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An in vitro metabolomics approach to identify hepatotoxicity biomarkers in human L02 liver cells treated with pekinenal, a natural compound

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Abstract

An in vitro cell metabolomics study was performed on human L02 liver cells to investigate the toxic biomarkers of pekinenal from the herb Euphorbia pekinensis Rupr. Pekinenal significantly induced L02 cell damage, which was characterised by necrosis and apoptosis. Metabolomics combined with data pattern recognition showed that pekinenal significantly altered the profiles of more than 1299 endogenous metabolites with variable importance in the projection (VIP) > 1. Further, screening correlation coefficients between the intensities of all metabolites and the extent of L02 cell damage (MTT) identified 12 biomarker hits: ten were downregulated and two were upregulated. Among these hits, LysoPC(18:1(9Z)/(11Z)), PC(22:0/15:0) and PC(20:1(11Z)/14:1(9Z)) were disordered, implying the initiation of inflammation and cell damage. Several fatty acids (FAs) (3-hydroxytetradecanedioic acid, pivaloylcarnitine and eicosapentaenoyl ethanolamide) decreased due to fatty acid oxidation. Dihydroceramide and Cer(d18:0/14:0) were also altered and are associated with apoptosis. Additional examination of the levels of intracellular reactive oxygen species (ROS) and two eicosanoids (PGE2, PGF2α) in the cell supernatant confirmed the fatty acid oxidation and arachidonic acid metabolism pathways, respectively. In summary, cell metabolomics is a highly efficient approach for identifying toxic biomarkers and helping understand toxicity mechanisms and predict herb-induced liver injury.

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Abbreviations

ALT:

Alanine transaminase

AST:

Aspartate transaminase

BPI:

Base peak intensity

CoA:

Coenzyme A

COX:

Cyclooxygenase

cPLA2:

Cytosolic phospholipase A2

CS:

Ceramide synthase

DCFH-DA:

2′,7′-Dichlorofluorescin diacetate

DHCer:

Dihydroceramide

DILI:

Drug-induced liver injury

DMEM:

Dulbecco’s modified eagle medium

DMSO:

Dimethyl sulfoxide

EPEA:

Eicosapentaenoyl ethanolamide

ER:

Endoplasmic reticulum

ESI:

Electrospray ionisation

FAs:

Fatty acids

FBS:

Fetal bovine serum

LPL:

Lipoprotein lipase

LysoPCs:

Lysophosphatidylcholines

MRM:

Multiple reaction monitoring

OPLS-DA:

Orthogonal projection to latent structure discriminate analysis

PCs:

Phosphatidylcholines

PLS-DA:

Partial least squares discriminant analysis

PPARα :

Peroxisome proliferator-activated receptor alpha

ROS:

Reactive oxygen species

S-plot:

Similarity plot

UPLC-QTOF/MS:

Ultra-performance liquid chromatography-quadrupole time of flight/mass spectrometry

UPLC-TQ/MS:

Ultra-performance liquid chromatography-triple quadrupole/mass spectrometry

VIP:

Variable importance in the projection

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Acknowledgments

The project is supported by National Basic Research Program of China (973 Program) (2011CB505300, 2011CB505303), National Natural Science Foundation of China (81274199, 81102762, 30901894 and 81403041), Open Fund of Collaborative Innovation Center of Chinese Medicinal Resources Industrialization (ZDXM-1-14), Six talent peaks project in Jiangsu Province (YY-015),the Natural Science Foundation of Jiangsu Province (BK20140961) and a project founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Authors and Affiliations

  1. Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Pharmacy College, Nanjing University of Chinese Medicine, 138 Xianlin Road, Nanjing, 210023, China

    Jiexia Shi, Jing Zhou, Hongyue Ma, Jin’ao Duan, Weiwei Tao & Dawei Qian

  2. Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, ON, M5S3E1, Canada

    Hongbo Guo & Zuyao Ni

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  1. Jiexia Shi
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  2. Jing Zhou
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  3. Hongyue Ma
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  6. Jin’ao Duan
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  7. Weiwei Tao
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Correspondence to Hongyue Ma or Jin’ao Duan.

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Jiexia Shi and Jing Zhou contributed equally to this work.

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Shi, J., Zhou, J., Ma, H. et al. An in vitro metabolomics approach to identify hepatotoxicity biomarkers in human L02 liver cells treated with pekinenal, a natural compound. Anal Bioanal Chem 408, 1413–1424 (2016). https://doi.org/10.1007/s00216-015-9202-4

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