Detection of endocrine and metabolism disrupting xenobiotics in milk-derived fat samples by fluorescent protein-tagged nuclear receptors and live cell imaging
Creators: name: Thakur, Keshav
name: Goud, Emmagouni Sharath Kumar
name: Jawa, Yashika
name: Keswani, Chetan
name: Onteru, Suneel
name: Singh, Dheer
name: Singh, Surya P.
name: Roy, Partha
name: Tyagi, Rakesh K.
Publisher: name: Taylor & Francis
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Cancer
subject: Inorganic Chemistry
subject: Computational Biology

description: 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.HighlightsDiverse 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. 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.