Dissertation

Chlordecone pretreatment promoted subcellular distribution of scavenger receptor class B type II to murine hepatic microsomes; mass spectrometry detected hepatic soluble cholesterol binding proteins and comparison of protein iTRAQ ratios using ESI QTOF and MALDI TOF/TOF

Scheri, Richard C

2008

Overview

Chlordecone belongs to the class of persistent organochlorine pesticides that are remarkably resistant to environmental degradation. Even though their use was banned in the United States in 1978, these compounds can still be detected in both humans and wildlife throughout the world. Previous work has shown that the pretreatment of male C57BL/6 mice with low doses of the persistent organochlorine (OC) pesticide, chlordecone (CD) stimulated biliary excretion of exogenous CH up to 3-fold, and further, that increased biliary excretion was not associated with changes in ATP-binding cassette transporter G8 (ABCG8) or scavenger receptor class B type I (SR-BI). In rodents, hepatic basolateral SR-BI is important in controlling plasma lipoprotein levels and cholesterol (CH) homeostasis, with major roles in reverse CH transport (RCT) and biliary excretion. The hepatic ABCG5/G8 heterodimer is a membrane transporter present on the apical surfaces of hepatocytes, and also plays a key role in biliary CH secretion. Scavenger receptor class B type II (SR-BII) was identified as a splice variant from the SR-BI gene and is expressed in a variety of tissues. Although the function of SR-BII is not clear it was proposed to play a role in CH homeostasis and trafficking that was distinctly different than SR-BI. In the present study, western blotting was used to show that a single dose of CD promotes subcellular distribution of SR-BII to murine hepatic microsomes while having no effect on liver crude membrane SR-BII. Western blotting also indicated CD pretreatment had no effect on the levels of liver fatty acid binding protein (L-FABP) in cytosol, but an increase in myosin-9 was observed with mass spectrometry. Myosin-9 may play a role in intracellular vesicular transport. This may at least partially explain the previously observed alterations in CH homeostasis produced by CD pretreatment. Changes in relative protein levels using a CH binding protein enriched fraction prepared from hepatic cytosol were detected with mass spectrometry using isobaric tagging for relative and absolute quantification (iTRAQ) methodology. Many factors can affect the accuracy of quantification with this technique. It has been observed that the low collision energies normally used in ESI QTOF can result in low iTRAQ reporter ion abundances. After evaluating protein changes in response to CD pretreatment, two-way ANOVA was used to compare the mean protein iTRAQ results from mass spectrometers using different collision energies in the same samples. It appears that iTRAQ analyses performed on an ESI QTOF without any special modifications in instrumental parameters produce essentially the same protein ratios as those obtained on a MALDI TOF/TOF.

• Publisher: ProQuest Dissertations & Theses
• Subject: Analytical chemistry / Environmental science / Toxicology
• ISBN: 9780549725534, 0549725539