Metabolic activation of myristicin and its role in cellular toxicity

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Metabolic activation of myristicin and its role in cellular toxicity Xu Zhu, yikun wang, Xiao Nan Yang, Xue-Rong Xiao, Ting Zhang, Xiu-Wei Yang, Hong-Bo Qin, and Fei Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Journal of Agricultural and Food Chemistry

Metabolic activation of myristicin and its role in cellular toxicity

Xu Zhua,b,†, Yi-Kun Wanga,b,†, Xiao-Nan Yanga,c, Xue-Rong Xiaoa, Ting Zhanga,b, Xiu-Wei Yangd, Hong-Bo Qina,*, Fei Lia,e,*

aStates

Key Laboratory of Phytochemistry and Plant Resources in West China,

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China bUniversity

of Chinese Academy of Sciences, Beijing 100049, China

cGuangxi

Key Laboratory of Medicinal Resources Protection and Genetic

Improvement, Guangxi Botanical Garden of Medicinal Plant, Nanning 530023, China dSchool

of Pharmaceutical Sciences, Peking University Health Science Center, Peking

University, Beijing 100191, China eJiangxi †

University of Traditional Chinese Medicine, Nanchang 330004, China

These authors contributed equally to this work.

1 ACS Paragon Plus Environment


1

ABSTRACT

2

Myristicin is widely distributed in spices and medicinal plants. The aim of this study

3

was to explore the role of metabolic activation of myristicin in its potential toxicity

4

through metabolomic approach. The myristicin-N-acetylcysteine adduct was

5

identified by comparing the metabolic maps of myristicin and 1'-hydroxymyristicin.

6

Supplement of N-acetylcysteine could protect against the cytotoxicity of myristicin

7

and 1'-hydroxymyristicin in primary mouse hepatocytes. When the depletion of

8

intracellular N-acetylcysteine was pretreated with diethyl maleate in hepatocytes, the

9

cytotoxicity induced by myristicin and 1'-hydroxymyristicin was deteriorated. It

10

suggested that N-acetylcysteine adduct resulting from myristicin bioactivation was

11

closely associated with myristicin toxicity. Screening of human recombinant

12

cytochrome P450s (CYPs) and treatment with CYPs inhibitors revealed that CYP1A1

13

was mainly involved in the formation of 1'-hydroxymyristicin. Collectively, this study

14

provided a global view of myristicin metabolism and identified N-acetylcysteine

15

adduct resulting from myristicin bioactivation, which could be utilized for

16

understanding the mechanism of myristicin toxicity.

17 18

KEYWORDS: myristicin, 1'-hydroxymyristicin, metabolic activation, metabolomics

19 20 21 22

INTRODUCTION 2 ACS Paragon Plus Environment

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23

Myristicin (5-allyl-1-methoxy-2,3-methylenedioxybenzene) is widely distributed in

24

flavoured foods and dietary supplements, including dill oil (7% myristicin) 1, parsley

25

oil (6% myristicin) 2, dry nutmeg (4% myristicin) 3, dry celery (2% myristicin) 4, and

26

fresh carrot (0.003% myristicin) 5. Myristicin is also found in multiple medicinal

27

plants around the world, such as Todaroa aurea

28

glochidiatus, Pseudorlaya pumila and Pseudorlaya minuscula 7. It is reported that

29

myristicin exhibits various pharmacological activities, including anti-inflammatory 8,

30

anti-microbial and anti-proliferative activity 9. Some studies also reported that

31

myristicin may cause potential toxicity 10, especially for hallucinatory side-effects 11.

32

However, the mechanism of myristicin toxicity remained unclear.

6,

Piper mullesua, Daucus

33

It is known that myristicin is one of major constituents in nutmeg. Several studies

34

reported that high content of myristicin in nutmeg was responsible for its poisoning

35

cases

36

incubation and rat urine, and identified as 5-allyl-1-methoxy-2,3- dihydroxybenzene

37

and 1'-hydroxymyristicin 14. It was found that cytochrome P450 3A4 (CYP3A4) and

38

CYP1A2

39

3-dihydroxybenzene from myristicin

40

CYPs involved in the generation of 1'-hydroxymyristicin, and whether metabolic

41

activation of myristicin was related to its toxicity.

11,12,13.

Two metabolites of myristicin were produced in rat liver microsomal

majorly

contributed

to

the 15.

generation

of

5-allyl-1-methoxy-2,

There are only few studies investigating

42

It has been demonstrated that mass spectrometry-based metabolomics is a

43

powerful tool to determine the metabolic characterization of xenobiotics via

44

systematically elucidating its metabolites

16-17.

In the present study, mass



45

spectrometry-based metabolomics was applied to determine reactive metabolites

46

associated with myristicin toxicity by comparing the metabolic maps of myristicin and

47

1'-hydroxymyristicin.

48 49

MATERIALS AND METHODS

50

Reagents

51

Myristicin was isolated from nutmeg and provided by Xiu-Wei Yang’s laboratory 18.

52

1'-Hydroxymyristicin was chemically synthesized by Hong-Bo Qin’s laboratory as

53

described in the supplementary materials. Reduced nicotinamide adenine dinucleotide

54

phosphate (NADPH), formic acid and chlorpropamide were from Sigma-Aldrich (St.

55

Louis, USA). N-acetylcysteine, methoxsalen, ticlopidine and ketoconazole were from

56

Meilun chemical reagent company (Dalian, China). Trimethoprim, 4-methylpyrazole,

57

diethyl maleate (DEM), α-naphthoflavone and quinidine were from Macklin reagent

58

company (Shanghai, China). Sulfaphenazole was from MCE (Med Chem Express

59

LLC, USA). The mouse liver microsomes (MLMs) and human liver microsomes

60

(HLMs) were purchased from Bioreclamationivt Inc. (Hicksville, NY, USA).

61

Recombinant human CYPs were from Xenotech, LLC (Kansas City, KS, USA). All

62

other reagents and organic solvents were commercially sold and of analytical or

63

chromatographic grade.

64 65

In vivo metabolism of myristicin and 1'-hydroxymyristicin


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66

The in vivo metabolism of myristicin and 1'-hydroxymyristicin was studied in male

67

C57BL/6 mice (6-8 weeks old) from Slaccas Laboratory Animal Company (Changsha,

68

China). The animals were acclimatized to the facilities for one week in a room at 23 ±

69

1 ℃ with a light/dark cycle of 12/12 h and humidity 50-60%. The mice had free

70

access to distilled water and standard rodent chow. The experimental procedures were

71

performed according to the guidelines of the Chinese Academy of Sciences, Kunming,

72

China, and approved by the Institutional ethical committee (IEC) of Kunming

73

Institute of Botany. The mice were randomly divided into three groups (n = 4 in each

74

group). Myristicin and 1'-hydroxymyristicin were orally administered to mice at the

75

dose of 200 mg/kg suspended in 0.5% sodium carboxymethylcellulose (CMC-Na) 19,

76

respectively. Treatment with 0.5% CMC-Na solution was used as control group. Mice

77

were housed in metabolic cages for 24 h after treatments, and urine and feces samples

78

were collected. The blood was collected from the mice orbit at 1 h and 24 h after

79

treatment, respectively, and plasma samples were obtained by centrifugation (2000 g,

80

5 min, 4 °C). The samples for LC-MS analysis were prepared according to our

81

previous established method

82

described in the supplementary materials.

20.

The processing procedures of these samples are

83 84

Metabolism of myristicin and 1'-hydroxymyristicin in MLMs, HLMs and

85

recombinant CYPs

86

The metabolism of myristicin was conducted by the incubation with hepatic

87

microsomes according to the previous method

20.


Myristicin (50 μM) and


88

1'-hydroxymyristicin (50 μM) were incubated with pooled MLMs and HLMs in

89

potassium phosphate buffer (PBS, pH = 7.4), respectively. The incubation mixtures

90

were reacted in a final volume of 200 μL, containing 0.5 mg/mL MLMs or HLMs

91

protein. After pre-incubation at 37 °C for 5 min, the reactions were initiated by adding

92

20 μL freshly prepared NADPH (1 mM) into the system, and then continued to

93

incubate at 37 °C for 40 min with gentle shaking. The reactions were quenched by

94

adding 200 μL of ice cold acetonitrile, and supernatants were obtained by

95

centrifugation (18 000 g, 20 min, 4 °C). Each supernatant sample (5 μL) was injected

96

into the UPLC-MS/MS system for analysis.

97

The contribution of cDNA-expressed recombinant CYP enzymes to myristicin

98

metabolism was screened using a panel of CYPs. The incubation system included 50

99

μM myristicin, 2 pmol/mL of each recombined human P450 (control, CYP1A1,

100

CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C19, CYP2C8, CYP2C9, CYP2D6,

101

CYP2E1, CYP3A4, and CYP3A5) in 20 mM PBS (pH 7.4). The sample was

102

processed as described above, and 5 μL supernatant was injected for UPLC-MS/MS

103

analysis. Each reaction was conducted in triplicates.

104 105

Formation of reactive metabolite myristicin-derived NAC conjugate

106

Trapping experiments were conducted to detect the electrophilic metabolites of

107

myristicin with and without NADPH. The incubation system consisted of 50 μM

108

myristicin, 0.5 g/mL of MLMs in 20 mM PBS. The system was pre-incubated at 37

109

°C for 5 min, and then the reactions were initiated by the addition of 20 μL NADPH 6 ACS Paragon Plus Environment

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110

(or the same volume of PBS buffer). To determine whether the formation of reactive

111

metabolite was dependent on liver microsomes, the reaction system consisting of 50

112

μM myristicin and 1 mM NAC in PBS was incubated at room temperature for 40 min

113

in the absence of MLMs. The sample was processed as described above, and the

114

supernatant of reactive solution was subjected to UPLC-MS/MS to detect the

115

formation of the myristicin-derived NAC conjugate.

116 117

Effect of CYP chemical inhibitors on reactive metabolite formation

118

To further determine which CYPs involved in the biotransformation of myristicin, the

119

specific inhibitors of CYPs were separately added into the microsomal system which

120

contained 0.5 mg protein/mL pooled MLMs, 1 mM NAC and 1 mM NADPH. CYPs

121

chemical inhibitors included α-naphthoflavone (1.0 μM for P450 1A1/2),

122

sulfaphenazole (100 μM for P450 2C9), trimethoprim (2.5 μM for P450 2C8),

123

ticlopidine (100 μM for P450 2B6 and P450 2C19), quinidine (5.0 μM for P450 2D6),

124

4-methylpyrazole (100 μM for P450 2E1), methoxsalen (20 μM for P450 2A13 and

125

P450 2A6), and ketoconazole (100 μM for P450 3A family). The reaction conditions

126

and sample process were the same as described above.

127 128

UPLC–MS/MS analysis and screening potential metabolites

129

All samples were detected and recorded on Agilent 1290 infinity UPLC system

130

(Agilent Technologies, Santa Clara, CA) coupled with Agilent 6530 Q-TOF mass

131

spectrometric detector. The chromatographic separation was achieved using 7 ACS Paragon Plus Environment


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132

XDB-C18 chromatographic column (2.1×100 mm, 1.8 µm, Agilent). The mobile

133

phase consisted of ACN with 0.1% formic acid (A) and 0.1% formic acid in water (B),

134

and the column eluted with a gradient elution ranging from 2 % to 98 % with the flow

135

rate was 0.3 mL/min in 16 min run. The column constant temperature was kept at 45

136

°C. Samples were detected by full-scanning via electrospray ionization in the positive

137

ion mode (ESI+). Full scan HR-MS (high resolution mass spectrometry) spectra were

138

obtained over the mass range m/z 50-1000. The MS/MS spectrum of metabolites was

139

performed in the targeted mode by collision energy of 10-20 eV. The MS/MS spectral

140

data were processed in Agilent Mass Hunter Workstation data Acquisition software

141

(Agilent, Santa Clara, CA). The structural elucidation of myristicin and

142

1'-hydroxymyristicin metabolites were further determined on the basis of their

143

accurate masses and MS/MS fragmentation patterns. The strategy of screening for

144

potential metabolites was according to previous research 21.

145 146

Cytotoxicity assessment of myristicin and 1'-hydroxymyristicin

147

Mouse primary hepatocytes were isolated from male C57BL/6 mice (8-10 weeks) by

148

the collagenase perfusion method as described previously

149

primary hepatocytes were maintained in William'E medium supplemented with 10%

150

fetal bovine serum and 1% penicillin-streptomycin, and placed in a humidified

151

atmosphere of 5% CO2 at 37 °C. Cells were seed into a 96-well plate with the density

152

of 1×105/well. Mouse primary hepatocytes were treated with myristicin or


21.

The isolated mouse

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153

1'-hydroxymyristicin at various concentrations (62.5-500 μM) for 24 h, and then

154

incubated with MTT at 37 °C for 4 h. Cell viabilitiy was measured as absorbance at

155

570 nm using EL×808IU microplate reader (Bio-Tek Instruments Inc, Winooski, VT,

156

USA), and the results were presented as the percentage of the control group.

157

To determine the effect of NAC on the cytotoxicity induced by myristicin and

158

1'-hydroxymyristicin, NAC and DEM (thiol depletion) were separately added to the

159

culture medium of mouse primary hepatocytes. Cytotoxicity of myristicin and

160

1'-hydroxymyristicin was calculated as IC50 values. Data represent the averages of

161

five wells per group. Each experiment was performed in triplicate.

162 163

Statistical analysis

164

All values were expressed as means ± standard error of mean (SEM). Statistical

165

analyses were performed by using Prism v.6 (GraphPad Software, San Diego, CA,

166

USA). Differences among multiple groups were calculated using one-way ANOVA

167

followed by Dunnett’s post hoc comparisons. Differences between myristicin group

168

and NAC/DEM+myristicin group as well as 1'-hydroxymyristicin group and

169

NAC/DEM+1'-hydroxymyristicin groups were performed by Student’s t-test.

170

Differences were considered to be significant when P < 0.05.

171 172

RESULTS AND DISCUSSION

173

Myristicin is a common aroma component in dietary and food additives, and is also 9 ACS Paragon Plus Environment


174

distributed in many herbal medicines

175

Carum carvi seed 23. It was reported that the abuse of nutmeg may cause hallucinatory

176

effects in human 24-25. Myristicin was subsequently identified as the toxic substance in

177

nutmeg

178

toxicity, comparative metabolism of myristicin and 1'-hydroxymyristicin was

179

investigated using metabolomics approach. The data revealed that the reactive

180

metabolite myristicin-NAC adduct was associated with its toxicity, and CYP1A1 was

181

the primary enzyme involved in the formation of myristicin-NAC adduct.

11.

22,

such as mace, Anethum graveolens and

In order to determine the metabolic pathway associated with myristicin

182 183

Screening the metabolites of myristicin and 1'-hydroxymyristicin

184

The combination of high-resolution mass spectrometry analytical technologies with

185

multivariate statistical analysis provided a useful tool for screening the metabolites of

186

drugs and xenobiotics

187

metabolomics was applied for screening the metabolites of myristicin and

188

1'-hydroxymyristicin in mice and liver microsomal incubations. An unbiased PCA

189

model was used to analyze metabolites of myristicin and 1'-hydroxymyristicin.

190

Myristicin group, 1'-hydroxymyristicin group, and control group showed the

191

significant differences in PCA score plot (Fig. 1A). The ions of metabolites of

192

myristicin and 1'-hydroxymyristicin were labeled in the loading scatter plot (Fig. 1B).

193

The same metabolites of myristicin and 1'-hydroxymyristicin were M6/H2 and

194

M10/H11 (Fig. 1C and 1D). The respective metabolites of myristicin (M1) and

195

1'-hydroxymyristicin (H7) were displayed in Fig. 1E and 1F, respectively. Among 18

16, 20.

In this study, comparative UPLC-QTOF-MS-based


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196

metabolites

197

1'-hydroxymyristicin, 12 metabolites were firstly reported (Supplementary Table 1

198

and Table 2). The relative abundance of myristicin and 1'-hydroxymyristicin

199

metabolites in vitro and in vivo are exhibited in Supplementary Fig. 2 and Fig. 3.

identified

by

UPLC-ESI-QTOF-MS

for

myristicin

and

200 201

Identification and formation of myristicin-NAC adduct

202

M10 and H11 were detected in the urine of mice treated with myristicin and

203

1'-hydroxymyristicin (Fig. 2B and 2C), respectively. M10 was eluted at 6.39 min and

204

showed pseudomolecular of [M+H]+ ion at m/z 370.0947, and H11 was eluted at 6.40

205

min and showed pseudomolecular of [M+H]+ ion at m/z 370.0952. Both metabolites

206

showed the similar MS/MS fragmentography models. They exhibited the

207

characteristic neutral losses of 163 Da NAC residue (parent ion m/z 370 → daughter

208

ion m/z 207) in MS/MS spectrum (Fig. 2D). These data demonstrated that M10 and

209

H11 were the same metabolite, which was identified as myristicin-NAC adduct. In

210

order to determine how myristicin-NAC adduct was generated, NAC trapping

211

experiments and spontaneous reaction of myristicin were performed, respectively. The

212

myristicin-NAC adduct was observed in the MLMs incubations system in the

213

presence of NADPH (Fig. 3B), while it could not be detected without NADPH (Fig.

214

3A), indicating that the generation of NAC conjugate is dependent on NADPH. In

215

addition, 1'-hydroxymyristicin could spontaneously bind with NAC to form

216

myristicin-NAC adduct (Fig. 3C) without MLMs. It is known that metabolic

217

activation is responsible for the generation of reactive metabolites from drugs, such as 11 ACS Paragon Plus Environment


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218

acetaminophen, tamoxifen, diclofenac and troglitazone

219

metabolites are liable to covalently bind to glutathione (GSH), cysteine, and even

220

biological macromolecules

221

found in MLMs metabolism of myristicin

222

detected in the present study.

25, 27.

26.

The reactive electrophile

Although myristicin-GSH adduct was previously 25,

only myristicin-NAC adduct was

223 224

CYPs contributed to 1'-hydroxylation of myristicin

225

To determine which CYPs contributed to the formation of 1'-hydroxymyristicin, a

226

panel of CYPs was conducted to evaluate the contribution of CYP isoforms

227

data indicated that CYP1A1, CYP1B1, CYP2C9 and CYP3A5 are the main enzymes

228

contributing to the formation of 1'-hydroxymyristicin (Fig. 3D). The results are listed

229

in Supplementary Table 3.

230

Incubation of liver microsomes with specific inhibitors is an effective method for

231

systematically defining the contribution of individual CYPs 29. In MLMs incubations,

232

the formation of the 1'-hydroxymyristicin was significantly decreased by

233

α-naphthoflavone,

234

4-methylpyrazole, methoxsalen, and ketoconazole. Among these inhibitors, the

235

CYP1A inhibitor α-naphthoflavone reduced the formation of 1'-hydroxymyristicin by

236

57%, and its inhibitory ability was higher than other CYPs inhibitors (Fig. 3E). These

237

data suggested that CYP1A1 was mainly responsible for the production of

238

1'-hydroxymyristicin. Some studies reported that CYP1A2 majorly involved in the

sulfaphenazole,

trimethoprim,


ticlopidine,

28.

The

quinidine,

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239

bioactivation of estragole

240

slightly contributed to the bioactivation of mryisticin, and instead it was CYP1A1

241

with most activity. Food and drug interactions should be monitored when consuming

242

foods rich in myristicin accompanied the treatment with CYP1A inhibitors or

243

activators. CYP1 family inhibitors may lead to higher concentrations of myristicin. In

244

contrast, CYP1A inducers may facilitate the formation of reactive metabolites

245

(1'-hydroxymyristicin), and increase the risk of myristicin-related toxicity. In addition,

246

as shown in Supplementary Fig. 2C, myristicin accounted for 19.76% and 43.42% of

247

the metabolites in MLMs and HLMs, respectively, and the percentages of

248

1'-hydroxymyristicin (M8) were 5.67% and 3.26% in MLMs and HLMs, suggesting

249

that metabolic activation of myristicin was higher in mice. One recent study reported

250

that the formation of 1'-hydroxymyristicin in rat was 1.8-fold higher than that in

251

human

252

different species.

33,

30,

methyleugenol

31,

and safrole

32.

However, CYP1A2

suggesting that metabolic activation of myristicin is different among

253 254

Cytotoxicity assessment of myristicin and 1'-hydroxymyristicin

255

A previous study reported that 1'-hydroxymyristicin showed higher toxic effects than

256

myristicin towards HepG2 cells

257

cytotoxicity

258

1'-hydroxymyristicin was estimated in mouse primary hepatocytes. Cell viability

259

following

260

1'-hydroxymyristicin was determined

via

metabolic

exposure

with

34.

In order to elucidate whether myristicin induced

activation,

62.5,

125, 34.

the

250

cytotoxicity

and

500

of

μM

myristicin

myristicin

and

and

The results indicate that myristicin showed 13

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261

remarkable cytotoxicity at 250 μM (Fig. 4A) and 1'-hydroxymyristicin was at 62.5

262

μM (Fig. 4B). The IC50 value of myristicin was 356.4 ± 1.06 μM, which was much

263

higher than of 1'-hydroxymyristicin (132.90 ± 1.07 μM), suggesting that metabolic

264

activation of myristicin could enhance its toxicity. It is reported that 1'-hydroxylation

265

was the key step in the metabolic activation of alkenylbenzenes, including apiol,

266

estragole, methyleugenol and safrole

267

NAC significantly ameliorated the cytotoxicity of myristicin and 1'-hydroxymyristicin

268

(Fig. 4C and 4D). On the contrary, depletion of NAC by DEM could aggravate their

269

cytotoxicity of myristcin and 1'-hydroxymyristcin (Fig. 4C and 4D). These data

270

suggested that the formation of myristicin-NAC adduct was responsible for myristicin

271

toxicity.

35.

Further study revealed that supplement of

272 273

Structural identification of myristicin and its metabolites

274

All metabolites were identified and characterized using UPLC-QTOF-MS/MS

275

analysis. In total, 10 myristicin metabolites were screened and defined as its

276

metabolites in vitro and in vivo metabolism of myristicin. Among these metabolites,

277

M1-2, M6-7 and M10 are novel metabolites (Supplementary Table 1 and Fig. 5).

278

Metabolite M1 (tR = 6.12 min) generated precursor ion [M+H]+ at m/z 195.0973+, was

279

2 Da (H2) higher than parent, and further formed fragment ions: [(M+H)-CH2]+ at m/z

280

181.0517+, [(M+H)-CH2O]+ at m/z 165.0533+. Consequently, M1 was characterized

281

as ring-opening product of myristicin in site of the 1, 3-dioxolane (Supplementary Fig. 14 ACS Paragon Plus Environment

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282

4B). Metabolite M2 showed a protonated molecular ion at m/z 179.0683+ in ESI+

283

mode, which was 14 Da (CH2) lower than parent, indicating M2 may be

284

demethylation product of myristicin. Daughter ions at m/z 161.0531+, 151.0762+,

285

147.0443+, corresponded to neutral elimination of 18 Da (H2O), 28 Da (CH2=CH2)

286

and 32 Da (H2O+CH2) (Supplementary Fig. 4C). Metabolite M3 exhibited an accurate

287

mass of [M+H]+ ion at m/z 227.0903+, which gave a good match for the molecular

288

formula C11H14O5 with error of 7.59 ppm. The key neutral loss of 60 Da (C2H4O2, m/z

289

227 → m/z 167) in the MS/MS spectrum demonstrated that M3 was dihydroxylated

290

myristicin. In addition, the other major fragment ions of M3 were at m/z 109 (owing

291

to neutral loss of OCH2O+H2O moiety from m/z 167) and 81. Thereby it identified

292

M3 as 1',3'-dihydroxylated myristicin.

293

Metabolite M4 was eluted at 5.99 min, and showed pseudomolecular of [M+H]+ ion

294

at m/z 209.0812+ with identical elemental composition of C11H12O4, which was 16 Da

295

(O) higher than parent. It indicated that M4 may be hydroxylated metabolites of

296

myristicin. The typical daughter ions of M4 at m/z 179.0692+ and 168.0411+ resulted

297

from neutral cleavage of OCH2 moiety and allyl group (CH2=CH-CH), respectively.

298

The two possible structures of M4 were displayed. The molecular composition of M5

299

was calculated as C10H12O3, according to the protonated molecular ion at m/z

300

181.0862+. The molecular weight of M5 was lower 12 Da than myristicin, indicating

301

M5 may be demethylenelated product occurred in the position of 1,3-dioxolane.

302

Additionally, it afforded a series of product ions at m/z 154.0877+, 135.0823+, and

303

121.0991+, resulting from the neutral loss of C2H2, CH2O and C4H6O groups, 15 ACS Paragon Plus Environment


304

respectively. The molecular formula of M6 was deduced as C11H10O3 from

305

quasi-molecular ion at m/z 191.0686+, which was lower 2 Da than parents. It

306

suggested that M6 was dehydroxylated product. And its fragment pathways of m/z

307

191→161 and 161→133 were formed due to neutral elimination of 30 Da (CH2O) and

308

28 Da (CO) (Supplementary Fig. 4D). On the basis of [M+H]+ ion at m/z 165.0552,

309

the molecular formula of M7 was deduced as the C9H8O3. And the molecular weight

310

of M7 was lower 16 Da than myristicin, indicating M7 may be dehydroxylation of

311

M5. The MS/MS of M7 yielded major fragment ions at m/z 131.0014+ (neutral loss of

312

H2O+CH4 moieties) and 115.0349+ (neutral loss of 2H2O+CH4 ).

313

M8 was identified as 1'-hydroxymyristicin. Its prominent fragment ions contained m/z

314

213.0433+ (C11H10O3Na+ portions, neutral loss of H2O), 191.0673+ (C11H11O3+

315

portions, cleavage of H2O+Na) and 165.0346+ (elimination of CO+Na). Phase II

316

metabolite M9 exhibited protonated molecular [M+H]+ ion at m/z 385.1092+, which

317

gave a precise match for the molecular formula of C17H20O10 with error of 5.51ppm.

318

The neutral elimination of 176 Da (m/z 385→209) in the MS/MS spectra suggested

319

the presence of glucuronic acid conjugate. The ions at m/z 165 and 147 were

320

generated from m/z 209 via neutral cleavage of C2H2+H2O and C2H2+2H2O moieties,

321

respectively. The ion of M9 at m/z 209 was elucidated as 1'-hydroxymyristicin moiety.

322

Therefore, M9 was deuced as 1'-hydroxymyristicin plus glucuronic acid adduct.

323 324

Comparison of myristicin and 1'-hydroxymyristicin metabolism

325

In this study, 10 and 11 metabolites were identified for myristicin (Supplementary 16 ACS Paragon Plus Environment

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326

Table 1 and Fig 5) and 1'-hydroxymyristicin using mass spectrometry-metabolomics

327

(Supplementary Table 2 and Fig 6), respectively. The metabolic profiling of

328

myristicin is displayed in Fig. 5. Myristicin, 1'-hydroxymyristicin and most of their

329

metabolites were primarily excreted in urine. Allyl and methylenedioxy were the

330

major metabolic sites of myristicin and 1'-hydroxymyristicin. The phase I metabolic

331

reactions of myristicin included dehydration, hydroxylation, ring-opening of

332

dioxolane, while the phase II metabolic reaction of myristicin contained NAC and

333

glucuronic acid conjugation. Similarly, the phase I metabolic reaction of

334

1'-hydroxymyristicin consisted of hydroxylation, demethylation, dehydrogenation and

335

dehydration, and its phase II metabolic reaction involved in the conjugation with Cys

336

and

337

(2',3'-dehydro-myristicin) and M10/H11 (myristicin-NAC adduct) were commonly

338

generated from the metabolism of myristicin and 1'-hydroxymyristicin.

339

In summary, the current study assessed the role of metabolic activation in the toxicity

340

of myristicin. The results indicated that metabolic activation of myristicin could

341

generate 1'-hydroxymyristicin, which further covalently bind with NAC. Supplement

342

of NAC could alleviate the cytotoxicity of myristicin, and depletion of NAC would

343

potentiate the cytotoxicity of myristicin. CYP1A1 was elucidated as the prominent

344

human CYPs responsible for the formation of 1'-hydroxymyristicin. There will be

345

potential damage to the body after long term and excessive consumption of foods and

346

herbs rich in myristicin, especially for co-treatment with some drugs that could affect

347

the expression of CYP1A1.

NAC

(Fig

6).

Among

these

metabolites,


the

metabolite

M6/H2


348 349

ABBREVIATIONS

350

CDCl3, deuterochloroform; CMC-Na, sodium carboxymethylcellulose; CYPs, human

351

recombinant cytochrome P450s; Cys, cysteine;

352

dimethyl sulfoxide; EtOAc, ethyl acetate; FBS, fetal bovine serum; GSH, glutathione;

353

HLMs, human liver microsomes; HR-MS, high resolution-mass spectrum; MLMs,

354

mouse

355

2,5-diphenyl-2H-tetrazolium bromide; NAC, N-acetylcysteine; NADPH, reduced

356

nicotinamide-adenine dinucleotide phosphate; PBS, potassium phosphate buffer; PCA,

357

principal component analysis; SEM, standard error of mean; UPLC-QTOF-MS,

358

ultra-performance liquid chromatography combined with quadrupole flight-triple

359

mass spectrometry.

liver

microsomes;

DEM, diethyl maleate; DMSO,

MTT,

360


3-(4,5-dimethyl-2-thiazolyl)-

Page 18 of 34

Page 19 of 34


362

AUTHOR INFORMATION

363

Corresponding authors

364

Fei Li, Ph.D., Kunming Institute of Botany, Chinese Academy of Sciences, Kunming

365

650201, China. Tel: +86-871-65216953, Email: [email protected];

366

Hong-Bo Qin, Ph.D., Kunming Institute of Botany, Chinese Academy of Sciences,

367

Kunming 650201, China. Tel: +86-871-65238010, Email: [email protected].

368 369

ORCID

370

Fei Li: 0000-0001-6911-2033

371 372

Author Contributions

373

†

Yi-Kun Wang and Xu Zhu contributed equally to this work.

374



376

Funding

377

This work was supported by the National Key Research and Development Program of

378

China (2017YFC0906903, 2017YFC1700906), CAS "Light of West China" Program

379

(Y72E8211W1), Kunming Institute of Botany (Y76E1211K1, Y4662211K1), State

380

Key Laboratory of Phytochemistry and Plant Resources in West China

381

(52Y67A9211Z1), and the Open Fund of State Key Laboratory of Pharmaceutical

382

Biotechnology, Nan-jing University (KF-GN-201705).

383 384

Notes

385

The authors declare no conflicts of interest.

386


Page 20 of 34

Page 21 of 34


388

Supporting Information

389

The supporting information includes chemical syntheses of 1'-hydroxymyristicin;

390

sample preparation method of urine, feces and blood; structural identification of

391

1'-hydroxymyristicin and its metabolites; NMR spectral of 1'-hydroxymyristicin; the

392

relative abundance of myristicin and its metabolites in urine, in plasma and in

393

microsomes; the relative abundance of 1'-hydroxymyristicin and its metabolites in

394

urine, in plasma and in microsomes; MS2 spectrums and fragmentation pathways of

395

M0, M1, M2 and M6; MS2 spectrums and fragmentation pathways of H0, H1, H7

396

and H10; summary of metabolites of myristicin produced in vivo and in vitro

397

metabolism; summary of metabolites of 1'-hydroxymyristicin produced in vivo and in

398

vitro metabolism; and roles of CYP450s in the formation of myristicin metabolites.



Page 22 of 34

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Bioactivation

of

Methyleugenol

to

the


Proximate

Carcinogen


Figure Captions

Fig. 1. Metabolomic analysis of myristicin and 1'-hydroxymyristicin metabolites in mouse urine. (A) Unsupervised PCA score plot of urine samples from control (●), myristicin (■) and 1'-hydroxymyristicin (▲) treated mice (n=4). (B) Loading plot of PCA for screening possible metabolites in urine. (C) Trend plot of common metabolite M6/H2. (D). Trend plot of common metabolite M10/H11. (E) Trend plot of unique metabolites M1. (F) Trend plot of unique metabolites H7.

Fig. 2. Characterization of myristicin-NAC adduct. Target extracted ions (m/z 370.0954) in chromatogram in (A) control mouse urine samples, and (B) mouse urine samples of myristicin. (C) mouse urine samples of 1'-hydroxymyristicin. (D) MS/MS spectrum and fragmentation pathways of M10 /H11.

Fig. 3. Formation of myristicin-NAC adduct in MLMs. Target extracted ions (m/z 370.0954) in chromatogram in MLMs incubations, (A) in the absence of NADPH, or (B) in the presence of NADPH. (C) 1'-Hydroxymyristicin captured with NAC without liver microsomes. (D) The formation rate of 1'-hydroxymyristicin in individual CYP450s. (E) Inhibitory effects of individual chemical CYPs inhibitors on the formation rate of 1'-hydroxymyristicin in MLMs.

Fig. 4. Assessment of myristicin and 1'-hydroxymyristicin cytotoxicity on 26 ACS Paragon Plus Environment

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primary mouse hepatocytes. (A) Effects of myristicin from 62.5 to 500 μM on the viability of primary mouse hepatocytes. (B) Effects of 1'-hydroxymyristicin from 62.5 to 500 μM on the viability of primary mouse hepatocytes. (C) Effect of NAC or DEM on myristicin (M) induced cytotoxicity towards primary mouse hepatocytes. (D) Effect of NAC or DEM on 1'-hydroxymyristicin (M') induced cytotoxicity towards primary mouse hepatocytes. *P < 0.05, **P < 0.01,

***P

< 0.001 compared to vehicle

control; #P < 0.05, ##P < 0.01, ###P < 0.001 compared to M or M'.

Fig.5 Proposed metabolic pathways of myristicin.

Fig.6 Proposed metabolic pathways of 1'-hydroxymyristicin.




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Metabolic activation of myristicin and its role in cellular toxicity

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