MSACL 2025 Abstract

Proteomic Analysis of Human Cells Exposed to Mono-n-butyl Phthalate (MnBP) using DIA-MS

Hwangkyo Jeong (1), Seungyoon Seo (1), Sujeong Yu (1), Seung-Hwa Lee(3), Hyun Ju Yoo (4), Hosub Im (5), Hoon Je Seong (6), Soo-Jong Hong (2), Yong Joo Park (7) and Jeonghun Yeom (1)
(1) Prometabio Research Institute, prometabio co., ltd. Hanam-si, Gyeonggi-do, Republic of Korea, (2) Department of Pediatrics, Humidifier Disinfectant Health Center, National Medical Center, Seoul, Republic of Korea, (3) Department of Medicine, University of Ulsan College of Medicine, Seoul, Republic of Korea, (4) Department of Convergence Medicine, Asan Institute for Life Sciences, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, (5) Institute for Life & Environmental Technology, Smartive Corporation, Hanam-si, Gyeonggi-do, Republic of Korea, (6) Department of Biological Science, Kunsan National University, Gunsan, Republic of Korea, (7) College of Pharmacy, Kyungsung University, Busan, Republic of Korea

Hwangkyo Jeong (Presenter) Prometabio Research Institute

Relevant Financial Disclosures (within past 24 months, reported on Aug 05, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Exposome is a concept that explains how exposure to internal and external substances, such as chemicals, in people's daily lives affects their health. Mono-n-butyl phthalate (MnBP), one of the environment pollutants, is easily exposed in daily life because it is widely used in plastic containers, etc. Although MnBP is known to be associated with respiratory diseases, studies on biological pathways and mechanisms are lacking. Therefore, we performed proteomics analysis using DIA-MS technology to identify differentially expressed proteins (DEPs) in monocytic cell line (THP-1) treated with MnBP and to elucidate the complex interactions related to MnBP exposure.

METHODS:
The control group was not treated with MnBP, while THP-1 cells were exposed to different concentrations of MnBP. All samples were incubated for 72 hours. Following incubation, the collected cell pellets and supernatants were lysed using 5% SDS. The extracted proteins were digested with trypsin for subsequent proteomic analysis. For proteomic analysis, DIA-MS was performed using the Exploris 480. Cell (500 ng) and supernatant (250 ng) samples were injected and analyzed in triplicate using a 35-minute gradient at a flow rate of 0.4 L/min. The DIA files from each sample were processed using DIA-NN (version 1.8.1) in library-free mode, with Human SwissProt (Version 2022.04) as the reference database. Data interpretation was conducted using PLS-DA and statistical analysis.

RESULTS:
MnBP, a component of the environment pollutants, may contribute to various diseases, including respiratory and skin disorders. However, the impact of environmental factors on biological changes remains poorly understood. In this study, we investigated the effects of MnBP on THP-1 cells using a proteomics-based approach. THP-1 cells were treated with MnBP at 250, 500, and 1,000 μg/mL. Control groups were maintained without MnBP treatment. After 72 hours of incubation, cell pellets and supernatants were collected. DIA-MS was performed for both qualitative and quantitative proteomic analysis across all samples.

A total of 6,281 proteins were identified from the cell pellet samples. Comparative analysis between MnBP-treated and untreated HaCaT cells revealed 164 DEPs. For THP-1 cells, comparative analysis across different MnBP concentrations identified 51 DEPs in the 250 μg/mL-treated group, 170 DEPs in the 500 μg/mL-treated group, and 537 DEPs in the 1,000 μg/mL-treated group, compared to the untreated control. Further analysis of DEPs identified in HaCaT and THP-1 cells led to the identification of 12 significant proteins. GO analysis indicated that the enriched biological processes included protein polymerization, intermediate filament organization, and protein repair. A total of 2,433 proteins were identified from the supernatant samples. Comparative analysis between MnBP-treated and untreated HaCaT supernatants revealed 267 DEPs. For THP-1 supernatants, comparative analysis across different MnBP concentrations identified 141 DEPs in the 250 μg/mL-treated group, 224 DEPs in the 500 μg/mL-treated group, and 312 DEPs in the 1,000 μg/mL-treated group, compared to the untreated control. GO analysis using 87 overlapping proteins among the DEPs identified by comparing THP-1 supernatan showed that Staphylococcus aureus infection was enriched in the KEGG pathway. These findings provide insights into MnBP-induced proteomic alterations and their potential biological implications.

CONCLUSION:
Our findings demonstrate that MnBP induces significant, dose-dependent changes in the THP-1 proteome, affecting both intracellular and secreted protein profiles. These alterations suggest disruption in structural protein integrity and cellular repair systems.

DISCUSSION:
Using the differentially expressed proteins (DEPs) commonly identified in both the monocytic cell line (THP-1) and keratinocyte cell line (HaCaT), GO analysis suggests that MnBP induces a conserved stress-related cellular response across different cell types. Furthermore, analysis of the THP-1 supernatant indicates that MnBP may influence immune regulatory mechanisms at the secretory level. These data collectively provide mechanistic insights into cell-type-specific immune responses to MnBP exposure and may serve as a foundation for future investigations into its immunotoxic effects.

Acknowledgement:
This work was supported by Korea Environment Industry & Technology Institute(KEITI) through Core Technology Development Project for Environmental Diseases Prevention and Management, funded by Korea Ministry of Environment(MOE)(RS-2022-KE002048) and the Research Program funded Korea National Institute of Health (2008-E33030-00, 2009-E33033-00, 2011-E33021-00, 2012-E33012-00, 2013-E51003-00, 2014-E51004-00, 2014-E51004-01, 2014-E51004-02, 2017-E67002-00, 2017-E67002-01, 2017-E67002-02, 2020E670200, 2020E670201).