Intrinsic Clearance of Xenobiotic Chemicals by ... - ACS Publications

Subscriber access provided by ORTA DOGU TEKNIK UNIVERSITESI KUTUPHANESI

Article

Intrinsic clearance of xenobiotic chemicals by liver microsomes: Assessment of trophic magnification potentials Guomao Zheng, Yi Wan, and Jianying Hu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b01178 • Publication Date (Web): 06 May 2016 Downloaded from http://pubs.acs.org on May 8, 2016

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 free 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 accessible to all readers and 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.

Environmental Science & Technology 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 34

Environmental Science & Technology

1

Intrinsic clearance of xenobiotic chemicals by liver microsomes: Assessment of trophic

2

magnification potentials

3

Guomao ZHENG, Yi WAN*, Jianying HU

4 5 6 7

1

Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences,

Peking University, Beijing 100871, China (Received

)

*Address for Correspondence:

8 9 10 11 12 13 14 15 16

Address for Correspondence

17

Dr. Yi WAN

18

College of Urban and Environmental Sciences

19

Peking University

20

Beijing 100871, China

21

TEL & FAX: 86-10-62759126

22

Email: [email protected]

1

ACS Paragon Plus Environment


23

ABSTRACT

24

The use of trophic magnification factors (TMFs) to characterize the bioaccumulation

25

potentials of chemicals was encouraged, however the method for assessment of trophic

26

magnification potentials is still lacking. We optimized the in vitro assays used for the

27

measurement of intrinsic clearance in liver microsomes by incorporating benzo[a]pyrene

28

[B(a)P] as a benchmark compound. The intrinsic clearance of 40 compounds was then

29

measured in microsomes from fish (weevers) and birds (quail); the characteristics of the

30

trophic transfer of these 40 compounds were previously investigated in an aquatic food web in

31

Bohai in northern China. Chemicals that are biotransformed at a rate similar to or higher than

32

that of B[a]P in the microsomes of both weevers and quail (CL/CLB[a]P: 0.1 to 2.4) generally

33

exhibited no significant trophic magnification or dilution in the food web (TMF ≈1 or 1).

36

The in vitro intrinsic clearance values of the target chemicals were found to be consistent with

37

their respective trophic transfer behavior in the aquatic food web. Significant negative

38

correlations were also found between the TMFs and intrinsic clearance values of all target

39

chemicals obtained in microsomes from both weevers and quail. Multiple linear regression

40

analysis showed that biotransformation rates (CL/CLB[a]P) is a more important factor

41

compared with log Kow in the assessment of the trophic magnification of chemicals in the

42

aquatic food web.

43

Keyword: Trophic Magnification, Food web, Bioaccumulation, Weever, Quail

2


Page 2 of 34

Page 3 of 34

44


INTRODUCTION

45

The persistent organic pollutants (POPs) protocol endorsed by more than 100 countries in

46

2004 has led to significant activities in the assessment of persistent, bioaccumulative and

47

toxic substances throughout the world.1 Bioaccumulation is a fundamental process in

48

environmental toxicology and risk assessment because it determines the internal dose of

49

environmental pollutants.2 Bioaccumulation potentials have been routinely scrutinized by

50

regulatory agencies across the globe as part of the risk assessment of chemicals.3

51

Governments worldwide have sought to evaluate all commercial chemicals to identify

52

substances that undergo trophic magnification in the food web and achieve harmful

53

concentrations in organisms.4

54

Screening and assessment of bioaccumulation generally rely on laboratory-derived

55

bioconcentration factors (BCFs) or field-determined bioaccumulation factors (BAFs).5 In the

56

past few decades, studies of pollutants in food webs have demonstrated that bioaccumulation

57

in food webs is not solely a lipid-water partitioning process. During a global “Lab-field

58

Bioaccumulation Workshop” held in New Orleans, Louisiana, the use of trophic

59

magnification factors (TMFs) to characterize the bioaccumulation potentials of chemicals was

60

encouraged because they provide a characterization of the average degree of trophic

61

magnification that occurs across an entire food web.4, 6-8 The hydrophilicity (water solubility)

62

of compounds is considered to be an important determinant of the trophic magnification of

63

chemicals in aquatic environments. A good correlation between TMFs and log Kow has been

64

reported for recalcitrant polychlorinated biphenyls (PCBs), chlorobenzenes (ClBs),

65

chlordanes, hexachlorocyclohexanes (HCHs), dichlorodiphenyltrichloroethane (DDTs), mirex,

3



Page 4 of 34

66

and dieldrin in the North Water Polynya marine food web.9 However, most trophodynamic

67

studies have focused on POPs or emerging POPs, which generally showed trophic

68

magnification in aquatic food webs. Some studies also found that compounds with a similar

69

log Kow did not biomagnify (e.g., 4-nonylphenol [NPs], phthalate esters [PEs]) or even

70

exhibited trophic dilution (e.g., polycyclic aromatic hydrocarbons [PAHs] or hydroxylated

71

polybrominated diphenyl ethers [OH-PBDEs]) in aquatic food webs, which suggests that

72

other factors also affect TMFs.10-13 Biotransformation has long been recognized as an

73

important source of uncertainty in predictions of bioaccumulation,14, 15 and biodegradation

74

testing is required for bioaccumulation assessments under the Chemical Substance Control

75

Law in Japan.3 However, the influence of biotransformation on the trophic transfer of

76

chemicals is unknown due to the limited availability of biological biotransformation rates and

77

their relationships with reported TMFs.

78

Currently, the methods most often adopted for accurately and efficiently determination of

79

biotransformation rates are still lacking. Among the various in vitro methods, subcellular

80

fractions of the liver (e.g., microsomes or S9) have been shown to be best suited to the

81

high-throughput screening of chemicals’ biotransformation potentials.14,

82

demonstrated that cryopreserved trout hepatocytes can be used to reliably obtain in vitro

83

intrinsic clearance of xenobiotics.17 The results of that study also suggested that incorporation

84

of one or more “benchmarking” chemicals into in vitro chemical depletion could potentially

85

reduce the variation between different batches.17 However, information regarding the nature

86

of an ideal benchmarking compound (e.g., the desired pathway for metabolism and ease of

87

analysis) is still lacking.

4


16

A recent study

Page 5 of 34


88

Bohai is an enclosed inland sea in northern China. In recent decades, the trophodynamics

89

of about 40 chemicals have been studied in the Bohai aquatic food web, which includes

90

phytoplankton/seston, zooplankton, invertebrates, fish, and birds.10, 12, 13, 18-21 The resulting

91

TMFs database of the 40 chemicals obtained from the same aquatic food web provides an

92

excellent opportunity to examine the influence of biotransformation and log Kow on the

93

trophic magnification of various chemicals. In this study, microsomes of the marine weever

94

(Lateolabras japonicus) and common quail (Coturnix coturnix) were used to obtain the

95

intrinsic biotransformation rates. The weever is a typical predatory fish that has a large body

96

size and a high trophic level (3.88±0.49) in the Bohai aquatic food web. 10, 19 Quail were used

97

to replace seagulls because fresh seagull liver tissue could not be obtained. An in vitro method

98

for the determination of repeatable biotransformation rates was developed and applied to

99

compounds with field-derived TMFs values. The relationships between the log Kow,

100

biotransformation rates, and TMFs were explored to establish a method to predict the trophic

101

magnification of chemicals in aquatic food webs.

102 103

MATERIALS AND METHODS

104

Chemicals and Reagents. Thirteen individual PAHs standards (acenaphthene [ACE],

105

fluorene [FE], phenanthrene [PH], anthracene (AN), fluoranthene [FL], pyrene [PY], chrysene

106

[CH], benzo[k]fluoranthene [B(k)F], benzo[a]pyrene [B(a)P], benzo[ghi]perylene [BP],

107

dibenz[a,h]anthracene [DA], benzo[b]fluoranthene [B(b)F], and benz[a]anthracene [B(a)A])

108

and four surrogate standards (acenaphthene-d10, phenanthrene-d10, chrysene-d12, and

109

perylene-d14) were obtained from AccuStandard (New Haven, CT). Eight individual PBDEs

5



110

(BDE28, BDE47, BDE99, BDE100, BDE119, BDE153, BDE154, and BDE183), mirex,

111

seven individual PCBs (PCB105, PCB114, PCB118, PCB123, PCB157, PCB169, and

112

PCB189), and

113

ON, Canada). 2'-OH-6'-Cl-BDE7, 6-OH-BDE47, 6'-MeO-BDE17, and 6-MeO-BDE47 were

114

synthesized in the Department of Biology and Chemistry, City University of Hong Kong.

115

HCB and p,p'-DDE were purchased from Chemservice (Chester, England), p,p'-DDMU was

116

obtained from Sigma (St. Louis, MO), and their surrogates (PCB121 and PCB 198) were

117

obtained from AccuStandard. 4-NP (technical grade) and 4-n-NP were purchased from Kanto

118

Chemicals (Tokyo, Japan). NPEOs was purchased from Hayashi Pure Chemicals (Tokyo,

119

Japan). Monobutyltin trichloride (MBT) was purchased from Acros Organics (Geel, Belgium),

120

dibutyltin dichloride (DBT), tributyltin chloride (TBT), and triphenyltin chloride (TPT) were

121

purchased from Wako (Osaka, Japan). Deuterated organotins (MBT-d9, DBT-d18, TBT-d27,

122

and TPT-d15) were obtained from Hayashi Pure Chemicals. Dichloromethane (DCM),

123

n-hexane, acetone, acetonitrile, and methanol of pesticide residue grade were obtained from

124

Fisher Chemicals (Fair Lawn, NJ). Methyl chloroformate was obtained from Sigma-Aldrich.

125

O-bis (trimethylsilyl) trifluoroacetamide (BSTFA) was obtained from Supelco (Bellefonte,

126

PA). Sodium tetraethylborate (NaBEt4) was purchased from Wako. Water was prepared with

127

a Milli-Q Synthesis water puriﬁcation system (Millipore, Bedford, MA). A nicotinamide

128

adenine dinucleotide phosphate (NADPH) regenerating system and dithiothreitol (DTT) were

129

purchased from Promega (Madison, WI). Sodium sulfate (analytical reagent grade, Beijing

130

Chemicals) was baked at 450 °C for 4 hours and stored in a drying oven before use.

13

C-labeled PCBs were obtained from Wellington Laboratories Inc. (Guelph,

6


Page 6 of 34

Page 7 of 34


131

Monopotassium phosphate, dipotassium phosphate, ethylene diamine tetraacetic acid (EDTA),

132

glycerol, and sucrose were purchased from Beijing Chemicals.

133

In Vitro Microsomal Incubations.

134

B[a]P was added to the incubation mixtures as a benchmark compound to normalize the

135

variation in the transformation rates that occurred in different batches of analyses. The details

136

of the preparation of microsomes are provided in the supporting information (SI). The protein

137

concentrations were determined with the Bradford method using bovine serum albumin as a

138

standard and according to the manufacturer’s protocol (Sigma-Aldrich). Hepatic microsomal

139

CYP450 enzymes and four specific CYP enzymes (7-ethoxyresorufin O-deethylase [EROD],

140

7-pentoxyresorufin O-demethylase [PROD], 7-methoxyresorufin O-demethylase [MROD]

141

and 7-benzyloxyresorufin O-dealkylatase [BROD]) from weevers and quail were measured

142

with fluorescence kits (Genmed Scientific Inc., USA). The EROD, PROD, MROD and

143

BROD were markers for CYP1A1, CYP2B1, CYP1A2, and CYP3A1 activity, respectively.

144

For in vitro incubations of weever microsomes, the reactions were performed in 50 mM

145

phosphate buffer (pH 7.4) containing 1 mM EDTA, 1 mM DTT, and 20% (v/v) glycerol. The

146

reaction mixtures (200 µL) consisted of 100 µL liver microsomes (2 mg/mL protein), 60 µL

147

NADPH regenerating system, and 1 µL substrate (0.5 µM for both individual compounds and

148

B[a]P) were incubated in 1.5-mL amber glass vials. Reactions were initiated by adding a

149

NADPH-generating system (NADP, 6.5 mM; glucose 6-phosphate, 16.5 mM; MgCl2, 16.5

150

mM; and glucose 6-phosphate dehydrogenase, 2 U/mL). Incubations were carried out in

151

triplicate in separate glass vials at 25°C, and 200 µL ice-cold acetone was added into each vial

152

to terminate the reaction after 0, 1, 3, 5, 9, 12 and 24 h.

7



153

For in vitro incubations of quail microsomes, 50 mM phosphate buffer (pH 7.4)

154

containing 5 mM MgCl2 and 0.5 mM EDTA was added into the incubation vials. The reaction

155

mixtures (200 µL) consisted of 100 µL liver microsomes (2 mg/mL protein), 60 µL NADPH

156

regenerating system, and 1 µL substrate (0.5 µM for individual compounds and 2 µM for

157

B[a]P) and were incubated in 1.5-mL amber glass vials. Reactions were initiated by adding a

158

NADPH-generating system (NADP, 6.5 mM; glucose 6-phosphate, 16.5 mM; MgCl2, 16.5

159

mM; and glucose 6-phosphate dehydrogenase, 2 U/mL). Incubations were carried out in

160

triplicate at 39°C in separate glass vials, and 200 µL ice-cold acetone was added into each vial

161

to terminate the reaction after 0, 10, 20, 30, 40, and 60 minutes

162

The vials were stored at −20°C until chemical analysis. The microsomes were inactivated

163

by boiling for 10 min and were then cooled on ice. Incubations with deactivated microsomes

164

and standards were used as negative controls to assess potential background interference and

165

the possibility of non-enzymatic changes. The percentage variations of concentrations of

166

target compounds along with the incubation time in the deactivated liver microsomes were

167

shown in SI Figure S1 and Figure S2.

168

Sample Analysis.

169

The incubation mixture in each vial was added with 1 mL of water and surrogates, and

170

extracted three times with 1 mL hexane. The aquatic fraction was then passed through a

171

Pasteur pipe filled with sodium sulfate to remove moisture and eluted with 1 mL hexane and 1

172

mL DCM, which were also used to rinse the glass vial before elution. All of the extracts were

173

combined and concentrated to 50 µL for instrumental analysis. The organotins were

174

derivatized with NaBEt4 before GC-EI-MS analysis, 4-NP was derivatized with BSTFA

8


Page 8 of 34

Page 9 of 34


175

before GC-EI-MS analysis, and 6-OH-BDE47 was derivatized with methyl chloroformate

176

before GC-NCI-MS analysis. PAHs, PCBs, DDTs, HCB, mirex, organotins, and 4-NP were

177

directly analyzed by GC-EI-MS (Agilent Technologies); PBDEs, 6-MeO-BDE47 and

178

6-OH-BDE47 were analyzed by GC-NCI-MS (Shimadzu QP 2010 plus, Japan); and NPEOs

179

were analyzed with high-performance liquid chromatograph (Agilent 1100 Series, USA)

180

coupled with a fluorescence detector (Agilent 1200 Series, USA). Detailed information on the

181

instrumental conditions for each group of chemicals are provided in SI.

182

Quality Assurance/Quality Control.

183

Concentrations of PAHs in sample extracts were quantified relative to deuterated PAHs

184

(chrysene-d12, perylene-d14 and perylene-d14), PBDEs and 6-MeO-BDE47 were quantified

185

relative to 6’-MeO-BDE17, 6-OH-BDE47 were quantified relative to 2’-OH-6’-Cl-BDE7,

186

p,p’-DDE, p,p’-DDMU, and HCB were quantified relative to PCB 121, 4-NP were quantified

187

relative to 4-n-NP, organotins were quantified relative to deuterated organotins (MBT-d9,

188

DBT-d18, TBT-d27 and TPT-d15), mirex was quantified to PCB 198, and PCBs were quantified

189

relative to

190

1%, 85 ± 13%, 85 ± 6%, 72 ± 19%, 124 ± 7%, 93 ± 26%, and 119 ± 14% for deuterated PAHs,

191

6’-MeO-BDE17, PCB 121, PCB198, 2’-OH-6’-Cl-BDE7, 4-n-NP, deuterated organotins, and

192

13

193

Data analysis.

194 195 196

13

C-labeled PCBs. The recoveries of surrogates standards were 78 ± 19%, 100 ±

C-labeled PCBs, respectively.

The declining concentrations of substrate in the incubation mixtures over time followed the monoexponential decay model and were fitted to the linear equation (1): ࢒࢔࡯࢚ = ࢒࢔࡯૙ − ࡷ ∙ ࢚

(1)

9



197

where t is incubation time; C0 and Ct are the substrate concentrations in the incubation

198

medium at time zero and time t, respectively; and K is the apparent first-order

199

biotransformation rate constant (h-1). The in vitro intrinsic clearance values (CL, mL/h/mg

200

protein) were obtained according to the equation (2): ۱‫= ۺ‬

201

ࡷ ࡯࢖࢘࢕࢚ࢋ࢏࢔

(2)

202

where Cprotein is the protein concentration (mg protein/ml) of the incubation mixtures. The in

203

vitro intrinsic clearance value of each chemical was normalized to that of B[a]P according to

204

the equation (3): ۱‫ۺ‬′ =

205

࡯ࡸ ࡯ࡸ࡮ሾࢇሿࡼ

(3)

206

where CL' is the normalized intrinsic clearance value of target compounds and CLB[a]P is the

207

intrinsic clearance value of B[a]P.

208

Statistical analysis.

209

Multiple linear regression models were constructed to determine the contribution of log

210

Kow and the biotransformation rate on the trophic magnification of chemicals. TMFs were

211

slopes of the correlations between concentrations of chemicals in organisms and trophic levels.

212

The significances of the correlations were examined by Spearman’s rank correlation test. The

213

TMFs were log10-transformed before analysis, and the linear regression was regarded as

214

significant when the p value was less than 0.05. The correlations between Ln(Ct/C0) and the

215

incubation time were also examined with Spearman’s rank correlation test. The linear

216

regression was regarded as significant when the value of p was below 0.05. All statistical

217

analyses were performed with SPSS 13.0 statistical software (SPSS, Inc., Chicago, IL).

218

10


Page 10 of 34

Page 11 of 34


219

RESULTS AND DISCUSSION

220

Hepatic microsomal enzyme activities.

221

The activities of hepatic microsomal CYP450 enzymes and the four specific CYP

222

enzymes (EROD, PROD, MROD, and BROD) in weevers and quail are listed in Table 1. The

223

total and specific CYP450 enzyme activities in the microsomes of weevers were within the

224

ranges of those in marine fish reported previously,22, 23 and the profiles of EROD, MROD,

225

PROD, and BROD activities were also similar to those of the corresponding enzymes in

226

previous investigations.24-27 In the quail liver microsomes, the EROD, MROD, PROD, and

227

BROD activities were determined to be 6.8±0.1, 1.7±0.8, 10.5±2.2 and 10.7±0.3

228

pmol/mg/min, respectively, which were all within the normal ranges reported previously for

229

herring gulls, glaucous gulls, yellow legged gulls, chickens, black-legged kittiwake, European

230

quail, grey partridges and northern fulmar.28-34 Similar hepatic enzyme activities observed

231

between quail and other avian species suggested that quail could be used as an acceptable

232

model to replace seagulls in this study. The incubation of chemicals with quail microsomes

233

was conducted for 1 h due to the relatively high activities of hepatic microsomal CYP

234

enzymes, and incubations of chemicals with weever microsomes were extended to 24 h to

235

evaluate biotransformation potentials of persistent compounds (e.g., PCBs). A similar

236

incubation time (24 h) was also applied in the incubation of fish microsomes in previous

237

studies.35-37 The enzyme activities of EROD, PROD, MROD, and BROD along with the

238

incubation time were determined in microsomes of weevers and quail, and no significant

239

decline was observed for the target enzyme activities (SI Figure S3), which suggests that

240

biotransformation can continue for up to 24 h during in vitro incubations.

11



241

Method Optimization.

242

The reliability of the in vitro assays for assessment of bioaccumulation is substantially

243

hampered by the high variability in enzyme viability and metabolic capacity between different

244

batches. This variability might be accounted for with the use of benchmark chemicals with

245

known metabolic characteristics.14,

246

compound in in vitro assays to normalize the variation that occurred across different batches

247

of analyses. B[a]P was reported to undergo biotransformation mainly through CYP1A1,38 and

248

depletion of the chemical followed first-order kinetics with relatively constant first-order rate

249

constant in previous studies.15-17, 39, 40 Three chemicals (PH, 6-MeO-BDE47, and 4-NP) from

250

diverse chemical classes were used to assess the variability in the assays, because PH,

251

6-MeO-BDE47, and 4-NP undergo steadily biotransformation by CYP1A1, CYP2B and

252

CYP1A2, respectively.41-43 The activities of EROD, PROD, and MROD—markers for

253

CYP1A1, CYP2B1, CYP1A2, respectively—have been determined to be within normal

254

ranges in microsomes of weevers (Table 1). Incubation of B[a]P with chemicals

255

biotransformed by the same or different metabolic enzymes in microsomes could help to

256

clarify whether the benchmark chemical would reduce the variations within different enzymes.

257

Each chemical was tested relative to B[a]P in the microsomes of weevers in triplicate batches

258

at a low substrate concentration (0.5 µM). The low substrate concentration prevented enzyme

259

saturation and high cytotoxicities of the chemicals, and allowed for detection of significant

260

depletion of substrates. The microsomes for each batch were isolated at different times

261

(January, July, and November 2014). As shown in Figure 1, the in vitro intrinsic clearance

262

values of PH, 6-MeO-BDE47, and 4-NP were 0.007±0.0017, 0.027±0.005, and 0.021±0.013

17

In this study, B[a]P was tested as a benchmark

12


Page 12 of 34

Page 13 of 34


263

mL/h/mg protein with the variation (%CV) in intrinsic clearance values of 23%, 23%, and

264

61%, respectively. After correction by the intrinsic clearance value of B[a]P, the CL' values

265

for PH, 6-MeO-BDE47, and 4-NP were 0.22±0.037, 0.41±0.019, and 0.29±0.046,

266

respectively, and the variation (%CV) of the three compounds decreased to 5-17%. The

267

corrected variation was in the range of the reported intra-laboratory variability (4.1-30%)

268

achieved when the hepatocyte assay was isolated and cryopreserved in one location.17 The

269

study’s results demonstrate that the incorporation of B[a]P benchmarking could substantially

270

reduce the variability between batches. And the variations of enzyme activities caused by

271

cytotoxicity of chemicals and other abiotic factors during the incubations could also be

272

corrected by the depletion of B[a]P in the in vitro system.

273

Another critical issue that can cause great variation in in vitro systems is the adsorption of

274

chemicals into the reaction vessels or proteins.14 In this study, a separate amber glass vial (1.5

275

mL) was used for incubations at different reaction times. This approach differs from previous

276

methods in which an aliquot of the suspension was taken from the reaction vial each time.17, 44,

277

45

278

solutions or adsorption into vessels. The adsorption of chemicals during incubations would

279

also cause substrate loss during liquid-liquid extraction, possibly as a result of the nonspecific

280

binding of chemicals to proteins within the system, a process that could not be reduced by

281

liquid-liquid extraction. An extraction method was developed in this study in which

282

incubation mixtures were passed through a Pasteur pipette filled with anhydrous sodium

283

sulfate, which was then eluted by hexane and DCM. As a result of this method, the target

284

compounds showed 88-114% recovery in all of the incubated samples terminated by ice-cold

Using the amber glass vial helped to avoid the uneven distribution of chemicals in the

13



285

acetone at 0 h (SI Figure S4); these values were significantly higher than those in samples in

286

which liquid-liquid extraction was used (40-55%).

287

Intrinsic Clearances.

288

The substrate depletion approach has been well adopted for estimation of intrinsic

289

clearance for a single compound.15,17,40,46-48 In this study, the biotransformation of target

290

compounds followed first-order kinetics in both weever and quail liver microsomes. A

291

summary of the in vitro intrinsic clearance values for the 40 compounds examined are listed

292

in Table 2. Relatively high intrinsic clearance rates were observed for PAHs,

293

6-OH/MeO-BDE47, NPEOs, 4-NP, and butyltins, and the CL of these compounds was

294

determined to be 0.0022-0.17 mL/h/mg protein and 0.26-4.6 mL/h/mg protein in the liver

295

microsomes of weevers and quail, respectively. In comparison, no statistically significant

296

decline was found for PBDEs, DDTs, HCB, mirex, PCBs, or TPT during the incubation

297

periods in weever microsomes, and extremely slow depletion was observed for PBDEs and

298

p,p'-DDMU in quail microsomes, with a CL of 0.05-0.39 mL/h/mg protein. The available

299

substrate depletion studies focused mainly on the hepatic clearance of 4-NP, PAHs, and some

300

pharmaceuticals in fish.39, 46-51 The CL of 4-NP was reported to be 0.18 and 0.11 mL/h/mg

301

protein in trout liver microsomes and S9 fractions, respectively,39 which were relatively

302

higher than those found in the weever microsomes in this study (0.021 mL/h/mg protein). The

303

intrinsic clearance values of B[a]P reported in three separate studies were 0.024±0.003

304

mL/h/mg protein (dosed concentrations, 0.5 µM),51 0.032-0.088 mL/h/mg protein (dosed

305

concentrations, 2 µM),39 and 0.19-0.37 mL/h/mg protein (dosed concentrations, 1-2.5 µM).49

306

The differences between these reported intrinsic clearance rates might have resulted from the

14


Page 14 of 34

Page 15 of 34


307

use of different trout subspecies, S9 fraction protein concentrations, and beginning substrate

308

concentrations. The CL of B[a]P in weever microsomes (0.03±0.02 mL/h/mg protein) in this

309

study was in the range of the reported values cited above.

310

To the best of our knowledge, no information is available about the in vitro intrinsic

311

clearance of chemicals in birds. The extremely low intrinsic clearance values of persistent

312

chemicals (e.g., DDTs, HCB, mirex, PCBs, and TPT) in quail microsomes were similar to the

313

values obtained in weever microsomes, but the CLquail of the chemicals that undergo steady

314

biotransformation was found to be 14 to 1500 times higher than those in weever microsomes

315

(Table 2). It is interesting to note that the hepatic clearance values of PAHs increased in a

316

linear relationship with benzo-rings in incubations with weever microsomes (Figure 4). The

317

relationship was similar to the increasing degree of stereoselectivity with the size and shape of

318

the molecules in the metabolism of PAHs by bullhead liver microsomes: B[a]P (five

319

benzo-rings) > CH (four benzo-rings) > PH (three benzo-rings).52-54 However, a negative

320

correlation was observed between the CL'quail of PAHs and their benzo-rings in quail

321

microsomes (Figure 4). The discrepancy between quail and fish for the microsomal

322

metabolism of PAHs was also reported between rats and fish, possibly because of the

323

significantly different stereoselectivities of cytochrome P450 enzymes between the two

324

species.52-56 The results suggested that quail and rat liver microsomal enzymes may have

325

similar stereoselectivity in the metabolism of PAHs.

326

Assessment of Trophic Magnification Potentials.

327

It has been reported that two important physiological processes determine whether a

328

chemical will bioaccumulate in fish: the uptake of the chemical from the environment, and the

15



Page 16 of 34

329

biotransformation of the chemical into water-soluble metabolites.14, 51 Fish take up chemicals

330

through their gills and intestines, and these pathways depend mainly on the lipophilicity of the

331

chemicals (log Kow).14, 57 Thus, the relationships between field-derived TMFs, log Kow, and

332

in vitro intrinsic clearance values in microsomes from weevers and quail were explored

333

(Table 2). As shown in Figure 5a, TMFs increased with log Kow for all target compounds,

334

but the correlation between TMFs and log Kow was not statistically significant for the 40

335

target chemicals in this study (Figure 5a). Although positive correlations have been reported

336

between TMFs and log Kow for some POPs in previous studies,9, 58-60 negative relationships

337

or no relationships were observed for dioxins, PAHs, and PEs.11,

338

relationships between TMFs and log Kow suggested the existence of other, more important

339

factors.

19, 61-63

The different

340

The in vitro intrinsic clearance values of each group of chemicals were found to be

341

consistent with that group’s trophic transfer behaviors in the aquatic food web (Table 2).

342

Chemicals that undergo significant trophic dilution in the aquatic food web (TMF

Intrinsic Clearance of Xenobiotic Chemicals by ... - ACS Publications

Recommend Documents