Research Paper
In-depth profiling of di(2-ethylhexyl) phthalate metabolic footprints in rats using click chemistry-mass spectrometry probes

https://doi.org/10.1016/j.jhazmat.2023.131190Get rights and content

Highlights

  • CC-MS strategy was developed for in-depth profiling DEHP metabolites in rats.

  • An alkyne-modified DEHP analogue was synthesized as a tracer.

  • MS probes labeling facilitates the detection of DEHP metabolites in LC-MS analysis.

  • A total of 247 potential DEHP metabolites were successfully screened.

  • A possible complementary pathway for the metabolism of DEHP in rats was proposed.

Abstract

Di(2-ethylhexyl) phthalate (DEHP), the most widely used plasticizers in the world, has been regarded as an endocrine disrupting chemical with serious adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the metabolic footprints of DEHP in vivo are still unclear. Here we developed a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth profiling DEHP metabolites in rats. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to specifically label the alkyne-DEHP metabolites, and prominently improve their detection sensitivity and selectivity. Using the CC-MS strategy, we successfully screened 247 alkyne-DEHP metabolites from rat urine, feces, and serum, including many unrevealed metabolites, such as oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites. The discovery of new DEHP metabolites provides additional insights for understanding the metabolism of DEHP, which may be beneficial in exploring the mechanism underlying DEHP induced-toxicity in the future.

Introduction

Endocrine disrupting chemicals (EDCs) are a class of exogenous substances that are present in nature or artificially synthesized, and have the ability to mimic or block hormones [1], [2]. Human and wildlife populations are exposed to EDCs through ingestion of food, dust and water, inhalation of volatile and particle bound contaminants, and dermal uptake, causing serious adverse health effects, such as decreased reproductive capacity, infant death, and even extinction [3], [4], [5]. The health risks and environmental contamination caused by EDCs have become a major public health problem all over the world [6].

Di(2-ethylhexyl) phthalate (DEHP) is the most widely used plasticizers in the world and is considered as a typical EDC [7], [8], [9]. DEHP has also been listed as the top-priority environmental pollutant by environmental protection agencies of Canada, the United States, the European union and other countries [10], [11]. Prolonged or high-dose exposure to DEHP can cause reproduction and behavior defects, as well as other toxicities, including immunotoxicity, embryotoxicity, hepatotoxicity, neurotoxicity and carcinogenicity [12], [13], [14], [15]. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are highly likely to be mediated by its metabolites rather than itself [16], [17], [18]. For example, mono(2-ethylhexyl) phthalate (MEHP, the primary metabolite of DEHP) and its derivatives are 10 times more toxic than DEHP itself [19], [20]. However, little is known about DEHP metabolites, including their diversity and metabolic processes. Therefore, uncovering the metabolism of DEHP in vivo is essential for elucidating the underlying mechanisms associated with DEHP exposure-induced toxicity.

Isotope tracing is a commonly used technique for exploring the metabolism of DEHP in vivo, generally using isotope-encoded DEHP as the tracer and mass spectrometry (MS) as its detector [21], [22], [23]. However, the coverage of DEHP metabolites traced by this technique is limited. This is mainly due to the poor ionization efficiency of DEHP and its metabolites in electron spray ionization (ESI) source, which hinders the MS detection of its low-abundance metabolites. Additionally, the isotopic atoms of some DEHP isotopic tracers (di(2-ethylhexyl)-[7-14C]phthalate or di(2-ethylhexyl)phthalate-3,4,5,6 D4) are usually located in the phthalate moiety, which can only satisfy the research on the metabolic pathway of the phthalate monoester [21], [22], [24], [25], [26]. Restricted by the aforementioned drawbacks, the metabolic footprint of DEHP in vivo remains largely unclear.

Click chemistry is a class of chemical conjugation reactions that have good selectivity as well as biocompatibility, and can be performed in high yield under mild aqueous conditions [27], [28]. The combination of click chemistry and mass spectrometry has gradually become a promising tool in bioscience fields. For example, in the study of post-translational modifications, click chemistry is always conjugated to cleavable biotin affinity tags for the selective enrichment of proteins and peptides of interest, which are subsequently identified and characterized by MS to enable the tracking of targets [29], [30], [31], [32]. However, apart from proteomics studies, there are few reports on the application of click chemistry-based MS strategy to investigate the metabolism of small molecules in vivo. Recently, Thiele and co-workers reported a click-chemistry mass spectrometry reporter strategy for highly sensitive tracing the lipid anabolism and catabolism using a series of alkyne-modified fatty acids [33], [34]. Hoki et al. developed a click-chemistry-based enrichment strategy to investigate metabolism of the ascarosides, and discovered several hundred novel compounds originating from diverse biosynthetic transformations [35]. These studies suggest that click chemistry may have great application potential in the field of metabolic tracing of EDCs.

In this study, we aim to develop an in vivo metabolic tracing strategy for DEHP with high sensitivity and coverage by a click chemistry-assisted mass spectrometry (CC-MS) probe technique. An alkyne-modified DEHP analogue (alkyne-DEHP) was synthesized as a tracer for in vivo tracing, and a pair of MS probes (4-azido-nphenylbenzamide, 4-ANPA, and its deuterated reagent d5-4-ANPA) were prepared to selectively label the alkyne-DEHP metabolites via click chemistry. Using this strategy, we explored the metabolic transformations of DEHP in rat urine, feces, as well as serum, and successfully detected 247 alkyne-DEHP metabolites, among which the oxidized phthalate diester metabolites and glucuronides of phthalate monoester metabolites were identified for the first time in rats.

Section snippets

Chemicals and Reagents

Eight alkyne standards were obtained from Aladdin Reagent Co. (Shanghai, China), Energy Chemical (Shanghai, China) and Bide pharmatech Ltd. (Shanghai, China). DEHP was purchased from Adamas Reagent Ltd. (Shanghai, China). MS probes reagents (4-ANPA and d5-4-ANPA) and alkyne-modified DEHP metabolites (alkyne-DEHP, alkyne-MEHP, and alkyne-5oxo-MEHP) were synthesized in our laboratory. The list of detailed information (name, CAS number, molecular formula, molecular weight and company) of standards

Principle of CC-MS strategy

In this study, we proposed a CC-MS strategy for in-depth profiling DEHP metabolites in rats. The principle of the CC-MS strategy is to use an alkyne-DEHP (Fig. 1A) as a tracer for in vivo tracing, and then selectively label its metabolites in vitro with a pair of MS probes (4-ANPA and d5-4-ANPA) containing an azido group and isotope coding via the click reaction. MS probes labeling can facilitate the ionization of the labeled metabolites in ESI, improve their LC separation, and give a

Conclusions

In this work, we reported a CC-MS strategy for in-depth tracing DEHP metabolism in vivo. The synthesized alkyne-DEHP analogue was used as a tracer to mimic the in vivo metabolic process of DEHP, while 4-ANPA and d5-4-ANPA probe can selectively label alkyne-DEHP metabolites, and prominently improve their detection sensitivity. Using the CC-MS strategy, 247 DEHP metabolites were successfully detected from the urine, feces, and serum of rats orally administrated alkyne-DEHP, including many novel

Environmental implication

Di(2-ethylhexyl) phthalate (DEHP) is the most widely used plasticizers in the world, and has been considered as an endocrine disrupting chemical (EDC) with adverse health outcomes. Accumulating evidence strongly suggests that the undesirable biological effects of DEHP are meditated by its metabolites rather than itself. However, the current knowledge about metabolic footprints of DEHP is very limited. Here we reported a click chemistry-assisted mass spectrometry (CC-MS) strategy for in-depth

CRediT authorship contribution statement

Yu-Ning Hu: Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft. Jin-Tao Zhang: Formal analysis, Investigation, Methodology. Pei-Rong Bai: Formal analysis, Investigation. Na An: Data curation, Validation. Jun-Jie Tan: Investigation. Yan-Zhen Wang: Data curation. Quan-Fei Zhu: Conceptualization, Funding acquisition, Writing – original draft, Writing – review & editing. Yu-Qi Feng: Conceptualization, Supervision, Funding acquisition, Writing – review &

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reports in this paper.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (22274119, 21721005) and the Fundamental Research Funds for the Central Universities (2042022kf1030). We thank Jun-Di Hao and Peng-Cheng Mei for their help in rat sample collection and preparation.

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