Modulation of Apoptosis by the Dithiolethione ACDT as a Neuroprotective Mechanism Against Manganese‐Induced Toxicity
Authors: Zaveri Rohan Purvin , Gadde Rajitha , Betharia Swati 
Published: April 2020

Introduction

Manganese (Mn) is an essential trace metal involved in metabolic processes in both animals and plants. Overexposure to this metal can cause oxidative stress leading to a neurological disorder known as manganism. Excessive accumulation of Mn is believed to cause apoptosis in cells. Apoptosis is a tightly regulated process in which cell death is induced by the activation of specific signaling pathways through numerous regulatory proteins such as Bax and Bcl‐2. Dithiolethiones are a class of sulfur‐containing heterocyclic molecules. Among them, ACDT (5‐amino‐3‐thioxo‐3H‐(1,2) dithiole‐4‐carboxylic acid ethyl ester) has been shown to induce cellular production of the natural antioxidant glutathione. It has been proven that ACDT provides neuroprotection against Mn toxicity by activating the Nrf2‐Keap1 pathway. In this study, we hypothesized that ACDT also causes upregulation of anti‐apoptotic protein Bcl‐2 and downregulation of pro‐apoptotic protein Bax as an additional mechanism of neuroprotection.

Methods

SH‐SY5Y cells were treated with ACDT at concentrations of 50, 75 and 100 μM. After 24 hours, the cells were either treated with MnCl2 (300 μM) or media for an additional 24 hours. Eight experimental groups included ‐ Control, MnCl2 (300 μM) alone, ACDT (50, 75, and 100 μM) alone and MnCl2 + ACDT (50, 75, and 100 μM) in combination.

The BCA assay was conducted for the quantification of total protein in each sample. A Western blot analysis was performed to detect and measure the levels of Bcl‐2 and Bax proteins. Denatured samples were loaded onto polyacrylamide gels. Subsequently, gel electrophoresis was performed, and separated proteins were transferred onto a nitrocellulose membrane. The membrane was blocked with blocking buffer and then treated sequentially with primary and secondary antibodies, followed by a chemiluminescent substrate. The protein bands on the membranes were then detected using a C‐Digit scanner, and quantified using the ImageStudio Lite® software. Beta actin served as a loading control.

Results

A significant decrease in Bax protein levels (4‐fold) was observed in groups that were pretreated with ACDT (75 μM and 100 μM) and then exposed to Mn. A significant decrease in Bax was also observed in the ACDT (50 μM) + Mn group when compared to ACDT (50 μM) alone. No significant changes were observed in Bcl‐2 levels for any treatment group. However, a trend towards a dose‐dependent decrease in Bcl‐2 levels was observed in cells treated with the ACDT (50, 75 and 100 μM) and MnCl2 in combination.

Conclusions

ACDT was found to cause a downregulation of the pro‐apoptotic protein Bax, which likely contributed to its neuroprotective effects. As no changes in levels of the Bcl‐2 protein were observed, the data obtained only partially met our hypothesis. Additional research is currently underway to conclusively prove the role of apoptosis in ACDT’s neuroprotective pathway.

Support or Funding Information

MCPHS University School of Pharmacy‐ SURF Program