Arsenic-Induced Injury of Mouse Hepatocytes through Lysosome and Mitochondria: An In Vitro Study

Amal Santra; Debasree Bishnu; Suman Santra; Subhadip Ghatak; Partha Sarathi Mukherjee; Gopal Krishna Dhali; Abhijit Chowdhury

doi:10.1155/2022/1546297

Arsenic-Induced Injury of Mouse Hepatocytes through Lysosome and Mitochondria: An In Vitro Study

Int J Hepatol. 2022 Sep 8:2022:1546297. doi: 10.1155/2022/1546297. eCollection 2022.

Authors

Amal Santra^{1

2}, Debasree Bishnu¹, Suman Santra³, Subhadip Ghatak³, Partha Sarathi Mukherjee^{1

2}, Gopal Krishna Dhali^{2

3}, Abhijit Chowdhury^{1

2

3}

Affiliations

¹ JCM Centre for Liver Research and Innovations, Kolkata, India.
² Liver Foundation, Kolkata, West Bengal, India.
³ Centre for Liver Research, School of Digestive & Liver Diseases, Institute of Post Graduate Medical Education & Research, Kolkata, India.

Abstract

Background and aims: The cellular mechanism of liver injury related to arsenic toxicity is ill defined. It is thought that oxidative stress and mitochondrial dysfunction may play some role in arsenic-induced liver damage. In this study, we evaluated subcellular events within the primary cultured mouse hepatocytes when exposed to inorganic arsenic.

Methods: Primary cultured mouse hepatocytes were treated with 10 μM arsenic for different time periods. Reactive oxygen species (ROS) formation, functional changes of the lysosome and mitochondria, and mode of hepatocytes death were studied by laser confocal microscopy, fluorescence spectroscopy, and flow cytometry. Expression of proapoptotic member of the BCL-2 family of genes BAX and antiapoptotic BCL-2 mRNA expression were studied by real-time PCR. Cytochrome c expression was studied by Western blotting.

Results: Fluorescence spectroscopy as well as flow cytometric analysis revealed that arsenic-induced formation of ROS was time dependent. Confocal microscopy showed initiation of ROS formation from periphery of the hepatocytes at 30 min of arsenic exposure that progressed to central part of the hepatocytes at 3 h of arsenic exposure. The ROS formation was found to be NADPH oxidase (NOX) dependent. This low level of intracellular ROS induced lysosomal membrane permeabilization (LMP) and subsequently released cathepsin B to the cytosol. The LMP further increased intracellular ROS which in turn triggered induction of mitochondrial permeability transition (MPT). Pretreatment of hepatocytes with LMP inhibitor bafilomycin A (BafA) significantly decreased, and LMP inducer chloroquine (ChQ) significantly increased the production of ROS suggesting that LMP preceded enhanced ROS generation in response to arsenic. MPT was accompanied with increase in BAX : BCL2 mRNA ratio resulting in upregulation of caspase 3 and increased hepatocyte apoptosis.

Conclusion: Although arsenic-related oxidative liver injury is well established, neither the site of origin of ROS nor the early sequence of events in arsenic toxicity due to ROS is known. We believe that our study provides evidences elucidating the early sequence of events that culminates in the death of the mouse hepatocytes during arsenic exposure.