Academic article

Combining metabolomics with bioanalysis methods to investigate the potential toxicity of dihexyl phthalate

Song, Dan; Xu, Chao; Holck, Askild Lorentz; Liu, Rong

Publication details

Journal: Environmental Toxicology, vol. 36, p. 10, 2020

Issue: 2

International Standard Numbers:
Printed: 1520-4081
Electronic: 1522-7278

Open Access: none


Dihexyl phthalate (DHP) is one of the most commonly used phthalate esters in various
plastic and consumer products. Human are inevitably exposed to DHPs. Although
several animal and human experiments have revealed that DHP can cause multiple
toxicities, few studies have previously assessed the effects of DHP exposure by liquid
chromatography mass spectrometry (LC-MS) analysis combine with molecular
biology methods on human cells. Therefore, the purpose of our study was to investigate
the effect of DHP on human cell metabolism by systems biology methods. In
this study, U2OS cancer cells were treated with 10 μM DHP for metabolomics analysis
and apoptosis analysis at indicate time. Metabolomic study of the metabolic
changes caused by DHP in U2OS cells was performed for the first time using integrative
liquid chromatography coupled with quadrupole time-of-flight mass spectrometry
(LC-Q-TOF-MS). To investigate the possible reason of fatty acids level altered by
DHP, we measured some key fatty acid synthesis and oxidation-related enzyme
expression levels by quantitative real-time PCR (Q-PCR). Apoptotic cells were analyzed
by flow cytometry and apoptosis-related gene expressions were measured by
Q-PCR. 20,70-Dichlorofluorescein diacetate (DCFH-DA) staining was used to evaluate
ROS content. Partial least squares-discriminate analysis (PLS-DA) clearly showed that
significant differences in metabolic profiles were observed in U2OS cells exposed to
DHP compared with controls. A total of 58 putative metabolites in electrospray ionization
source (ESI) + mode and 32 putative metabolites in ESI-mode were detected,
the majority of the differential metabolites being lipids and lipid-like molecules.
Among them, the altered fatty acids level corresponded to expression levels of genes
encoding enzymes related to fatty acids synthesis and oxidation. Moreover, DHP
induced reactive oxygen species (ROS) accumulation, promoted cell apoptosis and
inflammation, and resulted in a significant increase in apoptosis and inflammationrelated
gene expression levels compared with controls. In summary, our results
suggested that metabolomics combined with molecular bioanalysis methods could be
an efficient tool to assess toxic effects, which contribute to explore the possible
cytotoxicity mechanisms of DHP, and provide a basis for further research.