System Biology-Guided Chemical Proteomics to Discover Protein Targets of Monoethylhexyl Phthalate in Regulating Cell Cycle

Xu, Tengfei; Chen, Liyan; Lim, Yan Ting; Zhao, Haoduo; Chen, Hongjin; Chen, Luyang; Huan, Tao; Huang, Yi; Sobota, Radoslaw M.; Fang, Mingliang

doi:10.1021/acs.est.0c05832

Cited by 19 publications

(10 citation statements)

References 57 publications

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“…Transcriptome analysis has emerged as a useful approach for identifying the key toxicity targets and mechanisms in risk assessment. − Various contaminant-induced transcriptome patterns can help identify the biological processes and pathways associated with the toxicities at the organ or organism levels, providing critical information about potential target biomacromolecules. Molecular modeling has also been used to decipher molecular recognition and conformational changes to characterize the MIEs. − The combination of in silico and in vitro assays can further establish the link between molecular alterations and toxic effects to realize the connection between MIE and KEs.…”

Section: Introductionmentioning

confidence: 99%

Carcinogenic Risk of 2,6-Di-tert-Butylphenol and Its Quinone Metabolite 2,6-DTBQ Through Their Interruption of RARβ: In Vivo, In Vitro, and In Silico Investigations

Cui

Zhan

et al. 2021

Environ. Sci. Technol.

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Thousands of contaminants are used worldwide and eventually released into the environment, presenting a challenge of health risk assessment. The identification of key toxic pathways and characterization of interactions with target biomacromolecules are essential for health risk assessments. The adverse outcome pathway (AOP) incorporates toxic mechanisms into health risk assessment by emphasizing the relationship among molecular initiating events (MIEs), key events (KEs), and adverse outcome (AO). Herein, we attempted the use of AOP to decipher the toxic effects of 2,6-di-tert-butylphenol (2,6-DTBP) and its para-quinone metabolite 2,6-di-tert-butyl-1,4-benzoquinone (2,6-DTBQ) based on integrated transcriptomics, molecular modeling, and cell-based assays. Through transcriptomics and quantitative real-time PCR validation, we identified retinoic acid receptor β (RARβ) as the key target biomacromolecule. The epigenetic analysis and molecular modeling revealed RARβ interference as one MIE, including DNA methylation and conformational changes. In vitro assays extended subsequent KEs, including altered protein expression of p-Erk1/2 and COX-2, and promoted cancer cell H4IIE proliferation and metastasis. These toxic effects altogether led to carcinogenic risk as the AO of 2,6-DTBP and 2,6-DTBQ, in line with chemical carcinogenesis identified from transcriptome profiling. Overall, our simplified AOP network of 2,6-DTBP and 2,6-DTBQ facilitates relevant health risk assessment.

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Section: Introductionmentioning

confidence: 99%

Carcinogenic Risk of 2,6-Di-tert-Butylphenol and Its Quinone Metabolite 2,6-DTBQ Through Their Interruption of RARβ: In Vivo, In Vitro, and In Silico Investigations

Cui

Zhan

et al. 2021

Environ. Sci. Technol.

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“… 15 In addition, phthalates can inhibit placental cell proliferation, 7 disrupt the endocrine system, 36 impair glucose metabolism, 8 , 37 and induce cell cycle dysregulation. 38 Our findings that maternal DEHP exposure caused poor muscle development in the offspring could explain the mechanisms behind a previous observation of DEHP exposure‐related sarcopenia. 3 …”

Section: Discussionmentioning

confidence: 58%

“…Foetuses and neonates are particularly sensitive to phthalate metabolites 35 because they have a deficiency in DNA repair and detoxification enzymes, and an underdeveloped blood–brain barrier 15 . In addition, phthalates can inhibit placental cell proliferation, 7 disrupt the endocrine system, 36 impair glucose metabolism, 8,37 and induce cell cycle dysregulation 38 . Our findings that maternal DEHP exposure caused poor muscle development in the offspring could explain the mechanisms behind a previous observation of DEHP exposure‐related sarcopenia 3 …”

Section: Discussionmentioning

confidence: 62%

Maternal rodent exposure to di‐(2‐ethylhexyl) phthalate decreases muscle mass in the offspring by increasing myostatin

Luo

Rong

et al. 2022

J cachexia sarcopenia muscle

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Background Di‐(2‐ethylhexyl) phthalate (DEHP) and its metabolites can cross the placenta and may cause birth defects and developmental disorders. However, whether maternal DEHP exposure affects skeletal muscle development in the offspring and the pathways involved are unknown. This study investigated the effects of maternal DEHP exposure and the contribution of myostatin (MSTN) to skeletal muscle development in the offspring. Methods Pregnant wild‐type and muscle‐specific myostatin knockout (MSTN KO) C57BL/6 mice were randomized to receive vehicle (corn oil) or 250 mg/kg DEHP by gavage every other day until their pups were weaned (postnatal day 21 [PND21]). Body weights of the offspring mice were measured longitudinally, and their hindleg muscles were harvested at PD21. Also, C2C12 cells were treated with mono‐2‐ethylhexyl phthalate (MEHP), the primary metabolite of DEHP, and proteolysis, protein synthesis, and myogenesis markers were measured. The contribution of myostatin to maternal DEHP exposure‐induced muscle wasting in the offspring was determined. Results Maternal DEHP exposure reduced body weight growth, myofibre size, and muscle mass in the offspring compared to controls (Quad: 2.70 ± 0.1 vs. 3.38 ± 0.23, Gastroc: 2.29 ± 0.09 vs. 2.81 ± 0.14, Tibialis: 1.01 ± 0.07 vs. 1.25 ± 0.11, mg/tibial length in mm, all P < 0.01, n = 35). Maternal DEHP exposure significantly increased Myostatin expression (2.45 ± 0.41 vs. 0.03 ± 0.00 DEHP vs. controls, P < 0.01, n = 5), Atrogin‐1(2.68 ± 0.65 vs. 0.63 ± 0.01, P < 0.05, n = 5), MuRF1 (1.56 ± 0.51 vs. 0.31 ± 0.01, P < 0.05, n = 5), and Smad2/3 phosphorylation (4.12 ± 0.35 vs. 0.49 ± 0.18, P < 0.05), and decreased MyoD (0.27 ± 0.01 vs. 1.52 ± 0.01, P < 0.05, n = 5), Myogenin (0.25 ± 0.03 vs. 1.95 ± 0.56, P < 0.05, n = 5), and AKT phosphorylation (4.12 ± 0.35 vs. 1.00 ± 0.06, P < 0.05, n = 5), in skeletal muscle of the offspring in MSTNflox/flox, but not in MSTN KO mice. Maternal DEHP exposure resulted in up‐regulation of CCAAT/enhancer‐binding protein δ (C/EBPδ, 4.12 ± 0.35 vs. 1.00 ± 0.19, P < 0.05, n = 5) in skeletal muscle of the offspring in MSTNflox/flox and MSTN KO mice (4.12 ± 0.35 vs. 4.35 ± 0.28, P > 0.05, n = 5). In vitro, C/EBPδ silencing abrogated the MEHP‐induced increases in Myostatin, MuRF‐1, and Atrogin‐1 and decreases in MyoD and Myogenin expression. Conclusions Maternal DEHP exposure impairs skeletal muscle development in the offspring by enhancing the C/EBPδ‐myostatin pathway in mice.

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“…In a third example, combining TPP, quantitative proteomics, metabolomics, and in silico molecular docking, a novel protein target and MOA were revealed for the metabolite of the plasticizer di(2-ethylhexyl) phthalate and monoethylhexyl phthalate (MEHP) ( Xu T. et al, 2021 ). In the TPP experiment conducted with the cell lysate, 74 proteins were shifted by MEHP, and in molecular docking, MEHP bound to all 36 proteins with a published crystal structure.…”

Section: Current Advances In Cetsa Msmentioning

confidence: 99%

“…When all the multi-omics data had been integrated into metabolite–gene and protein–protein interaction networks, MEHP seems to affect metabolites and proteins important in the cell cycle transition from G1 to S phase. MEHP was confirmed to hinder cell transit from the G1 phase to S phase with a flow cytometry assay ( Xu T. et al, 2021 ).…”

Section: Current Advances In Cetsa Msmentioning

confidence: 99%

Current Advances in CETSA

Tolvanen

2022

Front. Mol. Biosci.

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Knowing that the drug candidate binds to its intended target is a vital part of drug discovery. Thus, several labeled and label-free methods have been developed to study target engagement. In recent years, the cellular thermal shift assay (CETSA) with its variations has been widely adapted to drug discovery workflows. Western blot–based CETSA is used primarily to validate the target binding of a molecule to its target protein whereas CETSA based on bead chemistry detection methods (CETSA HT) has been used to screen molecular libraries to find novel molecules binding to a pre-determined target. Mass spectrometry–based CETSA also known as thermal proteome profiling (TPP) has emerged as a powerful tool for target deconvolution and finding novel binding partners for old and novel molecules. With this technology, it is possible to probe thermal shifts among over 7,000 proteins from one sample and to identify the wanted target binding but also binding to unwanted off-targets known to cause adverse effects. In addition, this proteome-wide method can provide information on the biological process initiated by the ligand binding. The continued development of mass spectrometry labeling reagents, such as isobaric tandem mass tag technology (TMT) continues to increase the throughput of CETSA MS, allowing its use for structure–activity relationship (SAR) studies with a limited number of molecules. In this review, we discussed the differences between different label-free methods to study target engagement, but our focus was on CETSA and recent advances in the CETSA method.

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System Biology-Guided Chemical Proteomics to Discover Protein Targets of Monoethylhexyl Phthalate in Regulating Cell Cycle

Cited by 19 publications

References 57 publications

Carcinogenic Risk of 2,6-Di-tert-Butylphenol and Its Quinone Metabolite 2,6-DTBQ Through Their Interruption of RARβ: In Vivo, In Vitro, and In Silico Investigations

Carcinogenic Risk of 2,6-Di-tert-Butylphenol and Its Quinone Metabolite 2,6-DTBQ Through Their Interruption of RARβ: In Vivo, In Vitro, and In Silico Investigations

Maternal rodent exposure to di‐(2‐ethylhexyl) phthalate decreases muscle mass in the offspring by increasing myostatin

Current Advances in CETSA

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