Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging

Abstract

Nuclear receptors (NRs) are ligand-modulated transcription factors that regulate multiple physiological functions in our body. Many NRs in their unliganded state are localized in the cytoplasm. The ligand-inducible nuclear translocation of NRs provides a valuable tool for studying the NR-ligand interactions and their downstream effects. The translocation response of NRs can be studied irrespective of the nature of the interacting ligand (agonist, antagonist, or a small molecule modulator). These nuclear translocation studies offer an advantage over promoter-reporter-based transcription assays where transcription response is observed only with the activating hormones or agonistic ligands. Globally, milk serves as a major dietary source. However, suspected presence of endocrine/metabolism-disrupting chemicals like bisphenols, parabens, organochlorine pesticides, carbamates, non-steroidal anti-inflammatory drugs, chloramphenicol, brominated flame retardants, etc. has been reported. Considering that these chemicals may impart serious developmental and metabolism-related health concerns, it is essential to develop assays suitable for the detection of xenobiotics present at differing levels in milk. Since milk samples cannot be used directly on cultured cells or for microscopy, a combination of screening strategies has been developed herein based on the revelation that i) lipophilic NR ligands can be successfully retrieved in milk-fat; ii) milk-fat treatment of cells is compatible with live-cell imaging studies; and finally, iii) treatment of cells with xenobiotics-spiked and normal milk derived fat provides a visual and quantifiable response of NR translocation in living cells. Utilizing a milk-fat extraction method and Green Fluorescent Protein (GFP) tagged NRs expressed in cultured mammalian cells, followed by an assessment of NR response proved to be an effective approach for screening xenobiotics present in milk samples.

Highlights

• Diverse endocrine and metabolism-disrupting chemicals are suspected to contaminate milk.

• Nuclear receptors serve as ‘xenosensors’ for assessing the presence of xenobiotics in milk.

• Nuclear import of steroid receptors with (ant)agonist can be examined in live cells.

• Lipophilic xenobiotics are extracted and observed enriched in milk-fat fraction.

• A comprehensive cell-based protocol aids in the detection of xenobiotics in milk.

Keywords:

• Nuclear receptors
• milk-fat
• endocrine disruptors
• metabolism disruptors
• small molecule modulators
• live cell imaging
• nuclear translocation assay

Acknowledgements

The authors thank the funding agency for the financial support extended jointly to BHU-Varanasi, NDRI-Karnal, JNU-New Delhi, and IIT-Roorkee.

Author contributions

All authors contributed to the study’s conception and design. This comprehensive method involving material preparation, data collection, and analysis was developed, performed, and characterized by Rakesh K Tyagi, Keshav Thakur, Emmagouni Sharath Kumar Goud, and Yashika Jawa. The first draft of the manuscript was written by Rakesh K Tyagi, Keshav Thakur, Emmagouni Sharath Kumar Goud, and Yashika Jawa. Chetan Keswani, Suneel Onteru, Dheer Singh, Surya P Singh, and Partha Roy suggested the revisions in the subsequent versions of the manuscript. The final manuscript was edited and approved by all the authors as part of this joint inter-institutional research project.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

Data were generated at Special Center for Molecular Medicine. Derived data supporting findings are available from the corresponding author (RKT) on request.

Additional information

Funding

This inter-institutional project study was supported by the Indian Council of Agriculture Research New Delhi- ICAR, Government of India [grant number NASF-ABA-7006].