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Bioactive Constituents, Metabolites, and Functions

In vitro Determination of Protein Conjugates in Human Cells by LC-ESI-MS/MS after Benzyl Isothiocyanate Exposure Carla Kuehn, Tobias von Oesen, Corinna Herz, Monika Schreiner, Franziska S. Hanschen, Evelyn Lamy, and Sascha Rohn J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01309 • Publication Date (Web): 07 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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Journal of Agricultural and Food Chemistry

In vitro Determination of Protein Conjugates in Human Cells by LC-ESI-MS/MS after Benzyl Isothiocyanate Exposure

Carla Kühna, Tobias von Oesena, Corinna Herzb, Monika Schreinerc, Franziska S. Hanschenc, Evelyn Lamyb, Sascha Rohna* a

Institute of Food Chemistry, HAMBURG SCHOOL OF FOOD SCIENCE, University of

Hamburg, Grindelallee 117, 20146 Hamburg, Germany b

Molecular Preventive Medicine, Institute for Infection Prevention and Hospital Infection

Control, Medical Center, University of Freiburg, 79106 Freiburg, Germany c

Leibniz Institute of Vegetable and Ornamental Crops, Theodor-Echtermeyer-Weg 1, D-

14979 Großbeeren, Germany

E-Mail:

[email protected],

[email protected],

[email protected],

[email protected],

[email protected],

[email protected], [email protected]

*Corresponding Author: Prof. Dr. Sascha Rohn, Grindelallee 117, 20146 Hamburg, Germany, Email: [email protected], phone +49 40/42838-7979, fax +49 40/42838-4342

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Abstract

2

Glucosinolates and their breakdown products, especially isothiocyanates (ITC), are

3

hypothesized to exert a broad range of bioactivity. However, physiological mechanisms

4

are not yet completely understood. In this study, formation of protein conjugates after

5

incubation with benzyl isothiocyanate (BITC) was investigated in vitro. Survey of protein

6

conjugates was done by determining BITC cysteine and lysine amino acid conjugates

7

after protein digestion. Therefore, a liquid chromatography-tandem mass spectrometry

8

(LC-ESI-MS/MS) method was developed and validated. Stability studies showed that

9

cysteine conjugates are not stable under alkaline conditions, whereas lysine conjugates

10

did not show any correlation to pH values, although stability increased at low

11

temperatures. Lysine conjugates were the preferred form of protein conjugates and

12

longer BITC exposure times led to higher amounts. Knowledge about the reaction sites

13

of ITC in eukaryotic cells may help to understand the mode of action of ITC leading to

14

health promoting as well as toxicological effects in humans.

15

16

Keywords

17

HepG2 cells, Benzyl isothiocyanate, Protein conjugates, LC-ESI-MS/MS, Metabolism

18


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1 Introduction

20

Glucosinolates are secondary plant metabolites occurring in plants belonging to the

21

Brassicales family such as cabbage, broccoli, mustard and nasturtium. As a result of

22

tissue damage, caused for instance by cutting or chewing, the plant-endogenous

23

enzyme myrosinase, hydrolyzes the glucosinolates, releasing several breakdown

24

products. From those, the isothiocyanates (ITC) gained particular importance over the

25

years1. After ingestion, ITC are mainly absorbed passively into the epithelial cells of the

26

gastrointestinal tract. Subsequently, ITC are conjugated with glutathione (GSH),

27

metabolized along the mercapturic acid pathway and finally excreted via the urine as the

28

corresponding mercapturic acid2. Numerous in vitro studies suggest various health-

29

benefits of ITC such as anti-bacterial, anti-inflammatory, and anti-diabetogenic activity3-6.

30

Moreover, these compounds seem to have cancer preventive effects: in cell culture

31

experiments, an effective growth suppression of primary human ovarian carcinoma cells

32

was observed following a treatment with BITC (chemical structure in Figure 1) in

33

concentrations between 5 and 50 µM7. Further, a mediation of the cell cycle arrest and

34

the induction of apoptosis in human hepatoma cells was detected in an in vitro model

35

after treatment with 4-(methylthio)butyl isothiocyanate (MTBITC) exceeding 10 µM8. The

36

anti-carcinogenic activity of ITC presumably bases on different mechanisms of action, as

37

tumor formation and proliferation is influenced at different stages of cancer

38

development9, 10. In contrast to potential health-beneficial properties, some studies also

39

indicated negative effects of ITC such as triggering of skin sensitization by interaction

40

with peptides or even genotoxic effects resulting from a reaction with DNA11, 12. Although

41

ITC have been studied for decades, the exact mode of action is still discussed

42

controversially. A lot is known about the metabolism of ITC, but still there is lack of 3 ACS Paragon Plus Environment


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knowledge on side-reactions. One possible reason for the wide-ranging and inconsistent

44

effectiveness of ITC is their high reactivity against nucleophiles during the uptake into or

45

within the cell. Protein side chains and DNA seem to be the most important reaction

46

sites, as they occur in large quantities (as for proteins) or might lead to lasting adverse

47

effects (DNA adducts).

48

Due to the high reactivity, the reaction is not limited to the thiol group of GSH, but also

49

occurs with thiol groups of cysteine residues in proteins and further peptides forming

50

dithiocarbamates13. Based on the chemical resemblance of thiol and amino groups,

51

reactions of the electrophilic ITC with amino groups can occur as well forming thiourea

52

derivatives14,

53

targets for ITC also in eukaryotic cells or fractions thereof. Although this seems to have

54

a significant impact, only very rare data about the interaction of ITC with cell proteins is

55

available16. While there is some information on the formation of protein conjugates in

56

protein-rich food and albumin conjugates in human blood samples, it is not clear if and to

57

which extent ITC interact also with cell proteins17, 18.

58

Consequently, the aim of this study was to develop and validate a high performance

59

liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) method for

60

investigating the formation of protein conjugates in human cells after treatment with

61

BITC in vitro. Thus, the formation of BITC-cysteine (BITC-Cys) and BITC-lysine (BITC-

62

Lys) was investigated after digestion of the proteins. BITC was chosen exemplarily, as it

63

is naturally released by brassicaceous plants and its metabolism is well known19.

64

Furthermore, the stability of the formed conjugates was investigated in dependence of

15

. Therefore, cysteine and lysine side chains of proteins are presumable


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the pH value and temperature in order to get information about possible loss of analytes

66

during sample storage and sample preparation.

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2 Materials and Methods

68

2.1 Chemicals and Materials

69

1,4-dioxane, ammonia solution (25%), disodium hydrogen phosphate (98%), ethanol

70

(EtOH; HPLC grade), ethyl acetate (99.5%), and L-cysteine (99%), was purchased from

71

Carl Roth GmbH & Co. KG (Karlsruhe, Germany), Dulbecco's Modified Eagle Medium

72

(DMEM) with phenol red and L-glutamine, DMEM without phenol red and L-glutamine,

73

fetal calf serum (FCS), L-glutamine, penicillin-streptomycin solution, phosphate buffered

74

saline (PBS, without Ca and Mg) were obtained from Life Technologies (Darmstadt,

75

Germany), benzyl isothiocyanate (98%), Boc-L-lysine (99%), citric acid (99.5%),

76

methanol (MeOH; ultra LC-MS grade), and pronase E (from Streptomyces griseus, EC

77

3.4.24.4) were obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany),

78

ammonium chloride (99.8%), potassium chloride (99.5%), potassium dihydrogen

79

phosphate (99.5%), and sodium sulfate (99%) were obtained from Merck KGaA

80

(Darmstadt, Germany), formic acid (FA; 98%), and sodium chloride (99.5%) was

81

obtained from VWR International GmbH (Darmstadt, Germany), trifluoroacetic acid

82

(TFA; 99.5%) was purchased from AppliChem GmbH (Darmstadt, Germany), dimethyl

83

sulfoxide-d6 (DMSO-d6) was purchased from Eurisotop GmbH (Saarbrücken, Germany),

84

C18ec solid phase extraction cartridges (3 mL, 200 mg) were obtained from Macherey-

85

Nagel GmbH & Co. KG (Düren, Germany), All aqueous solutions were prepared with

86

water of Milli-Q quality.



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2.2 Chemical synthesis and characterization of the analytical standards

88

The analytical standards (Figure 2) were synthesized as described by Brüsewitz et al.

89

and Kumar et al. with minor modifications17,

90

dissolving L-cysteine (4 mmol) in water (30 mL) and then dropwise adding BITC (4

91

mmol), which was previously dissolved in 10 mL of EtOH:water (80:20, v/v). The

92

reaction mixture was stirred for 72 h at 20 °C. The obtained precipitation was filtered,

93

washed (with water, 10 mL then with EtOH, 10 mL), and subsequently recrystallized

94

from ethyl acetate. BITC-Lys was synthesized by adding BITC (4 mmol in 1,4-dioxane)

95

dropwise to Boc-L-lysine (4 mmol in sodium bicarbonate buffer (5 mL, pH 8,4)). After

96

incubating (10 h, 30 °C), the reaction mixture was extracted with ethyl acetate (3 x 5

97

mL). The remaining water phase was modulated to pH 4 and extracted again with ethyl

98

acetate (3 x 5 mL). After combining the organic phases, they were dried using

99

anhydrous sodium sulfate. The filtered solution was evaporated to dryness and the

100

residue was incubated with TFA (1 h, 20 °C) in order to remove the protection group.

101

Then, TFA was removed by a vacuum concentrator (Christ RVC 2-25 CDplus, Martin

102

Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) and the resulting

103

product was recrystallized in ethyl acetate for purification. Subsequently, the purity was

104

investigated using HPLC-UV resulting in values of 99% and 88% for BITC-Cys and

105

BITC-Lys, respectively. Further, the structures of the resulting compounds were

106

determined by NMR analysis.

107

BITC-Cys: 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.31 (m, 5H, aromatic), 4.83 (m, 2H,

108

CH2(c)), 3.69 (dd, 1H, CH (a)), 3.58 (dd, 1H, CH (b)), 3.33 (dd, 1H, CH (b))

19

. In brief, BITC-Cys was synthesized by


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BITC-Lys: 1H NMR (400 MHz, DMSO-d6) δ ppm: 8.20 (s, 1H, NH), 7.85 (s, 1H, NH),

110

7.29 (m, 5H, aromatic H), 4.65 (s, 2H, CH2 (f)), 3.47 (m, 5H, CH (a), CH2 (e), NH2), 1.78

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(m, 2H, CH2 (d)), 1.45 (m, 2H, CH2 (b)), 1.35 (m, 2H, CH2 (c))

112

2.3 Cell cultures

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The HepG2 cell line (ACC-180) was purchased from the German Collection of

114

Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany). The cells were

115

cultured

116

penicillin/streptomycin solution (1%). The incubation was performed in a 95% humidified

117

incubator at 37 °C and 5% CO2. For the experiments, 2 x 106 cells were seeded in T75

118

cell culture flasks and incubated for 48 h. Then, cells were washed twice with PBS and

119

exposed to 10 µM or 30 µM BITC for the indicated time points in cell culture medium

120

without FCS and without phenol red. Afterwards, cells were harvested using a cell

121

scraper with subsequent centrifugation at 300 x g for 5 min at 4 °C, supernatant medium

122

was removed and cell pellets were washed twice with cold PBS. The cell pellet and

123

supernatant were frozen at -80 °C.

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2.4 Sample preparation

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Sample preparation was conducted referring to a previously published method.18 For

126

determination of protein conjugates in the supernatant of the cell cultures, the thawed

127

samples were vortexed (1 min) and an aliquot of 1 mL was used for analysis. Cell pellets

128

were thawed, suspended in 500 µL of deionized water and lysed by ultrasonic for 20

129

min. After that, 500 µL PBS (pH 7.4) was added and the samples were vortexed for 1

130

min. For protein digestion 100 µL of protease solution was added (10 mg/mL) and the

in

DMEM

with

phenol

red

supplemented

with

FCS

(15%)

and



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samples were incubated overnight (37 °C, 15 h). For enzyme inactivation, the samples

132

were acidified with TFA (50 µL), vortexed (30 sec) and centrifuged (4 °C, 10 min, 20854

133

g). The supernatant was applied to previously prepared SPE cartridges. Preparation was

134

done by conditioning the cartridges with MeOH (3 mL) and equilibrating with FA (3 mL,

135

0.1% in water). After the samples passed through, the cartridges were washed with FA

136

(3 mL, 0.1% in water) and analytes were eluted with FA (3 mL, 0.1% in MeOH).

137

Subsequently, the samples were evaporated to dryness and the residues were re-

138

dissolved in FA (100 µL, 0.1% in MeOH:water, 80:20, v/v). For analysis, aliquots of 3 µL

139

were injected to the LC-ESI-MS/MS system. For the determination of BITC conjugates

140

with free amino acids in the incubation medium, the above mentioned method was

141

applied without protein digestion.

142

2.5 LC-ESI-MS/MS analysis

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LC-ESI-MS/MS analysis was conducted on a 4000 QTrap triple quadrupole MS/MS

144

system (AB Sciex Germany GmbH, Darmstadt, Germany) equipped with an Agilent

145

1200 series HPLC system (Agilent Technologies Deutschland GmbH & Co. KG,

146

Waldbronn, Germany). The data were acquired and processed using the software

147

Application Analyst 1.6.1 (AB Sciex Germany GmbH, Darmstadt, Germany). The

148

separation was performed on a Kinetex C18 column (5 µm, 100 Å, 150 x 2.1 mm;

149

Phenomenex Ltd., Aschaffenburg, Germany), equipped with a guard column of the

150

same material (2.1 x 4.6 mm). The column was equilibrated in a column oven to 20 °C

151

and the autosampler was set to 4 °C. After injection of 3 µL of the sample,

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chromatographic separation was done by a mobile phase consisting of 0.1% FA in water

153

(A) and 0.1% FA in MeOH (B) and a flow rate of 250 µL/min. The gradient started with 8 ACS Paragon Plus Environment

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90% of A and was held for 3 min. After that, B was increased from 10% to 90% within 8

155

min and then held for 6 min. In a final step the column was equilibrated at 10% B for 8

156

min. The quantitation of BITC-Cys and BITC-Lys was done by an external calibration

157

curve in a concentration range between 0.5 µM and 50 µM.

158

2.6 Method validation

159

In order to obtain the calibration model, accuracy, precision, specificity, and selectivity

160

the analysis of BITC-Cys and BITC-Lys adducts was validated based on the guidance of

161

bioanalytical method validation of the U.S. Food and Drug Administration (FDA). The

162

linearity of the method was confirmed by analyzing three twelve-point standard curves in

163

a concentration range of 0.05 to 200 µM. Therefore, calibration standards were prepared

164

in MeOH/H2O (80/20) with 0.1% FA added. The determination of the limit of detection

165

(LOD) and the lower limit of quantification (LLOQ) was performed by analyzing ten blank

166

samples. The samples for the determination of the precision were measured fivefold for

167

each concentration (0.5 µM, 5 µM and 50 µM).

168

2.7 Influence of the pH value and temperature on the stability of BITC-Cys and

169

BITC-Lys

170

The stability of BITC-Cys and BITC-Lys was investigated by incubating the standard

171

substances under varying pH values and temperatures. Using various buffer solutions,

172

the stability was determined under acidic (citrate phosphate buffer, pH 2), neutral

173

(phosphate buffer, pH 7), and basic (ammonium chloride/ammonia buffer, pH 10)

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conditions. The impact of temperature was investigated at 37 °C (mimicking

175

incubation/digestion temperature), 22 °C (room temperature), and 4 °C (autosampler 9 ACS Paragon Plus Environment


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temperature). For the analysis, BITC-Cys and BITC-Lys were dissolved at a

177

concentration of 100 µM in the various buffer solutions. Aliquots (500 µL) of the samples

178

were analyzed in duplicate after 2, 5, 8, 15, 24, and 48 h.

179

3 Results

180

A method for the determination of ITC protein conjugates was developed and validated.

181

Further, the formation of protein conjugates in human HepG2 cells was investigated

182

after treatment with BITC.

183

3.1 Method development and validation

184

In order to optimize the mass spectrometry parameters, solutions of the analytes were

185

introduced into the ESI source by direct-flow injection. Sufficient ionization efficacy was

186

obtained for both analytes in the positive ionization mode. Therefore, the declustering

187

potential, collision energy, and collision cell exit potential was optimized (Table 1).

188

Validation data of the analyzed compounds are shown in Table 2. Linearity range of

189

both analytes was from 0.05 to 100 µM. The linearity of the calibration curves was

190

proven by a coefficient of determination (R2) of 0.9985 or higher. BITC-Cys (5 nM)

191

showed a lower LLOQ value than BITC-Lys (38 nM). The precision of this method was

192

within the accepted range of 15% according to the FDA guidelines.

193

3.2 Influence of pH value and temperature on the stability of BITC-Cys and BITC-

194

Lys

195

Some studies indicate that dithiocarbamates are not stable under specific, especially

196

basic conditions15,

197

investigated and compared in order to estimate the possible loss of analytes during

20, 21

. Therefore, the stability of BITC-Cys and BITC-Lys was


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sample preparation procedures and storage for proving the reliability of the conjugates

199

to act as markers for protein modification by ITC.

200

Stability of BITC-Cys

201

BITC-Cys showed a reduced stability under basic (pH 10) and neutral (pH 7) conditions.

202

At pH 10 and 4 °C, only 57% of the initial concentration was left after 2 h. At higher

203

temperatures, the degradation was even more rapidly, showing only 20% of the initial

204

concentration at 37 °C and pH 10, after 2 h. No BITC-Cys was detectable at 37 °C after

205

8h and at 22 °C after 24 h. After 48 h, no BITC-Cys was detectable at pH 10 for any

206

temperature investigated. At neutral pH value, the stability increased compared to basic

207

conditions: at 4 °C, the concentration decreased by 20% and 30% within 2 h and 48 h,

208

respectively. At higher temperatures, the decrease was more rapidly, remaining only

209

10% and 5% for 22 °C and 37 °C, respectively. Acidic conditions clearly improved the

210

stability of BITC-Cys. All temperatures showed a similar decrease of 17-20% within 48 h

211

(Figure 3).

212

Stability of BITC-Lys

213

BITC-Lys showed different stability rates compared to BITC-Cys. The results indicated

214

no influence of the pH value and only a minor temperature influence. The recovery rates

215

of BITC-Lys at all pH values investigated did not differ much at one temperature level. At

216

37 °C, the recovery rate after 48 h was in the range of 83-85%. At 22 °C, the recovery

217

ranged between 89 and 91% and at 4 °C 89-93% of the initial concentration was

218

determined after 48 h. Thus, the temperature seems to have a significant influence on

219

the stability of BITC-Lys compared to the pH value. 11 ACS Paragon Plus Environment


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3.3 Determination of protein conjugates in human liver (HepG2) cells

221

Many studies investigated the metabolism of ITC in human cells, but there is only rare

222

data available about the formation of protein conjugates, so far. Therefore, cells have

223

been exposed to BITC and the occurrence of protein conjugates in the cell pellet has

224

been investigated. BITC-Cys and BITC-Lys were determined after protein digestion with

225

pronase E. As the incubation medium is supplemented with amino acids, the formation

226

of BITC conjugates in the incubation medium was investigated as well in order to

227

determine to which extent BITC is already bound in the incubation medium and thus, not

228

available for the cells.

229

Incubation supernatant

230

BITC-Cys and BITC-Lys were determined prior to and after protein digestion in order to

231

investigate the formation of conjugates with free amino acids and proteins that are

232

present in the supernatant. The incubation was performed with BITC at concentrations

233

of 10 µM and 30 µM for 2 h and 6 h. In the incubation supernatant, no BITC-Cys was

234

present, whereas BITC-Lys was detectable for both concentrations and exposure times.

235

The amounts of BITC-Lys deriving from protein conjugates ranged between 0.9 µM (2 h,

236

10 µM BITC) and 0.54 µM (2 h, 30 µM BITC), whereas the amount of BITC-Lys deriving

237

from free amino acids ranged between 1.2 µM (2 h, 10 µM BITC) and 3.7 µM (6 h, 30

238

µM BITC). Obviously, concerning the formation of BITC-Lys deriving from amino acids, a

239

longer exposure time of 6 h led to the formation of more BITC-Lys compared to a shorter

240

exposure time of 2 h at both BITC concentrations. Although the formation of BITC-Lys

241

deriving from protein conjugates increased after exposure with 30 µM BITC compared to

242

the exposure of 10 µM BITC, no significant difference was detected between 2 h and 6 h 12 ACS Paragon Plus Environment

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of exposure at 30 µM BITC (Figure 4). Differences were analyzed statistically by a t-test

244

with a confidence interval of 95% with IBM SPSS Statistics software.

245

Cell pellet

246

In the cell pellet, BITC-Cys and BITC-Lys adducts were detected. BITC-Cys showed

247

lower concentrations (0.03-0.11 µM) compared to BITC-Lys (3-21.4 µM). Further, BITC-

248

Cys concentrations decreased with increasing BITC concentration. However, an

249

extended exposure time of 6 h resulted in higher BITC-Cys concentrations compared to

250

the same BITC level at shorter incubation times (2 h). The highest amount of BITC-Lys

251

(21.4 µM) was determined after 6 h of BITC exposure and a concentration of 30 µM.

252

BITC exposure for 2 h with 30 µM resulted in a considerably lower BITC-Lys

253

concentration (5.4 µM). Exposure to 10 µM BITC resulted in concentrations of 3.0 µM

254

and 12.5 µM for 2 h and 6 h, respectively (Figure 5).

255

4 Discussion

256

In this study, a selective and sensitive LC-ESI-MS/MS method was developed and

257

validated for a rapid and reliable determination of BITC protein conjugates in eukaryotic

258

cells. The validation data indicate a reliable and reproducible method according to the

259

FDA guidelines. It was demonstrated in an in vitro model, that protein conjugates in

260

human cells are formed after exposure to BITC. Though this reaction is not directed,

261

because of the high reactivity of ITC, valuable information about the effect of ITC in the

262

cells can be derived.

263

Knowledge about the mode of ITC interaction with nucleophilic compounds is a

264

fundamental need for understanding their bioactivity in the human cells as well as in the 13 ACS Paragon Plus Environment


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human body. The metabolism of ITC in eukaryotic cells is mainly conducted through

266

non-enzymatically and enzymatically catalyzed conjugation with glutathione (GSH). After

267

passive diffusion of ITC into the cell, they undergo significant metabolic conversion. With

268

increasing ITC concentration, GSH is subsequently depleted in the cell. The decreasing

269

amount of GSH leads to reactions with larger molecules containing electrophilic

270

substitutes like thiol, amino, and hydroxyl groups22. For bovine serum albumin, it was

271

already shown that at physiological pH, ITC react primarily with the thiol groups of

272

cysteine residues of amino acids or proteins23. While the ITC-conjugation with thiol

273

moieties is reversible, reactions with amino groups (e.g. the ε-amino group of lysine)

274

forming thiourea derivatives seems to be irreversible24. The formation of ITC-protein

275

conjugates leads to structural and functional changes of the protein part. Earlier studies

276

demonstrated a reduced solubility of derivatized proteins in aqueous solutions, which

277

was ascribed to a replacement of hydrophilic lysine side chains by the more hydrophobic

278

BITC25. Further, modified proteins are less digestible by some of the proteolytic

279

enzymes located in the gastrointestinal tract and modified enzymes show a reduced

280

activity26,

281

functional changes might therefore play a key role in the effects that have been

282

observed after treatment with ITC. More than thirty proteins of redox-regulation, the

283

cytoskeleton, cell survival and apoptosis signaling have been identified for ITC

284

interaction in proteomic analysis so far23,

285

action at dietary relevant doses include blocking of phase-I cytochrome P450 xenobiotic

286

metabolizing

287

KEAP1/Nrf2/ARE pathway. Here, a direct interaction of ITC with multiple sulfhydryl

288

groups of KEAP1 is probably the initiating step as already described30. At

27

. The formation of ITC protein conjugates and the caused structural and

enzymes

and

activation

28, 29

. Best investigated mechanisms of ITC

of

phase-II

enzyme

expression

via


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supraphysiological doses, an increased sensitivity of cancer cells to growth arrest at the

290

G1, S, or G2/M phase and apoptosis induction was observed8,

291

apoptosis and cell cycle arrest, binding to cysteine residues of tubulin could be one

292

relevant step, as this has already been described for BITC at 10-20 µM in lung cancer

293

cells34,

294

used in HepG2 cells1, 36, 37 at two time points which reflect different stages of the cellular

295

process triggered.

296

BITC-Cys and BITC-Lys adducts have been determined after digestion of the modified

297

proteins. Further, the supernatant of the incubation was investigated prior to and after

298

protein digestion in order to estimate a potential loss of available BITC by binding to

299

amino acids and proteins contained in the incubation medium. There was no BITC-Cys

300

detectable in the incubation medium, neither prior to nor after digestion. This may be

301

due to the instability of cysteine conjugates under neutral and basic conditions21.

302

Furthermore, L-cystein was present in the incubation medium in the form of cystine,

303

thus, the thiol groups were not available as reaction site. Although there is evidence that

304

aliphatic ITC like allyl isothiocyanate (AITC) are able to cleave disulfide bonds of cystine,

305

there is no data available for aromatic ITC38. The analysis of GSH conjugates of various

306

ITC indicated that cysteine conjugates are of low stability in aqueous solutions15, 20,

307

However, there is no data available about the influence of pH value and temperature on

308

the stability of BITC-Lys conjugates compared to BITC-Cys conjugates. Knowledge

309

about the stability of the analytes is very important, as sample collection often takes

310

some days and a storage time up to several weeks is often required. Further, the sample

311

preparation procedure can be improved in order to ensure the highest possible stability

312

of the analytes. As already described in some publications, BITC-Cys was proven to be

31-33

. For induction of

35

. In the present study, also cell death/arresting concentrations of BITC were

39

.



Page 16 of 32

313

not stable under neutral and basic conditions, whereas acidic conditions and low

314

temperatures increase the stability21. On the contrary, BITC-Lys showed no correlation

315

between stability and pH value, why it is particularly appropriate as a marker for protein

316

modifications by BITC. This increased stability has already been shown for the reaction

317

of AITC with lysine under physiological conditions24. During sample preparation, an

318

acidic pH value and low temperatures cannot always be kept due to the required

319

conditions of the digestion enzyme of 37 °C and pH 7.4. Low temperatures would lead to

320

less activity and thus, an incomplete digestion. An acidic pH value would lead to protein

321

denaturation and consequently inactivate the enzyme. Therefore, it has to be considered

322

that a loss of BITC-Cys cannot be avoided to some extent. Consequently, BITC-Lys is

323

the more reliable marker for the determination of protein conjugates due to its high

324

stability concerning pH value and temperature.

325

In order to minimize protein conjugates in the medium, use of fetal calf serum (FCS) was

326

avoided during BITC exposure in the present study. The comparison of different

327

exposure durations and BITC concentrations showed that the reaction of BITC with

328

lysine residues of proteins seems to proceed very slowly. This observation is in

329

agreement with the literature, where it is postulated that ITC react up to one thousand

330

times faster with thiol groups than with amino groups40. Consequently, long exposure

331

times will lead to an increase of lysine conjugates compared to short exposure times.

332

Due to the reversibility of the cysteine conjugate and the irreversibility of the lysine

333

conjugate, a slow transformation of cysteine conjugates into lysine conjugates at long

334

exposure times has already been observed24. In the cell pellet, both, BITC-Cys and

335

BITC-Lys, were detectable. However, the amounts of BITC-Cys were very low compared

336

to BITC-Lys. Taking the results of the stability tests into account, there was only about 16 ACS Paragon Plus Environment

Page 17 of 32


337

20% of the initial amount of BITC-Cys left, as for sample preparation the digestion of

338

proteins took place at a buffered pH value of 7.4 for 15 h (compare to section 3.2).

339

Considering a loss of 80% of the BITC-Cys conjugates, there were still more BITC-Lys

340

conjugates formed. The formation of protein conjugates is dependent on the primary

341

structure of the protein as well as on possible modifications such as disulfide bonds or

342

glycosylations. Although some ITC are able to cleave disulfide bonds, these

343

modifications are likely to prevent the reaction of ITC with amino acid residues of

344

proteins. In the cells again the formation of BITC-Lys showed the same distribution as in

345

the incubation medium, as the formation of lysine conjugates occurred preferentially at

346

the longer exposure duration of 6 h.

347

The results may help understanding the mode of action of ITC in cells and for drawing

348

conclusions on health beneficial as well as toxicological effects in humans. Moreover, it

349

is possible to extend the assay to future clinical intervention studies of ITC conjugates in

350

vivo. In order to locate the exact reaction site in the cell (membranes, cytosol,

351

organelles), cellular compartments can be separated prior to analyzing the protein

352

conjugates. The reaction of BITC with proteins is not limited to the amino acids

353

investigated. Though only limited data is available, reaction products with amino acids

354

with similar chemical structure, namely containing amino groups, may occur as well41.

355

Therefore, the investigation of protein conjugates after BITC exposure should be

356

extended to other amino acids such as arginine, asparagine, glutamine, histidine, proline

357

and tryptophan. A reaction of BITC with the hydroxyl group of threonine or tyrosine is

358

even conceivable.



Page 18 of 32

359

Due to the multi-target character of ITC and their potential to interfere in multiple steps of

360

carcinogenesis, protein interactions are a promising field in investigating the mechanism

361

of chemoprevention.

362

363

Abbreviations

364

AITC,

365

isothiocyanate-cysteine; BITC-Lys, benzyl isothiocyanate-lysine; DMEM, Dulbecco’s

366

Modified Eagle Medium; EtOH, ethanol; FA, formic acid; FCS, fetal calf serum; GSH,

367

Glutathione; HPLC-UV, high performance liquid chromatography-ultraviolet; ITC,

368

Isothiocyanate; LC-ESI-MS/MS, liquid chromatography tandem mass spectrometry;

369

MeOH, methanol; MTBITC, 4-methylthiobutyl isothiocyanate; NMR, nuclear magnetic

370

resonance; PBS, phosphate buffered saline; TFA, trifluoroacetic acid

allyl

isothiocyanate;

BITC,

Benzyl

isothiocyanate;

BITC-Cys,

benzyl


Page 19 of 32


371

References

372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415

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18. Kühn, C.; von Oesen, T.; Hanschen, F. S.; Rohn, S., Determination of isothiocyanate-protein conjugates in milk and curd after adding garden cress (Lepidium sativum L.). Food Res Int 2018, 108, 621627. 19. Brüsewitz, G.; Cameron, B. D.; Chasseaud, L. F.; Gorler, K.; Hawkins, D. R.; Koch, H.; Mennicke, W. H., The metabolism of benzyl isothiocyanate and its cysteine conjugate. Biochem J 1977, 162, 99-107. 20. Kassahun, K.; Davis, M.; Hu, P.; Martin, B.; Baillie, T., Biotransformation of the naturally occurring isothiocyanate sulforaphane in the rat: identification of phase I metabolites and glutathione conjugates. Chem Res Toxicol 1997, 10, 1228-33. 21. Platz, S.; Kühn, C.; Schiess, S.; Schreiner, M.; Mewis, I.; Kemper, M.; Pfeiffer, A.; Rohn, S., Determination of benzyl isothiocyanate metabolites in human plasma and urine by LC-ESI-MS/MS after ingestion of nasturtium (Tropaeolum majus L.). Anal Bioanal Chem 2013, 405, 7427-36. 22. Murthy, N. V. K. K.; Rao, M. S. N., Interaction of Allyl Isothiocyanate with Mustard 12s Protein. J Agric Food Chem 1986, 34, 448-452. 23. Mi, L. X.; Wang, X. T.; Govind, S.; Hood, B. L.; Veenstra, T. D.; Conrads, T. P.; Saha, D. T.; Goldman, R.; Chung, F. L., The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Res 2007, 67, 6409-6416. 24. Nakamura, T.; Kawai, Y.; Kitamoto, N.; Osawa, T.; Kato, Y., Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine. Chem Res Toxicol 2009, 22, 536-542. 25. Rawel, H. M.; Kroll, J.; Haebel, S.; Peter, M. G., Reactions of a glucosinolate breakdown product (benzyl isothiocyanate) with myoglobin. Phytochemistry 1998, 48, 1305-11. 26. Rawel, H. M.; Kroll, J.; Schroder, I., In vitro enzymatic digestion of benzyl- and phenylisothiocyanate-derivatized food proteins. J Agric Food Chem 1998, 46, 5103-5109. 27. Rawel, H. M.; Kroll, J.; Schroder, I., Reactions of isothiocyanates with food proteins: Influence on enzyme activity and tryptical degradation. Nahrung 1998, 42, 197-199. 28. Mi, L.; Chung, F. L., Binding to protein by isothiocyanates: a potential mechanism for apoptosis induction in human non small lung cancer cells. Nutr Cancer 2008, 60 Suppl 1, 12-20. 29. Mi, L. X.; Hood, B. L.; Stewart, N. A.; Xiao, Z.; Govind, S.; Wang, X. T.; Conrads, T. P.; Veenstra, T. D.; Chung, F. L., Identification of Potential Protein Targets of Isothiocyanates by Proteomics. Chem Res Toxicol 2011, 24, 1735-1743. 30. Hong, F.; Freeman, M. L.; Liebler, D. C., Identification of sensor cysteines in human Keap1 modified by the cancer chemopreventive agent sulforaphane. Chem Res Toxicol 2005, 18, 1917-26. 31. Rose, P.; Whiteman, M.; Huang, S. H.; Halliwell, B.; Ong, C. N., beta-Phenylethyl isothiocyanatemediated apoptosis in hepatoma HepG2 cells. Cell Mol Life Sci 2003, 60, 1489-503. 32. Tang, L.; Zhang, Y., Dietary isothiocyanates inhibit the growth of human bladder carcinoma cells. J Nutr 2004, 134, 2004-10. 33. Zhang, Y.; Tang, L.; Gonzalez, V., Selected isothiocyanates rapidly induce growth inhibition of cancer cells. Mol Cancer Ther 2003, 2, 1045-52. 34. Mi, L. X.; Xiao, Z.; Hood, B. L.; Dakshanamurthy, S.; Wang, X. T.; Govind, S.; Conrads, T. P.; Veenstra, T. D.; Chung, F. L., Covalent binding to tubulin by isothiocyanates - A mechanism of cell growth arrest and apoptosis. J Biol Chem 2008, 283, 22136-22146. 35. Mi, L.; Gan, N.; Cheema, A.; Dakshanamurthy, S.; Wang, X.; Yang, D. C.; Chung, F. L., Cancer preventive isothiocyanates induce selective degradation of cellular alpha- and beta-tubulins by proteasomes. J Biol Chem 2009, 284, 17039-51. 36. Huang, S. H.; Wu, L. W.; Huang, A. C.; Yu, C. C.; Lien, J. C.; Huang, Y. P.; Yang, J. S.; Yang, J. H.; Hsiao, Y. P.; Wood, W. G.; Yu, C. S.; Chung, J. G., Benzyl isothiocyanate (BITC) induces G2/M phase arrest and apoptosis in human melanoma A375.S2 cells through reactive oxygen species (ROS) and both mitochondria-dependent and death receptor-mediated multiple signaling pathways. J Agric Food Chem 2012, 60, 665-75. 20 ACS Paragon Plus Environment

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37. Zhang, R. F.; Loganathan, S.; Humphreys, I.; Srivastava, S. K., Benzyl isothiocyanate-induced DNA damage causes G(2)/M cell cycle arrest and apoptosis in human pancreatic cancer cells. J Nutr 2006, 136, 2728-2734. 38. Kawakishi, S.; Namiki, M., Oxidative Cleavage of the Disulfide Bond of Cystine by Allyl Isothiocyanate. J Agric Food Chem 1982, 30, 618-620. 39. Al Janobi, A. A.; Mithen, R. F.; Gasper, A. V.; Shaw, P. N.; Middleton, R. J.; Ortori, C. A.; Barrett, D. A., Quantitative measurement of sulforaphane, iberin and their mercapturic acid pathway metabolites in human plasma and urine using liquid chromatography-tandem electrospray ionisation mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2006, 844, 223-34. 40. Brown, K. K.; Hampton, M. B., Biological targets of isothiocyanates. Biochim Biophys Acta 2011, 1810, 888-94. 41. Barknowitz, G.; Engst, W.; Schmidt, S.; Bernau, M.; Monien, B. H.; Kramer, M.; Florian, S.; Glatt, H., Identification and quantification of protein adducts formed by metabolites of 1-methoxy-3indolylmethyl glucosinolate in vitro and in mouse models. Chem Res Toxicol 2014, 27, 188-99.

479 480

Figure captions

481

Figure 1 Chemical structure of BITC

482

Figure 2 Synthesized conjugates of cysteine and lysine with BITC

483

Figure 3 Recovery rates of BITC-Cys after incubation at 4 °C, 22 °C and 37 °C for 48 h

484

at pH 2 (A), pH 7 (B) and pH 10 (C)

485

Figure 4 Total amount of BITC-Lys in the incubation medium subdivided into amino acid

486

conjugates and protein conjugates

487

Figure 5 Concentration of BITC-Cys (A) and BITC-Lys (B) in the cell pellet after

488

incubation with 10 µM and 30 µM of BITC for 2 and 6 h

489

490



Page 22 of 32

Tables Table 1 Optimized 4000QTrap tuning parameters for BITC-Cys and BITC-Lys analyte BITCCys BITCLys

a

b

Rt [min]

DP [V]

12.6

46

11.0

41

quantifier 271.1 -> 90.9 296.3 -> 108.0

c

d

dwell time [msec]

CE [V]

CXP [V]

350

33

14

350

21

8

qualifier 271.1 -> 122.0 296.3 -> 189.0

dwell time [msec]

CE [V]

CXP [V]

80

17

8

80

21

14

a

Rt retention time, bDP declustering potential, cCE collision energy, dCXP collision cell exit potential


Page 23 of 32


Table 2 Validation data for LC-ESI-MS/MS analysis of BITC-Cys and BITC-Lys linearity analyte

LLOQa

2

R

slope

range [µM]

precision RSDb [%]

[nM] 0.5 µM

5 µM

50 µM

BITC-Cys

0.05-100

0.9988

188,627

5

8.88

2.00

3.12

BITC-Lys

0.05-100

0.9985

57,406

38

11.15

7.31

6.87

a

LLOQ lower limit of quantification (the lowest concentration of analytes, which can be

quantitatively measured with suitable accuracy and precision); bRSD relative standard deviation



Page 24 of 32

Figure graphics

Figure 1

Figure 2


Page 25 of 32


Figure 3



Page 26 of 32

Figure 4

Figure 5


Page 27 of 32


For Table of Contents Only



Chemical structure of BITC 39x17mm (600 x 600 DPI)

ACS Paragon Plus Environment

Page 28 of 32

Page 29 of 32


Synthesized conjugates of cysteine and lysine with BITC 29x6mm (300 x 300 DPI)



Recovery rates of BITC-Cys after incubation at 4 °C, 22 °C and 37 °C for 48 h at pH 2 (A), pH 7 (B) and pH 10 (C) 174x340mm (300 x 300 DPI)


Page 30 of 32

Page 31 of 32


Total amount of BITC-Lys in the incubation medium subdivided into amino acid conjugates and protein conjugates 59x40mm (600 x 600 DPI)



Concentration of BITC-Cys (A) and BITC-Lys (B) in the cell pellet after incubation with 10 µM and 30 µM of BITC for 2 and 6 h 99x117mm (600 x 600 DPI)


Page 32 of 32

In Vitro Determination of Protein Conjugates in ... - ACS Publications

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