Hepatotoxicity of Pyrrolizidine Alkaloid Compound Intermedine: Comparison with Other Pyrrolizidine Alkaloids and Its Toxicological Mechanism.

Chen Wang,Haolei Han,Ziqi Wang,Xiangchun Zhang,Hongping Chen,Qinqin Zheng,Ruyan Hou

doi:10.3390/toxins13120849

Abstract

Pyrrolizidine alkaloids (PAs) are common secondary plant compounds with hepatotoxicity. The consumption of herbal medicines and herbal teas containing PAs is one of the main causes of hepatic sinusoidal obstruction syndrome (HSOS), a potentially life-threatening condition. The present study aimed to reveal the mechanism underlying the cytotoxicity of intermedine (Im), the main PA in Comfrey. We evaluated the toxicity of the retronecine-type PAs with different structures to cell lines derived from mammalian tissues, including primary mouse hepatocytes, human hepatocytes (HepD), mouse hepatoma-22 (H22) and human hepatocellular carcinoma (HepG2) cells. The cytotoxicity of Im to hepatocyte was evaluated by using cell counting kit-8 assay, colony formation experiment, wound healing assay and dead/live fluorescence imaging. In vitro characterization showed that these PAs were cytotoxic and induced cell apoptosis in a dose-dependent manner. We also demonstrated that Im induced cell apoptosis by generating excessive reactive oxygen species (ROS), changing the mitochondrial membrane potential and releasing cytochrome c (Cyt c) before activating the caspase-3 pathway. Importantly, we directly observed the destruction of the cell mitochondrial structure after Im treatment through transmission electron microscopy (TEM). This study provided the first direct evidence of Im inducing hepatotoxicity through mitochondria-mediated apoptosis. These results supplemented the basic toxicity data of PAs and facilitated the comprehensive and systematic evaluation of the toxicity caused by PA compounds.

Highlights

Herbal medicines and plant-related products are indispensable for human life in many countries
The cytotoxicity of intermedine (Im), intermedine N-oxide (ImNO), lycopsamine (La), lycopsamine N-oxide (LaNO), retrorsine (Re), retrorsine N-oxide (ReNO), senecionine (Sc) and senecionine N-oxide (ScNO) on primary mouse hepatocytes, HepD cells, H22 cells and HepG2 cells was firstly investigated by the cell counting kit-8 (CCK-8) assay
After cells were exposed to different concentrations (0, 20, 50, 75 and 100 μg/mL = 0, 67, 167, 250 and 334 μM) of Im, ImNO, La, LaNO, Re, ReNO, Sc and ScNO for 24 h, the cell survival was recorded by CCK-8 and a microplate reader

Summary

Introduction

Herbal medicines and plant-related products are indispensable for human life in many countries. Hepatic sinusoidal obstruction syndrome (HSOS) has been induced after ingestion of herbals and plant-related products in many countries and has attracted global attention [1,2]. HSOS is clinically characterized by hepatic congestion and swelling, blockage of liver blood vessels, detachment of sinusoidal endothelial cells and hepatic dysfunction [2,3]. Through an in-depth study of these HSOS patients, it was found that most of them were exposed to pyrrolizidine alkaloids (PAs) through the consumption of herbals and herbal teas [2,4]. Despite the great advances in medical technology in the past few decades, there is still no effective clinical therapy for PA-induced HSOS cases. Extensive and in-depth research on PA-induced hepatotoxicity and the development of effective treatments is urgently needed

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Discussion

Conclusion