


Page 30 of 36

661

(34) Bathelt, C. M.; Ridder, L.; Mulholland, A. J.; Harvey, J. N. Aromatic hydroxylation by

662

cytochrome p450: Model calculations of mechanism and substituent effects. J. Am. Chem. Soc.

663

2003, 125, (49), 15004-15005.

664

(35) Reinhard, F. G. C.; Sainna, M. A.; Upadhyay, P.; Balan, G. A.; Kumar, D.; Fornarini, S.;

665

Crestoni, M. E.; de Visser, S. P. A systematic account on aromatic hydroxylation by a cytochrome

666

p450 model compound i: A low-pressure mass spectrometry and computational study. Chem. Eur.

667

J. 2016, 22, (51), 18608-18619.

668

(36) Li, C.; Zhang, L.; Zhang, C.; Hirao, H.; Wu, W.; Shaik, S. Which oxidant is really

669

responsible for sulfur oxidation by cytochrome p450? Angew. Chem. Int. Ed. 2007, 46, (43), 8168-

670

8170.

671 672 673 674 675 676

(37) Ji, L.; Schuurmann, G. Model and mechanism: N-hydroxylation of primary aromatic amines by cytochrome p450. Angew. Chem. Int. Ed. 2013, 52, (2), 744-748. (38) Rydberg, P.; Ryde, U.; Olsen, L. Sulfoxide, sulfur, and nitrogen oxidation and dealkylation by cytochrome p450. J. Chem. Theory Comput. 2008, 4, (8), 1369-1377. (39) Ji, L.; Schuurmann, G. Computational biotransformation profile of paracetamol catalyzed by cytochrome p450. Chem. Res. Toxicol. 2015, 28, (4), 585-596.

677

(40) Kumar, D.; Sastry, G. N.; de Visser, S. P. Effect of the axial ligand on substrate

678

sulfoxidation mediated by iron(iv)-oxo porphyrin cation radical oxidants. Chem. Eur. J. 2011, 17,

679

(22), 6196-6205.


30

Page 31 of 36


680

(41) Wang, X.; Wang, Y.; Chen, J.; Ma, Y.; Zhou, J.; Fu, Z. Computational toxicological

681

investigation on the mechanism and pathways of xenobiotics metabolized by cytochrome p450: A

682

case of bde-47. Environ. Sci. Technol. 2012, 46, (9), 5126-5133.

683

(42) Fu, Z. Q.; Wang, Y.; Chen, J. W.; Wang, Z. Y.; Wang, X. B. How pbdes are transformed

684

into dihydroxylated and dioxin metabolites catalyzed by the active center of cytochrome p450s: A

685

dft study. Environ. Sci. Technol. 2016, 50, (15), 8155-8163.

686

(43) Meyer, A. H.; Dybala-Defratyka, A.; Alaimo, P. J.; Geronimo, I.; Sanchez, A. D.; Cramer,

687

C. J.; Elsner, M. Cytochrome p450-catalyzed dealkylation of atrazine by rhodococcus sp strain

688

n186/21 involves hydrogen atom transfer rather than single electron transfer. Dalton Trans. 2014,

689

43, (32), 12175-12186.

690

(44) Ji, L.; Ji, S.; Wang, C.; Kepp, K. P. Molecular mechanism of alternative p450-catalyzed

691

metabolism of environmental phenolic endocrine-disrupting chemicals. Environ. Sci. Technol.

692

2018, 52, (7), 4422-4431.

693 694 695 696 697 698

(45) Humphrey, W.; Dalke, A.; Schulten, K. Vmd: Visual molecular dynamics. J. Mol. Graph. Model. 1996, 14, (1), 33-38. (46) Sohl, C. D.; Cheng, Q.; Guengerich, F. P. Chromatographic assays of drug oxidation by human cytochrome p450 3a4. Nat. Protoc. 2009, 4, (9), 1252-1257. (47) Cytochrome p450: Structure, mechanism, and biochemistry. In 3rd ed.; Ortiz de Montellano, P. R., Ed. Kluwer Academic/Plenum Publishers: New York, 2005.


31


Page 32 of 36

699

(48) Yang, F. X.; Ding, J. J.; Huang, W.; Xie, W.; Liu, W. P. Particle size-specific distributions

700

and preliminary exposure assessments of organophosphate flame retardants in office air particulate

701

matter. Environ. Sci. Technol. 2014, 48, (1), 63-70.

702 703 704 705 706 707

(49) Lee, C. T.; Yang, W. T.; Parr, R. G. Development of the colle-salvetti correlation-energy formula into a functional of the electron-density. Physical Review B 1988, 37, (2), 785-789. (50) Becke, A. D. Density-functional thermochemistry .3. The role of exact exchange. J. Chem. Phys. 1993, 98, (7), 5648-5652. (51) Hay, P. J.; Wadt, W. R. Abinitio effective core potentials for molecular calculations potentials for the transition-metal atoms sc to hg. J. Chem. Phys. 1985, 82, (1), 270-283.

708

(52) Kumar, D.; de Visser, S. P.; Shaik, S. How does product isotope effect prove the operation

709

of a two-state "rebound" mechanism in c-h hydroxylation by cytochrome p450? J. Am. Chem. Soc.

710

2003, 125, (43), 13024-13025.

711

(53) Porro, C. S.; Kumar, D.; de Visser, S. P. Electronic properties of pentacoordinated heme

712

complexes in cytochrome p450 enzymes: Search for an fe(i) oxidation state. Phys. Chem. Chem.

713

Phys. 2009, 11, (43), 10219-10226.

714 715 716 717 718 719

(54) Strickland, N.; Harvey, J. N. Spin-forbidden ligand binding to the ferrous-heme group: Ab initio and dft studies. J. Phys. Chem. B 2007, 111, (4), 841-852. (55) Altun, A.; Breidung, J.; Neese, F.; Thiel, W. Correlated ab initio and density functional studies on h2 activation by feo+. J. Chem. Theory Comput. 2014, 10, (9), 3807-3820. (56) Cao, X. Y.; Dolg, M.; Stoll, H. Valence basis sets for relativistic energy-consistent smallcore actinide pseudopotentials. J. Chem. Phys. 2003, 118, (2), 487-496.


32

Page 33 of 36


720

(57) Miertus, S.; Scrocco, E.; Tomasi, J. Electrostatic interaction of a solute with a continuum -

721

a direct utilization of abinitio molecular potentials for the prevision of solvent effects. Chem. Phys.

722

1981, 55, (1), 117-129.

723 724

(58) Grimme, S. Semiempirical gga-type density functional constructed with a long-range dispersion correction. J. Comput. Chem. 2006, 27, (15), 1787-1799.

725

(59) Zaretzki, J.; Rydberg, P.; Bergeron, C.; Bennett, K. P.; Olsen, L.; Breneman, C. M. Rs-

726

predictor models augmented with smartcyp reactivities: Robust metabolic regioselectivity

727

predictions for nine cyp isozymes. J. Chem. Inf. Model. 2012, 52, (6), 1637-1659.

728

(60) Rydberg, P.; Rostkowski, M.; Gloriam, D. E.; Olsen, L. The contribution of atom

729

accessibility to site of metabolism models for cytochromes p450. Mol. Pharm. 2013, 10, (4), 1216-

730

1223.

731

(61) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J.

732

R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.;

733

Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.;

734

Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.;

735

Vreven, T.; Montgomery, J. A.; Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers,

736

E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.;

737

Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J.

738

E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev,

739

O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.;

740

Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.;


33


Page 34 of 36

741

Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian, Inc.: Wallingford CT,

742

2013. Gaussian 09, Revision D.01.

743

(62) Cooper, E. M.; Stapleton, H. M., Metabolism of organophosphate flame retardants by

744

human liver microsomes and porcine esterase. In SETAC North America 32nd Annual Meeting,

745

2011.

746 747

(63) Guengerich, F. P.; Shimada, T. Oxidation of toxic and carcinogenic chemicals by human cytochrome p-450 enzymes. Chem. Res. Toxicol. 1991, 4, (4), 391-407.

748

(64) Ohe, T.; Mashino, T.; Hirobe, M. Substituent elimination from p-substituted phenols by

749

cytochrome p450. Ipso-substitution by the oxygen atom of the active species. Drug Metab. Dispos.

750

1997, 25, (1), 116-122.

751

(65) la Farre, M.; Perez, S.; Kantiani, L.; Barcelo, D. Fate and toxicity of emerging pollutants,

752

their metabolites and transformation products in the aquatic environment. TrAC, Trends Anal.

753

Chem. 2008, 27, (11), 991-1007.

754

(66) Bletsou, A. A.; Jeon, J.; Hollender, J.; Archontaki, E.; Thomaidis, N. S. Targeted and non-

755

targeted liquid chromatography-mass spectrometric workflows for identification of transformation

756

products of emerging pollutants in the aquatic environment. TrAC, Trends Anal. Chem. 2015, 66,

757

32-44.

758

(67) Strobel, A.; Letcher, R. J.; Willmore, W. G.; Sonne, C.; Dietz, R. Structure-dependent in

759

vitro metabolism of alkyl-substituted analogues of triphenyl phosphate in east greenland polar

760

bears and ringed seals. Environ. Sci. Technol. Lett. 2018, 5, (4), 214-219.


34

Page 35 of 36


761

(68) Greaves, A. K.; Su, G. Y.; Letcher, R. J. Environmentally relevant organophosphate triesters

762

in herring gulls: In vitro biotransformation and kinetics and diester metabolite formation using a

763

hepatic microsomal assay. Toxicol. Appl. Pharmacol. 2016, 308, 59-65.

764 765

(69) Gramec Skledar, D.; Peterlin Masic, L. Bisphenol a and its analogs: Do their metabolites have endocrine activity? Environ. Toxicol. Pharmacol. 2016, 47, 182-199.

766

(70) Nakamura, S.; Tezuka, Y.; Ushiyama, A.; Kawashima, C.; Kitagawara, Y.; Takahashi, K.;

767

Ohta, S.; Mashino, T. Ipso substitution of bisphenol a catalyzed by microsomal cytochrome p450

768

and enhancement of estrogenic activity. Toxicol. Lett. 2011, 203, (1), 92-95.

769

(71) Chen, D.; Kannan, K.; Tan, H.; Zheng, Z.; Feng, Y. L.; Wu, Y.; Widelka, M. Bisphenol

770

analogues other than bpa: Environmental occurrence, human exposure, and toxicity-a review.

771

Environ. Sci. Technol. 2016, 50, (11), 5438-5453.

772

(72) Tezuka, Y.; Takahashi, K.; Suzuki, T.; Kitamura, S.; Ohta, S.; Nakamura, S.; Mashino, T.

773

Novel metabolic pathways of p-n-nonylphenol catalyzed by cytochrome p450 and estrogen

774

receptor binding activity of new metabolites. J. Health Sci. 2007, 53, (5), 552-561.

775

(73) Ricken, B.; Corvini, P. F. X.; Cichocka, D.; Parisi, M.; Lenz, M.; Wyss, D.; Martinez-

776

Lavanchy, P. M.; Mueller, J. A.; Shahgaldian, P.; Tulli, L. G.; Kohler, H.-P. E.; Kolvenbach, B.

777

A. Ipso-hydroxylation and subsequent fragmentation: A novel microbial strategy to eliminate

778

sulfonamide antibiotics. Appl. Environ. Microbiol. 2013, 79, (18), 5550-5558.

779 780 781


35


782

Page 36 of 36

SYNOPSIS GRAPHICS

783

784 785


36

The Metabolic Mechanism of Aryl Phosphorus Flame Retardants by

Recommend Documents