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Published on: June 5, 2020
Senecio latifolius induces in vitro hepatocytotoxicity in a human cell line
Manuela G Neuman1, Angela Y Jia, Vanessa Steenkamp
1In Vitro Drug Safety and BioTechnology, Department of Pharmacology, Institute of Drug Research, University of Toronto, South Tower of the MaRS Discovery Centre, 101 College Street, Toronto, ON M5G 1L5, Canada. manuela.neuman@utoronto.ca
Abstract:
The objectives of this study were twofold: (i) to determine the mechanism(s) of Senecio-induced toxicity in human hepatoblastoma cells (HepG2) in vitro and whether such toxicity could be prevented using N-acetyl-cysteine (NAC), and (ii) to evaluate whether caspases are involved in Senecio-induced apoptosis. Cells were treated with aqueous extracts of Senecio (10 mg x mL-1) with and without NAC. Cytotoxicity was determined by using the MTT assay. Total glutathione (GSH) was measured by using the Tietze assay. Cells were also treated with aqueous extracts of Senecio in the presence or absence of 50 micromol/L caspase-3 inhibitor (IDN) for 24 h. Apoptosis was determined by transmission electron microscopy, and DNA fragmentation was determined by ELISA and terminal dUTP nick-end labelling (TUNEL). Senecio produced cytotoxicity and depleted GSH in a concentration- and time-dependent manner. A significant depletion in GSH was observed after 15 min (p < 0.001 vs. control), whereas significant cytotoxicity was only observed after 3 h (p < 0.001 vs. control). Treatment with NAC prevented Senecio-induced GSH depletion and resulted in a significant decrease in Senecio-induced cytotoxicity (p < 0.001 vs. NAC-untreated cells). Treatment with Senecio for 24 h resulted in 22% +/- 2.5% (p < 0.001) apoptosis (vs. control). Pretreatment with 50 mumol caspase inhibitor reduced Senecio-induced apoptosis significantly (vs. non-exposed to IDN) (12% +/- 1.5%; p < 0.05). Our results suggest the mechanism of Senecio-induced cytotoxicity in HepG2 cells in vitro involves depletion of cellular GSH. Cytotoxicity is reduced by supplementation with NAC, which thus prevents GSH depletion. Caspase activation is involved in Senecio-induced apoptosis.
Insights
Senecio plant extracts cause liver cell toxicity by depleting glutathione (GSH). N-acetyl-cysteine (NAC) prevents this GSH depletion and reduces toxicity, while caspase inhibitors lessen Senecio-induced apoptosis.
Area of Science:
- Hepatotoxicity and cellular mechanisms
- Toxicology and pharmacology
Background:
- Senecio species are implicated in liver toxicity.
- The exact mechanisms of Senecio-induced cytotoxicity and apoptosis remain unclear.
- The potential protective role of N-acetyl-cysteine (NAC) against Senecio toxicity warrants investigation.
Purpose of the Study:
- To elucidate the mechanism of Senecio-induced toxicity in human hepatoblastoma (HepG2) cells in vitro.
- To assess the protective effect of N-acetyl-cysteine (NAC) against Senecio toxicity.
- To determine the involvement of caspases in Senecio-induced apoptosis.
Main Methods:
- HepG2 cells were treated with Senecio extracts with and without NAC.
- Cytotoxicity assessed via MTT assay; glutathione (GSH) levels measured using Tietze assay.
- Apoptosis evaluated using transmission electron microscopy, DNA fragmentation assays (ELISA, TUNEL), and caspase-3 inhibitor treatment.
Main Results:
- Senecio induced concentration- and time-dependent cytotoxicity and GSH depletion.
- NAC treatment prevented GSH depletion and significantly reduced Senecio-induced cytotoxicity.
- Senecio treatment led to significant apoptosis, which was reduced by a caspase-3 inhibitor.
Conclusions:
- Senecio-induced cytotoxicity in HepG2 cells is mediated by cellular GSH depletion.
- NAC effectively prevents Senecio-induced GSH depletion and subsequent cytotoxicity.
- Caspase activation plays a role in the apoptotic pathway triggered by Senecio exposure.
