Clivorine

Hum Exp Toxicol. 2010 Apr;29(4):303-9.

Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes.[Pubmed: 20144959]

Clivorine is an otonecine-type hepatotoxic pyrrolizidine alkaloid (HPAs), to which humans are exposed when consuming herbs containing such components. In the present study, we investigated Clivorine-induced oxidative stress injury on primary cultured rat hepatocytes.
METHODS AND RESULTS:
Rat hepatocytes were treated with various concentrations of Clivorine (1-100 microM) for 48 hours, and then cell viability was detected by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, while lipid peroxidation (LPO) level, glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), catalase (CAT) and superoxide dismutase (SOD) activities were determined to evaluate the oxidative injury. The results of MTT assay showed that Clivorine decreased cell viability in a concentration-dependent manner. Clivorine also increased LPO amounts in rat hepatocytes at the concentrations of 50 microM and 100 microM. Further results showed that Clivorine decreased GPx, GST and GR activities, which are all reduced glutathione (GSH)-related antioxidant enzymes. CAT and SOD are both important antioxidant enzymes, and the results showed that Clivorine increased CAT activity at the low concentration of 5 muM and decreased cellular SOD activity at all concentrations.
CONCLUSIONS:
Taken together, our results demonstrated that Clivorine induced toxicity on primary cultured rat hepatocytes by causing the damage on cellular redox balance.

Exp Toxicol Pathol. 2008 Jun;60(1):87-93.

The toxic effect of pyrrolizidine alkaloid clivorine on the human embryonic kidney 293 cells and its primary mechanism.[Pubmed: 18249102]

Clivorine is an otonecine-type pyrrolizidine alkaloid (PA) isolated from the Chinese medicinal plant Ligularia hodgsonii Hook., and our previous reports have shown its toxicity on human normal liver L-02 cells. It is generally believed that biotransformation of PAs to its metabolites is required for their toxicity; thus, there is nearly no report about the toxicity of Clivorine on other non-hepatic cells in vitro.
METHODS AND RESULTS:
The aim of this study is to observe the toxicity of Clivorine on the non-hepatic human embryonic kidney 293 (HEK293) cell that is of epithelial origin, and its primary mechanism. Our results showed that Clivorine significantly reduced HEK293 cell viability, but there was no detectable apoptotic DNA ladder and cleaved fragments of caspase-3 and caspase-9 in Clivorine-treated cells, which indicates the toxicity of Clivorine is not due to inducing apoptosis. The results of western blot showed that Clivorine induced sustained p38, c-Jun N-terminal kinase (JNK) and extracellular signal-related kinases (ERK1/2) phosphorylation in a concentration- and time-dependent manner, and the JNK inhibitor SP600125 significantly augmented the toxicity of Clivorine.
CONCLUSIONS:
Our results suggest that Clivorine itself has direct toxicity on HEK293 cells, and phosphorylated JNK may play some role in counteracting the toxicity of Clivorine on HEK293 cells.

PLoS One. 2014 Jun 6;9(6):e98970.

Quercetin prevents pyrrolizidine alkaloid clivorine-induced liver injury in mice by elevating body defense capacity.[Pubmed: 24905073 ]

Quercetin is a plant-derived flavonoid that is widely distributed in nature. The present study is designed to analyze the underlying mechanism in the protection of quercetin against pyrrolizidine alkaloid Clivorine-induced acute liver injury in vivo.
METHODS AND RESULTS:
Serum transaminases, total bilirubin analysis, and liver histological evaluation demonstrated the protection of quercetin against Clivorine-induced liver injury. Terminal dUTP nick end-labeling assay demonstrated that quercetin reduced the increased amount of liver apoptotic cells induced by Clivorine. Western-blot analysis of caspase-3 showed that quercetin inhibited the cleaved activation of caspase-3 induced by Clivorine. Results also showed that quercetin reduced the increase in liver glutathione and lipid peroxidative product malondialdehyde induced by Clivorine. Quercetin reduced the enhanced liver immunohistochemical staining for 4-hydroxynonenal induced by Clivorine. Results of the Mouse Stress and Toxicity PathwayFinder RT2 Profiler PCR Array demonstrated that the expression of genes related with oxidative or metabolic stress and heat shock was obviously altered after quercetin treatment. Some of the alterations were confirmed by real-time PCR.
CONCLUSIONS:
Our results demonstrated that quercetin prevents Clivorine-induced acute liver injury in vivo by inhibiting apoptotic cell death and ameliorating oxidative stress injury. This protection may be caused by the elevation of the body defense capacity induced by quercetin.

Environ Toxicol. 2010 Jun;25(3):304-9.

Protection of epidermal growth factor against clivorine-induced mitochondrial-mediated apoptosis in hepatocytes.[Pubmed: 19437449]

Pyrrolizidine alkaloids (PAs) are well-known natural hepatotoxins. In this study, we investigated the protection of epidermal growth factor (EGF) against the hepatotoxicity of Clivorine, which is an otonecine-type PA from traditional Chinese medicine Ligularia hodgsonii Hook.
METHODS AND RESULTS:
Cell viability assay and cell morphology observation showed that EGF (1 ng/mL) reversed Clivorine-induced cytotoxicity on human normal liver L-02 cells. EGF (1 ng/mL) also inhibited Clivorine-induced DNA fragmentation and caspase-3 cleavage. Our previous study has showed that antiapoptotic Bcl-xL degradation and mitochondrial-mediated apoptosis was involved in Clivorine-induced hepatotoxicity. In this study, we found that EGF (1 ng/mL) inhibited Clivorine-induced antiapoptotic Bcl-xL protein decrease, caspase-9 activation, and release of cytosolic cytochrome C. We further investigated the effects of vascular epidermal growth factor, basic fibroblast growth factor, and insulin-like growth factor-1 on Clivorine-induced cytotoxicity, and there is no significant protection observed.
CONCLUSIONS:
Our results suggest that EGF exerts its protective effects against Clivorine-induced hepatotoxicity probably by modulating mitochondrial-mediated apoptotic signal pathway.

Drug Discov Ther. 2009 Dec;3(6):247-51.

Pyrrolizidine alkaloid clivorine-induced oxidative stress injury in human normal liver L-02 cells.[Pubmed: 22495657]

Clivorine is an otonecine-type pyrrolizidine alkaloid isolated from the traditional Chinese medicine Ligularia hodgsonii Hook. Pyrrolizidine alkaloids (PAs) are well-known hepatotoxins widely distributed around the world. The present study sought to evaluate Clivorineinduced oxidative injury in human normal liver L-02 cells.
METHODS AND RESULTS:
After cells were treated with various concentrations of Clivorine for 48 h, cellular total antioxidant capacity, glutathione-S-transferase (GST) and glutathione reductase (GR) were determined to evaluate oxidative injury. Results showed that cellular total antioxidant capacity and GST activity both increased in Clivorine-treated L-02 cells, while Clivorine decreased GR activity in cells. Further, the protective effects of some antioxidants such as ascorbic acid (vitamin C, Vc), Trolox, dithiothreitol (DTT) and mannitol against Clivorine-induced cytotoxicity were observed. Results showed that Trolox, which is an analogue of tocopherol (vitamin E, Ve), prevented Clivorine-induced cytotoxicity in L-02 cells.
CONCLUSIONS:
Taken together, these results revealed Clivorineinduced oxidative injury in human liver L-02 cells. These results also indicated the possible use of Trolox in the reduction of Clivorine-induced hepatotoxicity.