DEVELOPMENT OF AN AUTOMATED LIPIDOMICS APPROACH TO UNDERSTAND THE ACTION OF OBESOGENS

John A. Bowden, Raquel Chamorro-García, Bruce Blumberg, Louis Guillette, Michele Schantz, and John Kucklick

Current approaches for assessing potential exposure to endocrine disrupting compounds (EDCs) typically focus on characterizing the EDC-mediated effects by measuring hormone levels or gene expression profiles; however, some EDCs have been shown to possess multiple disruption mechanisms including those pathways integral in lipid metabolism. Further, recent reports have implicated specific contaminants as capable of exerting an obesogenic response (referred to as obesogens) by operating as ligand mimics to specific receptors (e.g., PPAR and RXR) critical in adipocyte generation, lipid biosynthesis/storage, adipose formation and appetite regulation. However, the exact nature and overall contribution of these obesogens to the incidence of obesity is still largely uncharacterized. An approach well-suited for an improved understanding of the myriad of metabolic changes associated with obesogen exposure would be the development and implementation of lipid-based methods (e.g., lipidomics), since lipid metabolism is intimately linked to obesogen targets.

In this preliminary obesogen/lipidomic study, livers (both male and female) were collected and flash frozen from 8-week mice offspring that were exposed in utero to control, the model obesogen tributyltin (TBT) or rosiglitazone (ROSI, PPAR agonist). The mice studies were performed at the University of California (by Dr. Bruce Blumberg). The pregnant females were exposed to the agonists (0.1mg/Kg TBT, 1 mg/Kg ROSI and CMC (carboxymethyl cellulose, vehicle)) by gavage feeding at E16.5 (embryonic day 16.5). The use of TBT as an obesogen is based on previous research which has shown that exposure to TBT during critical developmental stages (e.g., in utero) can have dramatic effects on adipogenesis and lipid accumulation. After the frozen livers were homogenized, the lipids (with an internal standard cocktail) were extracted by the Folch method (chloroform/methanol, 2:1 v/v). Lipid extracts were then directly infused (shotgun lipidomics) into a hybrid triple quadrupole/linear ion trap mass spectrometer (equipped with electrospray ionization), modified with the inclusion of a syringe pump in series (between the autosampler and the mass spectrometer) to allow for a high-throughput automated workflow. Lipid alterations were assessed in positive (or negative) survey scan mode and on a class and/or individual species level using previously optimized intra-source separation and tandem mass spectrometry (MS/MS) parameters.

To date, there are no published studies using mass spectrometry to assess the impact of obesogen exposure on lipid metabolism. Using developed lipidomic/mass spectrometry-based methods described here, specific global, class, and individual lipid alterations were highlighted between treatment groups and sexes. Qualitative and semi-quantitative trends were shown to be present in several lipid species, including fatty acids, cholesteryl esters, triacylglycerols, phospholipids and sphingolipids.