The role of tumor necrosis factor alpha in lipopolysaccharide/ranitidine-induced inflammatory liver injury.

Exposure to a nontoxic dose of bacterial lipopolysaccharide (LPS) increases the hepatotoxicity of the histamine-2 (H2) receptor antagonist, ranitidine (RAN). Because some of the pathophysiologic effects associated with LPS are mediated through the expression and release of inflammatory mediators such as tumor necrosis factor alpha (TNF), this study was designed to gain insights into the role of TNF in LPS/RAN hepatotoxicity. To determine whether RAN affects LPS-induced TNF release at a time near the onset of liver injury, male Sprague-Dawley rats were treated with 2.5 x 10(6) endotoxin units (EU)/kg LPS or its saline vehicle (iv) and 2 h later with either 30 mg/kg RAN or sterile phosphate-buffered saline vehicle (iv). LPS administration caused an increase in circulating TNF concentration. RAN cotreatment enhanced the LPS-induced TNF increase before the onset of hepatocellular injury, an effect that was not produced by famotidine, a H2-receptor antagonist without idiosyncrasy liability. Similar effects were observed for serum interleukin (IL)-1beta, IL-6, and IL-10. To determine if TNF plays a causal role in LPS/RAN-induced hepatotoxicity, rats were given either pentoxifylline (PTX; 100 mg/kg, iv) to inhibit the synthesis of TNF or etanercept (Etan; 8 mg/kg, sc) to impede the ability of TNF to reach cellular receptors, and then they were treated with LPS and RAN. Hepatocellular injury, the release of inflammatory mediators, hepatic neutrophil (PMN) accumulation, and biomarkers of coagulation and fibrinolysis were assessed. Pretreatment with either PTX or Etan resulted in the attenuation of liver injury and diminished circulating concentrations of TNF, IL-1beta, IL-6, macrophage inflammatory protein-2, and coagulation/fibrinolysis biomarkers in LPS/RAN-cotreated animals. Neither PTX nor Etan pretreatments altered hepatic PMN accumulation. These results suggest that TNF contributes to LPS/RAN-induced liver injury by enhancing inflammatory cytokine production and hemostasis.

Modeling inflammation-drug interactions in vitro: a rat Kupffer cell-hepatocyte coculture system.

Xenobiotic-inflammation interactions lead to hepatotoxicity in vivo. Selected xenobiotic agents (acetaminophen, APAP; chlorpromazine, CPZ; allyl alcohol, AlOH; monocrotaline, MCT) for which this occurs were evaluated for ability to elicit the release of Kupffer cell (KC)-derived inflammatory mediators and to modulate lipopolysaccharide (LPS)-stimulated release of these mediators. Using KCs and hepatocytes (HPCs) isolated from rat, KC/HPC cocultures were treated with either LPS, xenobiotic, vehicle or a combination. Six hours later, the release of inflammatory mediators was assessed. LPS alone caused a concentration-dependent increase in TNF-alpha release but had no significant effect on the release of PGE(2). APAP by itself did not alter release of TNF-alpha, PGE(2), IL-10, Gro/KC or IFN-gamma; however, in the presence of LPS, APAP enhanced LPS-induced TNF-alpha and Gro/KC release. APAP also attenuated LPS-induced increases in IL-10 and MCP-1. CPZ alone caused a concentration-dependent increase in TNF-alpha release, which was approximately additive in the presence of LPS. AlOH alone did not affect TNF-alpha release, but decreased TNF-alpha production in the presence of LPS. AlOH increased PGE(2) production, and this effect was potentiated in the presence of LPS. MCT by itself did not affect release of TNF-alpha but increased the response to LPS. Neither MCT, LPS, nor the combination affected production of PGE(2). These results demonstrate that KC/HPC cocultures can be used to evaluate interactions of xenobiotics with LPS. Furthermore, data from these studies qualitatively mirror reported data from whole animal studies, suggesting that this model could be useful for predicting aspects of xenobiotic-inflammation interactions in vivo.

Characterization of the role of glutathione in repin-induced mitochondrial dysfunction, oxidative stress and dopaminergic neurotoxicity in rat pheochromocytoma (PC12) cells.

Repin, a major constituent in extracts of the plant Centaurea repens is thought to be the active principal responsible for the development of equine nigropallidal encephalomalacia (ENE), a fatal Parkinson-like neurodegenerative disorder in horses. Although the exact mechanism by which ingestion of this weed causes ENE is uncertain, a limited body of experimental evidence suggests a critical role for the glutathione redox system. In the present study, the mechanism of repin neurotoxicity was examined in PC12 cells with a focus on determining the role of glutathione (GSH) in repin-induced mitochondrial dysfunction, oxidative stress and dopaminergic toxicity. The results demonstrate that repin reduced both cellular GSH levels and mitochondrial function in a manner that was time- and concentration-dependent. The repin-induced changes in GSH levels were found to precede the changes in mitochondrial function. Depletion of GSH with a potent GSH depletor (ethacrynic acid (EA)) and a GSH synthesis inhibitor (buthionine sulfoximine (BSO)) prior to repin treatment enhanced the repin-induced mitochondrial change. In addition, repin caused a concentration-dependent decrease in cellular dopamine levels in NGF-differentiated PC12 cells. Increases in intracellular GSH levels induced by pre-treatment with reducing agents (N-acetyl-L-cysteine or reduced glutathione) completely protected the cells from repin-induced mitochondrial and dopaminergic toxicity. Antioxidants, coenzyme-Q and ascorbic acid completely blocked repin-induced dopaminergic toxicity. These data suggest that GSH plays a critical role in repin-induced neurotoxicity and that the maintenance of neuronal redox status may prove to be a useful strategy for the prevention and/or treatment of ENE. The results support the view that GSH depletion, leading to oxidative damage and subsequent mitochondrial dysfunction, may serve as a trigger for neuronal cell death.

Inactivation of the cytotoxic activity of repin, a sesquiterpene lactone from Centaurea repens.

Prolonged ingestion of Yellow Starthistle (Centaurea solstitialis) and Russian Knapweed (Centaurea repens) by horses has been shown to result in a fatal neurodegenerative disorder called equine nigropallidal encephalomalacia (ENE). Bioassay-guided fractionation of extracts from Centaurea species using the PC12 cell line have led to the identification of one of several putative agents, which may contribute to ENE, namely, the sesquiterpene lactone (SQL) repin (1), previously linked to ENE due to its abundance in C. repens. To characterize the molecular basis of repin-induced neurotoxicity, the present study was designed to identify reactive functional groups that may contribute overall to its toxicity. The reaction of repin (1) with glutathione (GSH) led to the exclusive addition of GSH to the alpha-methylenebutyrolactone affording a GSH conjugate (3b) that lacked toxicity in the PC12 cell assay, while selective reduction of the alpha-methylenebutyrolactone double bond of 1 also resulted in an analogue (2) that was devoid of toxicity relative to the parent compound. Unlike repin, analogue 2 failed to decrease cellular dopamine levels in PC12 cells, further substantiating the requirement of the alpha-methylenebutyrolactone group. Results from this study are suggestive that GSH depletion by the SQL repin may be a primary event in the etiology of ENE, increasing the susceptibility to oxidative damage.