Hepatoprotective mechanism of silymarin: no evidence for involvement of cytochrome P450 2E1.

The involvement of the alcohol-inducible cytochrome P450 2E1 in the hepatoprotective mechanism of the plant flavonoid extract silymarin, and its main active component silybin, was investigated in isolated hepatocytes. Allyl alcohol toxicity, associated lipid peroxidation and GSH depletion was efficiently counteracted by silymarin (0.01-0.5 mM), and at higher concentrations by silybin. Cell damage by t-butyl hydroperoxide was also prevented by silymarin and silybin, but less efficiently. However, the covalent binding of the acetaminophen intermediate, formed via P450 2E1, was unaffected by addition of the flavonoids. Silybin did not influence microsomal 2E1-catalyzed demethylation of N-nitrosodimethylamine. Neither did demethylation of N-nitrosodimethylamine or aminopyrine by isolated microsomes affect the in vivo administration of silybin. Addition of silymarin or silybin to primary cultures of isolated hepatocytes did not prevent cell damage induced by exposure to the P450 2E1 substrate CCl4. In contrast, the mere presence of low concentrations (25-50 microM) of these compounds was found to inhibit cell attachment to the matrix and eventually resulted in cell damage. We conclude that contrary to earlier reports we found no evidence for an interaction of silymarin or silybin with cytochrome P450 2E1. This suggests that the antioxidant and free radical scavenging properties may account for most of the therapeutic effect of these compounds. The untoward effect of silymarin on cultured cells may have consequences when considering long-term prescription of this therapeutic agent.

The effects of silymarin on the attachment and viability of primary cultures of rat hepatocytes.

The effects of the hepatoprotective compound silymarin on hepatocytes in primary culture were studied. Exposure of cells in primary culture (both conventional cells and perivenous or periportal cells isolated by the digitonin-collagenase perfusion technique) to high concentrations of silymarin surprisingly demonstrated that silymarin per se was cytotoxic. Incubation of cells for 18 hr with silymarin at concentrations exceeding 25 mum abruptly increased cell damage, whereas viability decreased in a more linear fashion with increasing concentrations of its major constituent, silybin. Morphologically, cell cultures exposed to silymarin concentrations lower than 20 mum appeared normal, but at higher concentrations intercellular contacts were lost; cells appeared granulated and took up eosin. Silymarin and silybin at these doses were also found to prevent cell attachment. The mechanism responsible for this effect at relatively low concentrations of silymarin during prolonged exposure in the primary cell culture system is not clear at present. The effects of low doses on cell attachment to the matrix suggest an action on the cell membrane and/or on the cytoskeleton.

Comparative study of the hepatoprotective effect of silymarin and silybin on isolated rat hepatocytes.

The flavonoid silymarin and its main active component silybin have been used in the treatment of toxic liver diseases. In order to evaluate the hepatoprotective potency of both these compounds, their effects on the viability, lipid peroxidation and reduced glutathione (GSH) depletion induced by allyl alcohol (AA) and tert-butyl hydroperoxide (t-BuOOH) in suspensions of isolated hepatocytes were investigated. Cells were preincubated for 30 min with silymarin and silybin before the addition of AA or t-BuOOH. Samples were taken after 1-2 hr of incubation. Cell death, after 2 hr of incubation with 0.2 mm AA, was prevented by 0.01 mm silymarin; however, 2 mm silybin was required to give comparable protection. The presence of silymarin reduced AA-induced lipid peroxidation by more than 90%, whereas silybin was much less effective. The near-complete depletion of intracellular GSH by AA was restored in a dose-dependent manner by silymarin, but silybin did not have this effect. Protection by silymarin against the toxic effects of t-BuOOH was less pronounced than that against AA. In conclusion, silymarin was much more effective than silybin in preventing the toxic effects induced by two pro-oxidant toxins, AA and t-BuOOH.