Characterization of basal and di (2-ethylhexyl) phthalate altered fatty acid transport across the human immortalized blood brain barrier cell line, hCMEC/D3

Lori Beth Karpes, Purdue University

Abstract

Fatty acids (FA) are highly important in both the development of the fetal brain and its functioning in later stages of life. During fetal development, the blood brain barrier (BBB) is thought to play a large role in the regulation of lipid homeostasis within the brain and therefore the development of the CNS. Di-(2-ethylhexyl)-Phthalate (DEHP) is a widely used plasticizer and has been identified as a compound that can alter the lipid content of rat pup brains after maternal exposure apparently by altering fatty acid homeostasis at the placental level. While the effects at the placenta seem to be the primary site of DEHP's impact, the BBB also represents a point of regulation for the availability of FAs to the brain both before and after parturition. To investigate the effects of DEHP on the human BBB cell model hCMEC/D3, we first established the endogenous expression of mRNA levels of key FA transport conferring proteins and transcriptional regulatory factors that control their production via qRT-PCR. Additional qRT-PCR studies determined that the mRNA expression of these targets was altered upon exposure to DEHP, indicating a similar effect at the BBB as at the placenta. Protein expression of these targets was investigated via Western Blotting to confirm these findings and it was determined that while not all targets had corresponding protein expression alteration, upstream regulators of FA homeostasis were observed to undergo modest changes that may potentially signify the capacity of DEHP to alter homeostatic processes at the BBB. As the overall consequence of the changes is not readily apparent, the movement of radiolabeled FAs across the hCMEC/D3 line after DEHP exposure was also examined and it was determined that the directional movement of the essential FAs Linoleic and Linolenic acid, which are important precursors to many bioactive signaling molecules and modified FAs, was modestly reduced in the apical to basolateral direction. Mass balance investigations also indicate that exposure to DEHP results in the intracellular accumulation of these FAs, which along with the reduced transport suggests that the in vivo movement of fatty acids into the brain may potentially face disruption in a similar manner as observed at the placental level though of much lower magnitude.

Degree

Ph.D.

Advisors

Knipp, Purdue University.

Subject Area

Pharmaceutical sciences|Developmental biology|Surgery

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