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

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.

Mitochondrial Oxidative Stress Is the General Reason for Apoptosis Induced by Different-Valence Heavy Metals in Cells and Mitochondria

“…However, As(III) (As 2 O 3 and AsO 2 − ) brought about oxidative stress. The stress manifested in MPTP opening, mitochondrial swelling, ∆Ψ mito decline, a decrease in ATP, reduced glutathione, an ROS production increase, and cytochrome c release in investigations with PC12 cells, Hela cells, isolated rat hepatocytes, rat cardiomyocyte H9c2 cells, L02 hepatocytes, lung bronchial epithelial HBE cells, Hela cells, mouse insulinoma MIN6 cells, laryngeal squamous carcinoma Hep-2 cells, Chang human hepatocytes, mouse embryonic fibroblasts, mouse hepatocytes, human hepatocellular carcinoma HepG2 cells, colon cancer HT-29 cells, cervical cancer cells, primary cultured mouse hepatocytes, and human myeloid leukemia U937 cells [161,162,271,279,[281][282][283][284][285][287][288][289][290][291][292][293][294][295][296]. In addition, AsO 2 − in L02 hepatocytes induced ERK signaling to activate both apoptosis and mitophagy [282] along with ferroptosis in mouse insulinoma MIN6 cells or PC-12 cells [283,297].…”

TFEB and TFE3 cooperate in regulating inorganic arsenic-induced autophagy-lysosome impairment and immuno-dysfunction in primary dendritic cells

“…However, As is also found to inhibit the autophagosome-lysosome fusion in NIH 3T3 cells by co-tranfected with the monomeric red fluorescent protein (mRFP)-GFP-LC3 fluorescent tandem reporter and LAMP1-CFP constructs (Dodson et al 2018 ). Furthermore, as to the impaired lysosome-dependent degradation process, As-induced low levels of ROS could cause LMP and subsequent release of CTSB to the hepatic cytoplasm (Santra et al 2022 ). Wang et al have observed that trivalent arsenic exposure impairs lysosomal membrane stability in earthworms (Wang et al 2016 ).…”