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Factors affecting the bioaccessibility and intestinal transport of difenoconazole, hexaconazole, and spirodiclofen in human Caco-2 cells following in vitro digestion Yanhong Shi, Jin-Jing Xiao, Rong-Peng Feng, Yu-Ying Liu, Min Liao, Xiangwei Wu, Ri-Mao Hua, and Haiqun Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02781 • Publication Date (Web): 15 Sep 2017 Downloaded from http://pubs.acs.org on September 18, 2017

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Factors affecting the bioaccessibility and intestinal transport

2

of difenoconazole, hexaconazole, and spirodiclofen in human

3

Caco-2 cells following in vitro digestion

4 5

Yan-Hong Shi a,c †, Jin-Jing Xiao b,c†, Rong-Peng Feng a,c, Yu-Ying Liu a,c, Min

6

Liao b,c, Xiang-Wei Wu a,c, Ri-Mao Hua a,c, Hai-Qun Cao b,c*

7 8

a

9

Province 230036, China.

School of Resource & Environment, Anhui Agricultural University, Hefei, Anhui

10

b

11

230036, China.

12

c

School of Plant Protection, Anhui Agricultural University, Hefei, Anhui Province

Provincial Key Laboratory for Agri-Food Safety, Anhui Province, China.

13 14

† These authors contributed equally to this work.

15

* Corresponding author:

16

School of Plant Protection, Anhui Agricultural University

17

130 West Changjiang Road, Hefei, Anhui Province 230036P.R. China

18

Tel. /Fax: +86- 65785730

19

E-Mails: [email protected]

20 21 22 1


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Abstract

24

This study examined how gastrointestinal conditions affect pesticide bioaccessibility

25

and intestinal transepithelial transport of pesticides (difenoconazole, hexaconazole,

26

and spirodiclofen) in humans. We used an in vitro model combining human gastric

27

and intestinal digestion, followed with Caco-2 cell model for human intestinal

28

absorption. Bioaccessibility of three tested pesticides ranged from 25.2 to 76.3% and

29

10.6 to 79.63% in the gastric and intestinal phases, respectively. A marked trend

30

similar to the normal distribution was observed between bioaccessibility and pH, with

31

highest values observed at pH 2.12 in gastric juice. No significant differences were

32

observed with increasing digestion time; however, a significant negative correlation

33

was observed with the solid-liquid (S/L) ratio, following a logarithmic equation. R2

34

ranged from 0.9198 to 0.9848 and 0.9526 to 0.9951 in the simulated gastric and

35

intestinal juices, respectively, suggesting that the S/L ratio are also major factors

36

affecting bioaccessibility. Moreover, significant dose- and time-response effects were

37

subsequently observed for intestinal membrane permeability of difenoconazole, but

38

not for hexaconazole or spirodiclofen. This is the first study to demonstrate the uptake

39

of pesticides by human intestinal cells, aiding quantification of the likely effects on

40

human health and highlighting the importance of considering bioaccessibility in

41

studies of dietary exposure to pesticide residues.

42 43

Keywords: Bioaccessibility, apple, pesticide, Caco-2 model, risk assessment

2



44

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1. Introduction

45

Exposure to pesticide residues is a global concern due to their widespread

46

occurrence and the potential risks to human health. Dietary ingestion of pesticide

47

residues via fruits and vegetables, which on average constitute more than 30 % of our

48

diet, has been deemed the most important exposure pathway to foodborne

49

contaminants 1. Numerous studies have demonstrated that food processing technology

50

would contribute to pesticide dissipation 2. Ingestion of fruits and vegetables could

51

result in higher exposure to regulated compounds compared with other food

52

groups because of the method of consumption (i.e. raw or semi-processed) 3. This is

53

particularly relevant with regards to the unsafe levels of pesticide residues in apples,

54

reportedly the “dirtiest fruits”, with an average of 98 % of conventional apples

55

containing pesticide residues based on the list presented by the US Environmental

56

Working Group (EWG) 4. Understanding the mechanism of chemical absorption

57

following ingestion is therefore important for accurate assessment of the risk to

58

human health.

59

Bioaccessibility is a crucial parameter in determining systemic absorption 5. To

60

have a negative health effect in the human body, a contaminant must be bioavailable 6,

61

which relies on the degree of bioaccessibility

62

that the amount of compound absorbed may be less than the level of a contaminant in

63

the liquid or food

64

conservatively assume complete desorption of total concentrations

65

resulting in overestimates of the resulting risks. In recent years, bioaccessibility

7, 8

. Studies of bioaccessibility suggest

9, 10

. However, despite this, most human risk assessments 11, 12

, potentially

3


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studies have therefore received a great deal of attention, having been used to evaluate

67

the total content of heavy metals 13 and other contaminants in soil 14 and dust 15.

68

Various in vitro digestion/Caco-2 cell culture models have recently been

69

developed, allowing simpler, cheaper, and more rapid assessments of human exposure

70

to contaminants compared to in vivo studies, which are expensive, laborious, and

71

often pose ethical dilemmas

72

digestion times, and the solid-liquid (S/L) ratio) and biochemical conditions

73

encountered in the gastrointestinal tract can be simulated using in vitro digestion

74

models, and used to estimate the rate and extent of contaminant release during the

75

ingestion of liquids or food (bioaccessibility). They can also be used to indicate

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potential availability for uptake. Based on in vitro digestion models, studies suggest

77

that the bioaccessibility of contaminants ranges from 1.59 to 76.90 % 9, 17, depending

78

on several factors including physicochemical parameters of the human digestive

79

process mentioned above and the presence of various dietary components such as

80

cellulose, tannin, and phytate

81

highlight the importance of considering bioaccessibility during risk assessments.

82

However, no detailed study has yet documented the bioaccessibility of pesticides

83

during human digestion or the bioaccessible fractions, particularly in terms of

84

physicochemical predictors of pesticide bioaccessibility. Kang et al. 2016 20 suggested

85

that understanding the relative bioavailability of contaminants would provide more

86

precise information on their digestion and absorption, thereby enhancing the accuracy

87

of predicted risks to human health.

16

. Both physicochemical (e.g., gastrointestinal pH,

18, 19

, and other biochemical parameters. These studies

4



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The Caco-2 cell culture model is currently the most widely-used approach for

89

investigating the bioavailability of contaminants. Derived from human colon

90

adenocarcinoma, which is similar to the human small intestinal tract in terms of

91

enzymatic and morphological characteristics

92

transport characteristics of small intestinal cells. In addition, the Simulator of Human

93

Intestinal Microbial Ecosystem (SHIME) is one of the most common methods of

94

examining in vitro digestion, accurately stimulating the colonic environment

95

compared with other static or dynamic methods

96

also be used to accurately reflect the bioaccessibility of contaminants after

97

consumption.

21

, the model allows analysis of the

22

. Thus, the SHIME method could

98

In this study, we used an in vitro model combining human gastric and intestinal

99

digestion (SHIME) followed by a model of human intestinal absorption (Caco-2 cell

100

culture model) to (1) quantify the bioaccessibility of three pesticides commonly used

101

on apples (difenoconazole, hexaconazole, and spirodiclofen) and investigate related

102

parameters during human gastrointestinal digestion after ingestion; (2) determine

103

pesticide bioavailability and corresponding uptake characteristics of human intestinal

104

cells; and (3) provide a more realistic estimate of potential risks using a combination

105

of acceptable daily intake (ADI) values and bioaccessibility.

106 107

2. Materials and methods

108

2.1. Chemicals and samples

109

Pesticide standards (hexaconazole (99.90 %), spirodiclofen (99.30 %), and 5


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difenoconazole (99.20 %)) were purchased from the National Pesticide Quality

111

Supervision and Inspection Center (Beijing, China). Individual standard solutions for

112

optimization experiments and mixed standard solutions for calibration and validation

113

experiments were diluted in n-hexane, acetonitrile or dimethylsulfoxide (DMSO) and

114

stored at 4 °C.

115

HPLC grade n-hexane and acetonitrile were supplied by Thermo Fisher

116

Scientific (MA, USA) and Tedia Company, Inc. (OH, USA), respectively, and used

117

for preparation of the standards. A florisil solid-phase extraction (SPE) column (1000

118

mg / 6 mL) was purchased from Agela Technologies (Tianjin, China), and

119

arabinogalactan, peptone, xylan, pectin, soluble starch, mucin, cysteine, glucose, yeast,

120

pepsin, bile extract and pancreatin were purchased from Meifeng Chemical Industry

121

Co., Ltd. (Sichuan, China). Dulbecco's modified Eagle’s medium (high glucose, 4500

122

mg / dl) (DMEM), fetal bovine serum (FBS), trypsin-EDTA (0.25 % trypsin, 0.02 %

123

ethylenediaminetetraacetic

124

(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell proliferation and

125

cytotoxicity assay kit, and the human colon carcinoma cell line (Caco-2) (all

126

Sigma-Aldrich, St Louis, MO) were used to create the in vitro Caco-2 model system.

127

acid

[EDTA])

solution,

samples were homogenized and stored at −20 °C until analysis.

129

2.2. In vitro digestion model based on human SHIME

131

MTT

Fushi apples were purchased from local markets across Hefei City, China. All

128

130

the

The human SHIME protocol described by Van et al. and Yu et al.

23, 24

was

applied with slight modifications. The nutrition solution for digestion juice 6



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preparation contained per L: 1.00 g peptone, arabinogalactan and xylan, respectively,

133

2.00 g pectin, 3.00 g soluble starch, 4.00 g mucin, 0.50 g cysteine, 0.40 g glucose and

134

3.00 g yeast, and was autoclaved at 121 °C for 15 min before use.

135

Gastric phase. To simulate the gastric phase, a mixture of 5.00 g homogenized

136

sample and 20.00 mL gastric juice was added to a 50.00-mL centrifuge tube. The

137

gastric juice contained 200.00 mL nutrition solution and 25.00 mL gastric acid (0.09 g

138

pepsin per L and 0.10 mol/L HCl). The pH was adjusted to 4.00 using HCl solution to

139

represent recently-fed conditions, then samples were incubated in water at 37 °C (100

140

rpm) for 2 h.

141

Intestinal phase. To mimic the intestinal step, 5.00 g homogenized sample was

142

transferred to 20.00 mL of intestinal juice (12.50 g NaHCO3, 6.00 g bile extract, and

143

0.90 g pancreatin per L nutrition solution, pH 7.20), then incubated in a shaking

144

water bath at 37 °C (100 rpm) for 4 h. Aliquots (10.00 mL) of the supernatants were

145

collected after centrifugation for 10 min at 6000×g and used for gas chromatography

146

(GC) analysis.

147

To determine the parameters related to human gastrointestinal digestion after

148

ingestion, variations in the following parameters were modeled: gastrointestinal pH,

149

digestion time, and the S/L ratio. To analyze the effect of gastrointestinal pH, gastric

150

and intestinal phase digestion was examined at pH 1.13, 1.67, 2.12, 2.66, 3.01 and

151

6.01, 6.59, 7.01, 7.66 and 8.02, respectively. To determine the effect of digestion time,

152

samples were incubated for 10, 60, 90, 120 or 180 min and 10, 30, 60, 120, 210, 300,

153

360 or 480 min, respectively. The effects of the S/L ratio on the bioaccessibilities of 7


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the three pesticides were examined by altering the presence of apple samples (ratios

155

of 1/20, 2/20, 5/20, 8/20 and 10/20). All experiments were conducted independently

156

three times.

157

2.3. Caco-2 human intestinal cell culture The procedure for cellular uptake was developed according to D’Imperio et al.

158

25

159

2016

with slight modifications. Thawed Caco-2 cells were maintained and

160

expanded in a 75-cm2 flask using DMEM with 10.00 % FBS at 37 °C under an

161

atmosphere of 5.00 % CO2 and 95.00 % air at constant humidity. The expanded cells

162

(reaching 80.00 % confluence) were trypsinized by treatment with 0.25 %

163

trypsin-EDTA (ethylene diamine tetraacetic acid) solution at 37 °C, washed, diluted,

164

and resuspended in DMEM with 10.00 % FBS in a new 75 cm2 flask. For the

165

transport experiments, cells serially passaged 24 times were seeded into

166

polycarbonate membrane-coated transwell cell culture inserts (24-well plate, pore size

167

0.4 µm × 6.5 mm; Merck Millipore, Guyancourt, France) at a density of 100,000 cells

168

/ filter in 2.00 mL medium. The cellular monolayers were cultured for 21 d

169

post-confluence in DMEM supplemented with 1 % antibiotic solution, and the

170

DMEM replaced every other day until day 21.

171

2.4. Assessment of transepithelial electrical resistance, toxicity, and cell viability

172

The integrity of the monolayer was assessed and validated by measuring

173

transepithelial electrical resistance (TEER) using Millicell-ERS apparatus (Millipore,

174

cat.#: MERS00002). TEER values were obtained following the manufacturer’s

175

protocol and according to D’Imperio et al.

25

, with values>200 ohm/cm2 used as the 8



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threshold to determine tight junctions in the cell monolayer. Each experimental

177

treatment was performed in triplicate.

178

A MTT cell proliferation and cytotoxicity assay kit was used to determine cell

179

viability. Seeded cells (density of 100,000) in a 96-well plate were added to the test

180

compounds then incubated at 37 °C for 0, 12, 24, 48 or 72 h. Cell viability was

181

determined according to the manufacturer’s instructions, and absorbance read at 550

182

nm in a 96-well plate Multiskan GO reader (Thermo Scientific, Hudson, NH, USA).

183

Cells treated with (DMSO)were used as a control. Cellular viabilities under all

184

treatments were measured independently three times.

185

2.5. Analysis of pesticide transport using Caco-2 cells

186

The monolayers were washed twice in phosphate buffered solution (PBS)

187

(pH 7.3, 37 °C) then balanced for 15 min at 37 °C. A total of 0.60 mL of appropriate

188

dilutions (pH 6.5) of standard solution were mixed with PBS then placed in the apical

189

(AP) or basolateral chamber (BL) of the insert. The opposite chamber was

190

supplemented with 1.20 mL of DMSO dilution (diluted with PBS, pH 7.3). Cell

191

cultures were incubated at 37 °C (shaken at 50 rpm) under 5 % CO2 with 95 %

192

relative humidity for 0, 30, 60, 90, 120 or 150 min. Following incubation, 0.60 mL of

193

PBS solution from the BL or AP chamber was collected and stored at −80 °C until

194

liquid chromatography-mass spectrometry (LC-MS) analysis. Each experiment was

195

performed in duplicate.

196

2.6. Sample analysis

197

GC analysis. Samples extracted from gastrointestinal juice were analyzed using 9


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an Agilent 7890 GC (Agilent Technologies Inc., CA, USA; extraction and clean-up

199

methods are documented in Section 1 of the Supplemental Materials), equipped with a

200

63

201

0.25 µm film thickness). Operating conditions were: initial oven temperature, 80 °C

202

for 1 min followed by a linear increase to 220 °C at 20 °C/min then by 15 °C/min to

203

250 °C before holding at 290 °C for 5 min. Both injector and detector temperatures

204

were maintained at 250 °C. The carrier gas was nitrogen (99.999%) and the flow rate

205

was 1.2 mL/min. Pesticide concentrations were quantified using the external standard

206

method with a lower limit of reporting of 2 µg. Recovery of each pesticide was in the

207

range of 72.45–115.55 % (Table S1), fulfilling the requirements of pesticide residue

208

analysis.

Ni electron capture detector and capillary column with HP-5MS (30 m × 25 µm ×

209

LC-MS analysis. Samples collected from the transwell chamber were filtered

210

through a 0.22 µm nylon filter then analyzed using a Waters AcquityTM ultra

211

performance liquid chromatography interfaced to the XEVO Triple Quad mass

212

spectrometry system (UPLC-MS/MS) (Waters Co., Milford, MA, USA). A 2.10 ×

213

100 mm column packed with 1.70-µm particles (ACQUITY ® UPLC BEH

214

C18column, Waters) was employed and maintained at 35 °C. The mobile phase was

215

composed of HPLC grade H2O (A) and acetonitrile (B), with a flow rate of 0.30

216

mL/min used throughout and an injection volume of 5.00 µL. Elution conditions are

217

summarized in Table S2. The MS system was operated in multiple-reaction

218

monitoring (MRM) mode, equipped with an electrospray ionization source (ESI +).

219

Parameters were: source temperature, 150 °C; capillary voltage, 3.00 kV; cone 10



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220

voltage, 28 V; desolvation gas flow, 800 L / h; cone gas flow, 50 L / h; desolvation

221

temperature 400 °C. MRM conditions are detailed in Table S3.

222

2.7. Statistical analysis

223

Data are expressed as the mean ± standard deviation. Statistical analysis of each

224

parameter was performed using analysis of variance (ANOVA) followed by Tukey’s

225

test 26. All figures were drawn using Origin Pro 9.0 software (Origin Lab Corporation,

226

USA). Differences among means were considered statistically significant at a p-value

227

of 0.05.

228 229 230

Bioaccessibility of the apple samples (gastric and intestinal) was calculated using the following formula:

Bioaccessibility BA, % = × 100 %

231

where C1 is the concentration of the compound of interest in the intestinal or

232

gastric juice (mg / kg), V is the volume of intestinal or gastric juice (mL), C2 is the

233

concentration of the compound of interest in the apple sample (mg/ kg), and M is the

234

weight of the apple sample (g).

235

The bioaccessible concentration was calculated by the formula below:

236

Bioaccessible concentration mg/kg = W mg/kg × BA %

237

Where W is the concentration of compound of interest in apples.

238

The apical to basolateral (AP to BL) permeability coefficients (Papp) of the according

239

pesticides were calculated according to the equation:

240 241

P#$$ =

%& %'

(

× )×

*

where Q is the amount of the compound of interest appearing in the acceptor 11


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compartment as a function of time (t), A is the surface area of the Transwell

243

membrane (1.12 cm2), and C0 is the initial the compound of interest concentration in

244

the donor compartment.

245 246

3. Results and discussion

247

3.1. Effect of gastrointestinal pH on the bioaccessibility of pesticides

248

pH is thought to affect the solubility and release of certain contaminants from the

249

food matrix 27. However, Charman et al. 1997

28

250

the overall effect of increasing pH of the stomach on bioavailability in response to

251

food ingestion. Thus, in this study, fasting conditions (pH of approximately 2) were

252

imitated to investigate the effect of gastrointestinal pH on the bioaccessibility of

253

pesticides (Fig. 1).

found that it was difficult to predict

254

Bioaccessibility of hexaconazole and spirodiclofen changed little from pH 1.13

255

to 1.67, and difenoconazole bioaccessibility increased with increasing pH from 1.13

256

to 2.12, but decreased slightly thereafter from pH 2.12 to 3.01. Hexaconazole showed

257

relatively high bioaccessibility (67.01–80.00 %) in the gastric phase conditions, and

258

was significantly more bioavailable than in the intestinal phase (49.44–56.34 %). A

259

similar phenomenon was observed previously 20. This was possibly due to the stability

260

of hexaconazole in weakly acid to alkaline solutions, with aqueous hydrolysis

261

occurring at low pH 29. As a result, release of hexaconazole from the matrix is faster

262

in the gastric phase compared to the intestinal phase

263

hexaconazole shows higher affinity for pepsin than pancreatin, subsequently slowly

30

. It is also possible that

12



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release during the intestinal phase. In contrast, the increased pH from gastric to

265

intestinal phase conditions resulted in little variation in the bioaccessibility of

266

spirodiclofen or difenoconazole, possibly due to similar affinity for pepsin and

267

pancreatin in the gastric and intestinal phases. These results confirmed that pH value

268

is one of the main factors affecting bioaccessibility and absorption efficiency during

269

the gastric and intestinal phases.

270

3.2. Effect of digestion time on the bioaccessibility of pesticides

271

In general, the digestion time of the stomach under fed conditions is

272

approximately 2 h, and 6 h in the intestinal phase 31. Yu et al. 2009

32

273

release of contaminants from the food matrix to simulated gastric and intestinal juice

274

increased with increasing incubation time. Furthermore, in a previous digestion model,

275

digestion time in the gastric and intestinal phases were identified as a key factor in

276

the bioaccessibility of certain elements

277

and 10 - 480 min were therefore selected to investigate the bioaccessibility of the

278

three pesticides in the gastric and intestinal phases, respectively.

found that the

33, 34

. In this study, digestion times of 10–150

279

In gastric samples (Fig. 2a), bioaccessibility reached a maximum at 90 min in the

280

simulated gastric juice and did not change significantly with increasing digestion time.

281

At that digestion time, bioaccessibility of the three pesticides was in the order of

282

hexaconazole (70.43–76.28 %) > difenoconazole (53.70–62.57 %) > spirodiclofen

283

(25.20–30.14 %). A similar trend was observed with the gastric pH samples. Overall,

284

increased digestion time resulted in only a moderate increase in release.

285

From gastric to intestinal phase conditions, certain chemicals are absorbed by 13


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the blood via various channels, subsequently affecting release efficiency after

287

digestion in the intestinal phase

288

intestinal phase involves three phases: absorption, distribution, and elimination 9. In

289

this study, all three pesticides showed relatively high bioaccessibility at 210 min (Fig.

290

2b), when the bioaccessibilities of hexaconazole, difenoconazole, and spirodiclofen

291

were 79.45 %, 68.75 %, and 81.43 %, respectively. This was followed by a notable

292

drop to 300 min. A marked trend similar to a normal distribution was observed,

293

possibly because absorption by the human intestinal epithelium increased the release

294

efficiency after digestion in the intestinal system. Maldonado-Valderrama et al.

295

found that the presence of bile salts affected the release of heavy metals during the

296

intestinal phase. Here, bioaccessibility of difenoconazole and spirodiclofen was

297

significantly higher during the intestinal phase than the gastric phase, while

298

hexaconazole showed relatively low bioaccessibility. This was possibly due to the

299

effect of bile salts on the digestion of lipids, thereby increasing difenoconazole and

300

spirodiclofen bioaccessibility. In contrast, interaction with chemical composition and

301

the formation of insoluble complexes possibly led to a reduction in the release of

302

hexaconazole.

303

3.3. Effect of the S / L ratio on bioaccessibility

35

. The uptake profile of contaminants during the

36

304

The S / L ratio represents the amount of food matrix compared to the volume of

305

gastric or intestinal juice. In in vitro digestion models, the S/L ratio was found to be a

306

key factor in the bioaccessibility of heavy metals 37. Van et al. (2004) also observed a

307

decrease in bioaccessibility with an increasing S / L ratio in the range of 1 / 100 to 1 / 14



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308

1 38. In this study, a ratio of 1 / 20 to 10 / 20 of apple to gastric or intestinal juice was

309

therefore selected. A similar correlation was observed (Fig. 3), with a strong

310

logarithmic relationship between bioaccessibility and the S / L ratio. The coefficient

311

of R2 ranged from 0.9198 to 0.9848 and 0.9526 to 0.9951 in the simulated gastric and

312

intestinal phases, respectively, suggesting that the simulated relationship was an

313

accurate reflection of the relative bioavailability.

314

The S/L ratio had a marked effect on pesticide bioaccessibility, with significant

315

decreases in hexaconazole, difenoconazole and spirodiclofen availability with S/L

316

ratio in the range 1/20 to 5/20 (gastric phase) and 1/20 to 8/20 (intestinal phase).

317

These reductions were probably due increased adsorptivity and the subsequent effect

318

on the release efficiency. The adsorptivity effect of solids is known to increase with

319

increased amount of matrixes

320

increased solubilization of certain elements can eliminate certain compounds due to

321

the formation of insoluble complexes 19. However, in this study, the effect of the S/L

322

ratio slowed or disappeared within a range of 5/20 and 8/20 to 10/20 in the gastric and

323

intestinal phase, respectively. This lessening effect was thought to have been

324

caused by the presence of a saturated adsorption state, or perhaps the increased

325

sample size resulted in partial contact between the apple and the gastrointestinal juice,

326

subsequently resulting in insufficient release into the simulated gastrointestinal juice.

327

Overall,

328

affecting bioaccessibility, although there were no significant differences between the

329

gastric and intestinal phases.

therefore,

the

39

S/L

. In addition, Peixoto et al. (2016) revealed that

ratio

was

found

to

be

a

major

factor

15


Page 17 of 32


330

3.4. Analysis of pesticide transport using Caco-2 cells

331

Before analyzing bioavailability, the integrity and differentiation of the Caco-2

332

cell monolayer needs monitoring via measurements of TEER, with acceptable TEER

333

values for bioassays ranging from 200 to 1000 ohm / cm2

334

TEER values increased along an S-curve, reaching a stable phase at 21 d. These

335

findings show that there were sufficiently tight junctions between the cells. This trend

336

of increasing TEER values was also consistent with previous findings

337

values >200 ohm / cm2, showed that the cells were fused and differentiated into

338

intestinal epithelial cells. Moreover, the TEER values of the cell monolayer were

339

>500 ohm / cm2 at 16 d, but reaching a stable phase at 21 d, which less than 650

340

ohm/cm2, indicating good integrity. Non-differentiated Caco-2 cells have no similar

341

physiological characteristics of enterocytes and do not represent an appropriate cell

342

model for intestinal uptake studies 41,

343

used in the subsequent pesticide transport analysis.

25

. As Fig. 4a shows, the

40

. The TEER

thus, the 21-d differentiated Caco-2 cells were

344

In general, cell viability in a Caco-2 assay should be 1.00×10-4 cm/s 44. As

369

shown in Table 1, permeability coefficients of 0.86 - 6.40 × 10-3 cm / s were observed,

370

indicating that difenoconazole could be completely absorbed in humans. Furthermore,

371

the ratio of Papp (BL-AP) to Papp (AP-BL) can be used to predict the mode of transportation

372

45

373

transport but was transformed from BL to AP, is thought to be transported by a

. As a result, difenoconazole, which exhibited moderate recovery in AP to BL

17


Page 19 of 32


374

membrane transporter located on the AP-side of the membrane of the small intestines.

375

Drug transporters such as P-glycoprotein (P-gp) and Multidrug Related Protein (MRP)

376

efflux pumps are important factors in determining drug transport

377

further investigation.

378

3.5. Bioaccessible concentrations and health risk estimates

46, 47

, warranting

379

Based on negligible cell uptake efficiency, realistic predictions of potential risk

380

are difficult. In this study, calculations of bioaccessible concentrations were carried

381

out according to Wang et al. 2011 48, using the estimated bioavailability to compare

382

the estimated risk dose and acceptable daily intake. To do so, the following was

383

simulated: an S/L ratio of 1/10 with a digestion time of 2 h at pH 3.00 for gastric

384

samples and 4 h at pH 8.35 for intestinal samples, as well as an increase to the amount

385

of the maximum residue limit (MRL) accepted by the China to assume a worst-case

386

scenario

387

gastrointestinal digestion 19, 50.

49

. These reference values were based on simulations of normal human

388

Table 2 shows the bioaccessible concentrations of each pesticide, indicating low

389

percentages of exposure (within the range 14.08–29.30 %). Hexaconazole had the

390

lowest exposure level (0.0704 mg/kg), which was also lower than the MRL (0.50

391

mg/kg). ADI values are obtained based on experimental toxicological data of a

392

particular pesticide. Difenoconazole and spirodiclofen are reportedly have similar

393

ADI values (0.01 mg/kg bw), suggesting similar toxicology. However, in this study,

394

levels of difenoconazole were more than twice those of spirodiclofen, suggesting that

395

spirodiclofen creates significantly lower risk to human health than difenoconazole. 18



Page 20 of 32

396

Hexaconazole and spirodiclofen also presented similar exposure levels, but they have

397

significantly different ADI values, indicating the higher potential risk of hexaconazole

398

compared to spirodiclofen. Although hexaconazole exhibited the highest potential risk,

399

levels in the gastrointestinal juice were lower than the amount of ingested compound.

400

Moreover, it is important to remember that these calculations assumed the worst-case

401

scenario, and thus, the risk is perhaps overestimated. In this context, an exhaustive

402

study of the possible synergistic effects of pesticides is required in order to create a

403

more accurate understanding of the health risks.

404

Acknowledgments

405

This work was supported by the National Natural Science Foundation of China [grant

406

number 31601663] and the Anhui Natural Science Foundation [grant number

407

1508085MC50].

408

Supporting Information

409

Extraction and clean-up methods during processing of the apple samples (Section 1).

410

Analytical recovery, relative standard deviations (RSDs), correlation coefficients (r2)

411

and limits of quantification (LOQ) for the gastrointestinal juice samples studied.

412

(Table S1). Elution (Table S2) and MRM (Table S3) conditions for UPLC–MS/MS

413

analysis of hexaconazole, spirodiclofen, and difenoconazole.

414 415 416 417 418 419 420 421

References 1.

Martín Cerdeño, V. J., Consumo de frutas y hortalizas en España. Distribución y consumo 2009,

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Gonzalez-Barreiro,

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554 555

Page 24 of 32

five in vitro digestion models to study the bioaccessibility of soil contaminants. Environ Sci Technol 2002, 36, 3326-3334.

556 557 558 559 560 561

Figure captions

562 563

Fig. 1. Effect of pH in the gastric (a) and intestinal phase (b) on the bioaccessibility of

564

hexaconazole, spirodiclofen, and difenoconazole. Different lower-case letters at the

565

top of columns represent significant differences in bioaccessibility at a p-value of

566

0.05.

567 568

Fig. 2. Effect of digestion time in the gastric (a) and intestinal phase (b) on

569

the bioaccessibility of hexaconazole, spirodiclofen, and difenoconazole.

570 571

Fig. 3. Effect of the S/L ratio in the gastric (a) and intestinal phase (b) on

572

the bioaccessibility of hexaconazole, spirodiclofen, and difenoconazole. Fitted curves

573

were plotted using the Log2P1 function of Origin Pro 9.0 software. The horizontal

574

ordinate represents the ratio of apple sample to the volume of gastric or intestinal

575

juice.

576 23


Page 25 of 32


577

Fig. 4. Analysis of transepithelial electrical resistance (TEER) (a) and cell viability (b)

578

using

579

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay.

Millicell-ERS

apparatus

coupled

with

an

MTT

580

24



581

Page 26 of 32

Tables

582 583

Table 1. Concentration effects on difenoconazole transport across Caco-2 cell monolayers and the corresponding Papp value Concentration

a Papp AP→BL584

AP→BL transport (ng)

（µg/mL）

30 min

60 min

90 min

120 min

150 min

(cm/h)

0.01

3.20 ± 0.35 × 10-4

3.21 ± 0.17 × 10-4

1.52 ± 0.31 × 10-4

3.93 ± 0.25 × 10-4

5.45 ± 0.33 × 10-4

1.00 ± 0.08 × 10-3

0.05

1.00 ± 0.13 × 10-3

5.35 ± 0.43 × 10-3

4.62 ± 0.27 × 10-3

7.25 ± 0.42 × 10-3

4.08 ± 0.39 × 10-3

-2 6.40 ± 0.30 × 10586

0.1

1.87 ± 0.20 × 10-3

4.78 ± 0.39 × 10-3

5.76 ± 0.58 × 10-3

1.53 ± 0.36 × 10-2

1.22 ± 0.18 × 10-2

0.85 ± 0.09 ×10-3

585

587 588

a

Papp AP→BL: Transport from the apical to basolateral side. Data represent means ± standard deviation.

589 590 591 592

25


Page 27 of 32


593

Table 2. Risk assessment based on the acceptable daily intake (ADI) and the in

594

vitro bioaccessibility of hexaconazole, spirodiclofen, and difenoconazole in

595

gastrointestinal juice. MRL

Bioavailability in

Bioavailability in

Bioaccessible

a

gastric juice (%)

intestinal juice (%)

concentration (mg/kg)

Hexaconazole

0.5

34.68 ± 3.21 c

40.59 ± 1.24

0.0704 (14.08 %) b

Spirodiclofen

0.5

41.18 ± 1.58

40.23 ± 2.97

0.0828 (16.56 %)

Difenoconazole

0.5

48.93 ± 4.36

59.89 ± 3.10

0.1465 (29.30 %)

Pesticide

(mg / kg)

596

a

597

safety standard - maximum residue limits for pesticides in food).

598

the bioaccessible concentration to the MRL. c standard deviation.

Maximum residue limit, obtained from the GB 2763-2014, China (national food b

Ratio of

599 600 601 602 603 604 605 606 607 608 609 610 26



611

Page 28 of 32

Fig. 1.

612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 27


Page 29 of 32


633 634

Fig. 2.

635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 28



Page 30 of 32

655 656 657

Fig. 3.

658 659 660 661 662 663 664 665 666 667 668 669 670 671 672 673 674 675 676 29


Page 31 of 32


677

Figure. 4.

678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 30



693 694 695 696 697 698

Page 32 of 32

TOC Graphic

31


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