Effect of Pharmaceutical Potential Endocrine Disruptor Compounds on Protein Disulfide Isomerase Reductase Activity Using Di-Eosin-Oxidized-Glutathion

Abstract

BackgroundProtein Disulfide Isomerase (PDI) in the endoplasmic reticulum of all cells catalyzes the rearrangement of disulfide bridges during folding of membrane and secreted proteins. As PDI is also known to bind various molecules including hormones such as estradiol and thyroxin, we considered the hypothesis that adverse effects of endocrine-disrupter compounds (EDC) could be mediated through their interaction with PDI leading to defects in membrane or secreted proteins.Methodology/Principal FindingsTaking advantage of the recent description of the fluorescence self quenched substrate di-eosin-oxidized-glutathion (DiE-GSSG), we determined kinetically the effects of various potential pharmaceutical EDCs on the in-vitro reductase activity of bovine liver PDI by measuring the fluorescence of the reaction product (E-GSH). Our data show that estrogens (ethynylestradiol and bisphenol-A) as well as indomethacin exert an inhibition whereas medroxyprogesteroneacetate and nortestosterone exert a potentiation of bovine PDI reductase activity.ConclusionsThe present data indicate that the tested EDCs could not only affect endocrine target cells through nuclear receptors as previously shown, but could also affect these and all other cells by positively or negatively affecting PDI activity. The substrate DiE-GSSG has been demonstrated to be a convenient substrate to measure PDI reductase activity in the presence of various potential EDCs. It will certainely be usefull for the screening of potential effect of all kinds of chemicals on PDI reductase activity.

Potentiation of the reductase activity of protein disulphide isomerase (PDI) by 19-nortestosterone, bacitracin, fluoxetine, and ammonium sulphate

“…In a previous paper 5 , we reported that two non-oestrogenic steroids, MPA, and 19-NT potentiated PDI reductase activity. Even with a somewhat limited screening, in the present study we were able to identify a few other molecules also potentiating PDI reductase activity using the recently developed assay with DiE-GSSG as substrate 6 .…”

“…To our knowledge, this paper and the previous one from our laboratory 5 are the first to describe ligands that potentiate PDI activity. Molecules with such a potentiating effect on PDI activity would be of utmost interest for the treatment of pathologies originating from intracellular protein aggregation.…”

“…Likewise, the non-steroidal anti-inflammatory molecule indomethacin was also found to inhibit PDI reductase activity in the same assay. Surprisingly, we found that two non-oestrogenic steroids, medroxyprogesterone acetate (MPA) and , in contrast potentiated PDI reductase activity 5 . In order to get a better understanding of the ligands properties responsible for inhibition or augmentation of PDI reductase activity, we performed a preliminary screening with various other molecules in order to get a first guess on the molecular structural features of the ligands exhibiting either inhibitory or stimulatory effects, respectively.…”

“…The PDI substrate, DiE-GSSG, was synthesized and purified as previously described 6 with minor modifications 5 . PDI reductase activity was measured through abolishment of fluorescent self quenching when DiE-GSSG is reduced into two molecules of eosin-reduced glutathione (E-GSH).…”

“…In particular, PDI has been shown to bind oestradiol and this leads to a decrease in its isomerase activity as followed by its activity on RNAse renaturation 4 . Using di-eosin oxidized glutathione (DiE-GSSG) as substrate, it has been previously shown 5 that all the potent oestrogenic molecules tested [oestradiol, E2; 17α-ethynylestradiol (EE2) and diethylstilbestrol (DES)] also exhibited an inhibitory effect on PDI reductase activity. Likewise, the non-steroidal anti-inflammatory molecule indomethacin was also found to inhibit PDI reductase activity in the same assay.…”