Silymarin mediated differential modulation of toxicity induced by carbon tetrachloride, paracetamol and D-galactosamine in freshly isolated rat hepatocytes.

Influence of silymarin on the modulation of hepatotoxicity induced by carbon tetrachloride (CCl4), paracetamol (AAP) and D-galactosamine (GalN) was examined in freshly isolated rat hepatocytes in suspension culture. While the three hepatotoxicants produced differential biochemical response, the flavone was able to restore biochemical alterations only in hepatocytes exposed to CCl4 and AAP induced toxicity. Silymarin at 0.4 mM was able to counteract lipid peroxidation and enzyme leakage induced by 3 mM CCl4 The flavone also offered protection by more than 60% in hepatocytes isolated from PB pre-treated rats where CCl4 at 2 mM produced enhanced toxicity over hepatocytes isolated from untreated control rats. Similarly, the flavone protected AAP-induced GSH depletion by more than 75% in hepatocytes isolated from untreated and 3-methylcholanthrene treated rats. However, instead of protecting GalN-induced depletion of UDP-glucuronic acid in hepatocytes, the flavone itself reduced the nucleotide content very rapidly compared to GalN, the later exerted time dependent effect. Silymarin at 0.4 mM reduced UDPGA by more than 60%. The results suggested that freshly isolated hepatocytes in suspension culture offer a simple and convenient method for evaluation of pharmaceutical agents of antihepatotxic potentials against various hepatotoxicants.

Differential biochemical response of freshly isolated rat hepatocytes to paracetamol, carbon tetrachloride and D-galactosamine toxicity.

Differential response of freshly isolated rat hepatocytes to paracetamol (acetaminophen, AAP), carbon tetrachloride (CCl4) and D-galactosamine (GalN) was examined. The viability of the cells in suspension culture was not altered for 4 hr when incubated in Hank's balanced salt solution (HBSS) supplemented with 4.2 mM NaHCO3, 10 mM HEPES buffer and 0.5% bovine serum albumin. AAP induced time and dose dependent depletion of GSH as an early manifestation of AAP toxicity. Hepatocytes exposed to AAP exhibited lower lactate dehydrogenase (LDH) activity released into the medium than in controls. This was due to the interaction of a reactive metabolite of AAP, i. e. N-acetyl p-benzoquinoneimine (NAPQI) with cell proteins. Hepatocytes isolated from rats pretreated with 3-methylcholanthrene expressed higher sensitivity to AAP toxicity at least by a factor of 5. Furthermore, AAP-induced toxicity was not found to be related to any lipoperoxidative stress. CCl4 on the other hand elicited a highly lipoperoxidative response in hepatocytes and consequent leakage of cellular enzymes with lengths of incubation. Again the sensitivity of the response to CCl4 was enhanced remarkably in hepatocytes isolated from phenobarbital- pretreated rats; LDH leakage increased by 3-fold and thio-barbituric acid reactive substances by 25-fold. Unlike the two toxicants, galactosamine depleted UDP-glucuronic acid in a concentration and time-related manners. The differential biochemical response of hepatocytes to three hepatotoxicants investigated may prove useful as rapid in vitro screen for selection of compounds of hepatoprotective potentials.

Effects of silymarin on UDP-glucuronic acid and glucuronidation activity in the rat isolated hepatocytes and liver in relation to D-galactosamine toxicity.

Influence of silymarin on UDP-glucuronic acid (UDPGA) and glucuronidation activity of freshly isolated rat hepatocytes in suspension and in rat liver in vivo was examined. Viability of the hepatocytes (> 85%) was not altered in Hank's balanced salt solution at least for 4 hr at 37 degrees C under oxygen. Silymarin at 0.4 mM depleted UDPGA by more than 60% at the end of 4 hr of incubation, the fall in nucleotide pool was rapid and concentration (0.1-0.4 mM)-dependent. The rate of glucuronidation of 3-OH- benzo(a)pyrene (3-OH-BP) determined simultaneously was also reduced significantly; silybin being 3-times more effective than silymarin. Combination of flavonoids with D-galactosamine (GalN) further attenuated the glucuronidation functions of the cells. The flavonoids also offered strong inhibition of UDP-glucose dehydrogenase (UDP-GDH) activity in the liver cytosolic fraction while the activity in hepatocytes was not affected even after 4 hr of incubation. Interestingly, the GalN- induced strong inhibition of UDP-GDH in isolated hepatocytes was completely abolished by flavonoids. Decrease in UDPGA appeared neither due to the activation of UDPGA-pyrophosphatase activity nor to the inhibition of UDP-GDH activity in hepatocytes. Further, the flavonoids also inhibited hepatic UDP-glucuronyltransferase activity towards 3-OH-BP (UGT) both in vitro and in intact cells. On the contrary, silymarin administered (70 mg/kg body wt; i.p.) to rats for 3 hr increased the hepatic UDPGA by 2-fold while GalN (400 mg/kg body wt) reduced the nucleotide content to 50% of control. Coadministration of silymarin and GalN restored the UDPGA content significantly while the activities of UDP-GDH and UGT were comparable to the untreated control. The results indicated that silymarin elicits differential effects on the rate of glucuronidation and contents of UDPGA in the isolated rat hepatocytes and in liver. The flavonoid counteracted D-GalN-induced lowering of UDPGA presumably by relieving UDP-GDH of in vivo inhibition affected by GalN-metabolite.