Silymarin induces recovery of pancreatic function after alloxan damage in rats.

Alloxan has been widely used to produce experimental diabetes mellitus syndrome. This compound causes necrosis of pancreatic beta-cells and, as is well known, induces oxidant free radicals which play a relevant role in the etiology and pathogenesis of both experimental and human diabetes mellitus. Previously we have reported hypoglycemic and antilipoperoxidative actions of silymarin in serum and pancreatic tissue respectively. The aim of this study was to test whether silymarin could reduce the hyperglycemia and revert the pancreatic damage in alloxan treated rats, tested with silymarin in two protocols: using both compounds simultaneously for four or eight doses, or using the compound 20 days after alloxan administration for 9 weeks. Serum glucose and insulin were determined, and pancreatic fragments were used for histology and insulin immunohistochemistry. Pancreatic islets were isolated to assess insulin and Pdx1 mRNA expression by RT-PCR. Our results showed that 72 hours after alloxan administration, serum glucose increased and serum insulin decreased significantly, whereas pancreatic tissue presented morphological abnormalities such as islet shrinkage, necrotic areas, loss of cell organization, widespread lipoid deposits throughout the exocrine tissue, and loss of beta cells, but insulin and glucagon immunoreactivity was scattered if any. In contrast the pancreatic tissue and both insulin and glucose serum levels of rats treated with silymarin were similar to those of control animals. In addition, insulin and glucagon immunoreactive cells patterns in Langerhans islets were also normal, and normal insulin and Pdx1 mRNA expression patterns were detected during pancreatic recovery in Langerhans islets. The overall results suggest that silymarin induces pancreatic function recovery demonstrated by insulin and glucagon expression protein and normoglycemia after alloxan pancreatic damage in rats.

Prevention of alloxan-induced diabetes mellitus in the rat by silymarin.

Silymarin is a free-radical scavenger and a membrane stabilizer which prevents lipoperoxidation and its associated cell damage in some experimental models. It has been proposed that lipid peroxidation caused by free radicals may be involved in alloxan-induced diabetes mellitus. Alloxan elicits pancreatic lipid peroxidation which precedes the appearance of hyperglycemia in mice. We studied the effects of silymarin on rat pancreas, the effect of this flavonoid on pancreatic, hepatic and blood glutathione (GSH) together with the pancreatic malondialdehyde concentrations in response to alloxan. On its own, silymarin increases pancreatic and blood GSH without changes in either hepatic GSH or in blood glucose. Silymarin prevents the increase in lipid peroxidation produced by alloxan. It also blunts the sustained increment in plasma glucose induced by alloxan. We suggest that silymarin has a protective effect on the pancreatic damage in experimental diabetes mellitus. This may be related to its antioxidative properties and to the increase in concentrations of plasma and pancreatic glutathione.

Comparative effects of colchicine and silymarin on CCl4-chronic liver damage in rats.

The comparative effects of colchicine (10 micrograms day-1, p.o.) and silymarin (50 mg kg-1, p.o.) each given for 5 days a week on the chronic carbon tetrachloride (CCl4) liver damage were studied. Treatment with CCl4 resulted in a marked reduction of Na+, K+, and Ca2(+)-ATPases in plasma liver membranes as compared to vehicles or either silymarin or colchicine alone. Collagen content in livers of animals treated with CCl4 was increased about four-fold as compared to controls and histological examination of liver samples showed that collagen increase distorted the normal liver architecture. Colchicine or silymarin treatment completely prevented all the changes observed in CCl4-cirrhotic rats (namely, lipid peroxidation, Na+, K+ and Ca(2+)-ATPases), except for liver collagen content which was reduced only 55% as compared with CCl4-treated rats and for alkaline phosphatase and glutamic pyruvic transaminase which still remained above controls. In the CCl4 + silymarin group, the loss of glycogen content was completely prevented. However, when rats were treated with CCl4 + colchicine, liver glycogen content could not be restored. The hepatoprotective effects of colchicine or silymarin were very similar in regard to the prevention of chronic liver damage.

Effect of portal vein ligation and silymarin treatment on aspirin metabolism and disposition in rats.

The influence of portal hypertension on the metabolism and pharmacokinetics of aspirin was evaluated after the administration of a single oral dose of acetylsalicylic acid (20 mgkg(-1)) in portal-vein-ligated (PVL) rats. Experiments were also performed in control (sham-operated rats) and in rats that received an oral daily dose (150 mgkg(-1)) of silymarin from the tenth day after surgery for 7 d. Plasma concentration profiles of all groups exhibited monoexponential decay but with important changes in pharmacokinetic parameters. The aspirin elimination constant (k) for PVL rats was lower than for control rats, whereas the plasma half-life and area under the curve were greater than those in the control group. However, Cmax was comparable with that of the control rats. Urinary excretion of the metabolites (salicylic acid and glucuronides) was significantly altered in PVL rats: the urinary glucuronides were reduced and urinary salicylic acid was increased. The activities of plasma and liver esterases were increased significantly in PVL rats, while the activity of p-nitroanisole-O-demethylase was not affected. Depletion of cytochrome P 450 was also noted in the same group of rats. Silymarin markedly reversed the alterations found in the PVL group.

Relationship between naproxen plasma concentration and its anti-inflammatory effect in experimental hepatitis.

The relationship between the plasma concentration and the anti-inflammatory effect of naproxen (CAS 22204-53-1) after oral administration of a 6 mg.kg-1 dose was studied in rats with galactosamine-induced acute hepatitis and under control conditions. In control animals naproxen peak plasma levels of 35 +/- 0.4 micrograms.ml-1 were reached in 0.5 +/- 0 h. Concentration then decayed, half-life being 5.2 +/- 0.4 h. AUC was 131 +/- 5 micrograms.h.ml-1. In intoxicated rats peak plasma levels of 29 +/- 0.3 micrograms.ml-1 were reached in 0.7 +/- 0.1 h, half-life was increased to 11.1 +/- 1.3 h, and the AUC reached 259 +/- 21 micrograms.h.ml-1. In control rats the protective effect of naproxen against carrageenan-induced inflammation increased slowly, reaching a maximum of 38% in 4 h. The protective effect against plasma concentration curve exhibited a clear counterclockwise hysteresis, probably due to a slow naproxen transport from the circulation to its site of action. In animals with hepatitis, the protective effect remained quite constant at about 40% despite variations in plasma levels, probably because the maximal effect was reached. No clear hysteresis was observed in the effect-plasma concentration curve, suggesting that naproxen arrival to its site of action was faster. Results show that the relationship between naproxen plasma concentration and its anti-inflammatory effect is complex and therefore predictions on the pharmacological response in liver damage cannot be readily made by solely considering pharmacokinetic data.

Effects of leukotriene synthesis inhibition on acute liver damage induced by carbon tetrachloride.

Administration of the leukotriene synthesis inhibitors nordihydroguaiaretic acid (NDHGA, 30 mg/kg), caffeic acid (20 mg/kg) or nafazatrom (100 mg/kg) and of the phosphodiesterase inhibitor and free radical trapper dipyridamole (10 mg/kg) prevented the alterations in enzyme activity displayed by acute CCl4 administration. The effect was less evident in preventing hepatic glycogen depletion or lipid peroxidation. A synergistic protective effect between dipyridamole and NDHGA or caffeic acid was observed. In conclusion, the present results show that acute hepatic damage induced by CCl4 can be partially prevented by leukotriene synthesis inhibitors and the protection is enhanced with the simultaneous use of phosphodiesterase inhibitors.

Pharmacokinetics and pharmacodynamics of naproxen in acute experimental hepatitis.

The pharmacokinetics of intravenous naproxen (CAS 22204-53-1) 6 mg kg-1, were studied in Wistar rats under control conditions and 1, 3 and 10 days after the induction of acute hepatitis by a single intraperitoneal injection of galactosamine 375 mg kg-1. In non-intoxicated rats t1/2 was 5.31 +/- 0.90 h, Vd was 0.21 +/- 0.01 l.kg-1, Cl was 17 +/- 5 ml.h-1.kg-1, AUC was 354.4 +/- 8.8 micrograms.h.ml-1 and 99.18 +/- 0.09% of naproxen was bound to plasma proteins. One day after intoxication the liver was considerably damaged; cytochrome P450, the main enzymatic system for naproxen metabolism, was decreased in 90%. Naproxen t1/2 was increased to 11.9 +/- 1.2 h but Vd did not change significantly, therefore clearance was reduced to 37%. Binding to plasma proteins was decreased to 83.21 +/- 0.27%. At day 3 liver function and naproxen pharmacokinetics exhibited a partial recovery, t1/2 was 6.59 +/- 0.92 h and clearance was 12.2 +/- 5 ml.h-1.kg-1 being not significantly different from controls; notwithstanding AUC was still significantly different from that of non-intoxicated rats. 10 days after galactosamine injection all pharmacokinetic parameters, as well as those of liver function, returned to basal levels. The results indicate that acute hepatitis produces an important reduction in naproxen elimination. Changes in naproxen disposition, which exhibit a time course similar to that of liver damage, are mainly determined by a decrease in intrinsic hepatic clearance and in the binding to plasma proteins.

Erythrocyte alterations correlate with CCl4 and biliary obstruction-induced liver damage in the rat.

Erythrocyte and hepatocyte plasma membranes derived from CCl4-cirrhotic and bile duct ligated rats were studied. Six groups of animals were used: Group 1 received CCl4 for 8 weeks. Animals in group 2 received CCl4 for 12 weeks. Group 3 consisted in animals that received CCl4 for 8 weeks and then only vehicle for 4 weeks more. Group 4 was the control which received only vehicle. In group 5, obstructive jaundice was induced by bile duct ligation (BDL); animals in group 6 were operated without ligation of the bile duct (sham operated). The activity of Na+/K+ and Ca2+ dependent ATPases, as well as the cholesterol/phospholipid ratio (CH/PL) were determined in both, erythrocyte and hepatocyte plasma membranes. In the CCl4 groups, Na+/K+ and Ca(2+)-ATPases activity was decreased about 60-70% while the CH/PL ratio was increased significatively in both, erythrocyte and hepatocyte plasma membranes. Discontinuation of CCl4 treatment reverted the enzymatic and lipidic alterations to normal in both cell types. ATPases activity and CH/PL ratio in erythrocyte resembled to those of hepatocytes in BDL rats. A strong correlation (p < 0.005) was found when erythrocyte ATPases activity and CH/PL ratio were compared with those of hepatocytes. Moreover, histopathological analysis of liver sections correlated well with both, erythrocyte and hepatocyte biochemical findings. We concluded that determination of ATPases activity and CH/PL ratio in erythrocyte membranes could be a simple, safe and useful marker of cell liver damage in CCl4-induced liver cirrhosis and biliary obstruction in the rat.

Protective effect of colchiceine against acute liver damage.

Pretreatment of rats with colchiceine (10 micrograms/day/rat) for seven days protected against CCl4-induced liver damage. CCl4 intoxication was demonstrated histologically and by increased serum activities of alanine amino transferase (ALT), alkaline phosphatase (Alk. Phosph.) gamma glutamyl transpeptidase (GGTP), bilirubins and decreased activity of glucose-6-phosphatase (G-6Pase). Furthermore, an increase in liver lipid peroxidation and a decrease in plasma membrane GGTP and Alk. Phosph. activities were found. Colchiceine increased 1.5-fold the LD50 of CCl4 and prevented the release of intracellular enzymes as well as the decrease in GGTP and Alk. Phosph. activities in plasma membranes. It also completely prevented the lipid peroxidation induced by CCl4 and limited the extent of the histological changes.

Prevention of CCL4-induced liver cirrhosis by silymarin.

The efficacy of silymarin treatment in preventing biochemical and histological alterations in CCL4-induced liver cirrhosis in rats was studied. Four groups of rats were treated with: (1) CCL4; (2) mineral oil; (3) CCL4 + silymarin; and (4) silymarin. All animals were sacrificed 72 h after the end of treatments. The activities of alkaline phosphatase (alk. phosp.), gamma-glutamyl transpeptidase (GGTP), glutamic pyruvic transaminase (GPT) and glucose-6-phosphatase (G6Pase), and bilirubin content were determined in serum. Na+, K+-ATPase and Ca++-ATPase activities were measured in isolated plasma membranes. Lipoperoxidation, triglycerides (TG), and glycogen contents were also measured in liver homogenates. Liver cirrhosis was evidenced by significant increases in liver collagen, lipoperoxidation, serum activities of alk. phosp., GGTP, GPT, G6Pase, bilirubin content, and liver TG. Activities of ATPases determined in plasma membranes were significantly reduced, as was liver glycogen content. Silymarin cotreatment (50 mg/kg b.wt) completely prevented all the changes observed in CCL4-cirrhotic rats, except for liver collagen content which was reduced only 30% as compared to CCL4-cirrhotic rats. Silymarin protection can be attributed to the agent's antioxidant and membrane-stabilizing actions.

Protection against thallium hepatotoxicity by silymarin.

The effect of silymarin (100 mg/kg i.p.) on the biochemical indicators of liver damage induced by thallium (10 mg/kg p.o.) was studied in rats. The production of malondialdehyde and the content of reduced glutathione in the liver were measured as indicators of lipid peroxidation. Thallium intoxication increased the serum activities of glutamic pyruvic transaminase, gamma-glutamyl transpeptidase, alkaline phosphatase and the liver concentration of triglycerides. Thallium decreased the activity of alkaline phosphatase and increased that of gamma-glutamyl transpeptidase in the liver cell membrane. It also abolished the membrane activity of Na+/K+ ATPase. Lipid peroxidation was enhanced by thallium as malondialdehyde production was increased and the content of reduced glutathione was decreased in the liver. Silymarin completely prevented all these changes. It is suggested that thallium toxicity is due, at least in part, to the promotion of lipid peroxidation. The membrane stabilizing effect of silymarin observed in this and in other models of liver toxicity is due to some antioxidant property, possibly related to its ability to scavenge free oxygen radicals.

Silymarin improves metabolism and disposition of aspirin in cirrhotic rats.

The profile of urinary salicylate metabolites was determined after an i.p. administration of acetylsalicylic acid (ASA) to CCl4-cirrhotic rats to rats which in addition to CCl4 received an oral dose of silymarin throughout the CCl4 treatment to produce cirrhosis and to control groups. ASA esterase activity was determined in serum and livers. The time course of plasma concentration of salicylates in similar groups was followed after the i.p. injection of ASA. The cirrhotic animals showed a lack of urinary glucuronides and an increase in urinary gentisic and salicylic acids. The activities of plasma and serum ASA esterase were significantly increased in cirrhosis and the plasma half-life of ASA was reduced. The simultaneous administration of silymarin (50 mg/kg of b.w.) along with CCl4, completely prevented all the alterations. The mechanism by which silymarin prevented those alterations is not completely known but our results establish the potential use of silymarin in cirrhotic patients to prevent disorders in drug metabolism and disposition frequently found in patients with liver diseases.

Aspirin disposition in rats acutely intoxicated with CCl4.

The profile of urinary salicylate metabolites was determined after an oral administration of acetylsalicylic acid (ASA) to: 1, control rats; 2, rats treated with CCl4 and 3, rats intoxicated with CCl4 and also pretreated with colchicine for 7 days. The following enzymatic activities were determined: liver and plasma ASA-esterase, liver UDP-glucuronyltransferase and liver aniline hydroxylase. The time course of plasma concentration of salicylates in similar groups were followed after the intraperitoneal administration of acetylsalicylic acid (ASA), salicylic acid (SA) or gentisic acid (GA). The animals acutely intoxicated with CCl4 showed a reduction in urinary excretion of glucuronates and an increased urinary excretion of gentisic and salicylic acids. The activities of plasma and liver ASA-esterases were significantly increased in CCl4-treated rats while the aniline hydroxylase was reduced and the UDP-glucuronyltransferase remained unchanged. The plasma half lives of salicylates were reduced in CCl4-treated rats regardless of the administered parent compound. Colchicine pre-treatment completely prevented the alterations produced by acute intoxication with CCl4. The heterogeneity of liver metabolic dysfunctions present in acute liver damage was evidenced. It is emphasized that the pharmacokinetic alterations produced by acute liver injury can be the result of complex factors that may involve changes in circulation, hepatic binding protein and other routes of elimination.

Heterogeneity of changes on the disposition of aspirin in rats with CCl4-induced chronic liver damage.

The profile of urinary salicylate metabolites was determined after the oral administration of acetylsalicylic acid (ASA) to CCl4-cirrhotic rats, CCl4-cirrhotic rats treated with colchicine for 1 month, and control groups. The following enzymatic activities were determined: liver and plasma ASA-esterase, liver UDP-glucuronyltransferase, and liver aniline hydroxylase. The time-course of plasma concentration of salicylates in similar groups was followed after the intraperitoneal administration of salicylic acid (SA) or gentisic acid (GA). The cirrhotic animals showed a lack of urinary glucuronates and an increase in urinary gentisic and salicylic acids. The activities of plasma and liver ASA-esterases were increased significantly in cirrhosis, whereas aniline hydroxylase was reduced and UDP-glucuronyltransferase remained unchanged. The plasma half-lives of salicylates were reduced in the cirrhotic animals regardless of the administered parent compound. Colchicine treatment reversed almost completely the alterations. The heterogeneity of liver metabolic dysfunctions present in chronic liver disease was demonstrated. It is emphasized that the pharmacokinetic alterations produced by liver damage are the result of a complex set of factors involving changes in the hepatic circulation, protein binding, and the existence of other routes of elimination.

Acute thallium intoxication: kinetic study of the relative efficacy of several antidotal treatments in rats.

Rats were administered a sublethal dose of thallium (12.35 mg/kg as aqueous thallous sulfate, Tl2SO4, equivalent to 10 mg Tl+/kg per os) on day 0. Urine and feces were collected separately every day for 8 days and analyzed by atomic absorption spectroscopy. Based on information in the literature, five antidotal treatments (dithizone, activated charcoal, furosemide, Prussian Blue, and a combination of Prussian Blue and furosemide) were compared with controls for their efficacy in reducing the total thallium body load. In the control group (C,n = 10) of an approximate administered dose of 2 mg, after 8 days 411 micrograms (21%) had been eliminated in the urine and 641 micrograms (32%) in the feces, making a total of 1,052 micrograms (53%). In the treated groups the effects were very significant and in accord with the mode of action of the antidotes: furosemide (a diuretic) only enhanced urinary elimination; activated charcoal and Prussian Blue (unabsorbed adsorbents) only increased fecal elimination; dithizone increased only urinary elimination, whereas combined treatment with Prussian Blue and furosemide increased elimination by both routes. At the end of 8 days the control group had only eliminated 53% of the dose; this was increased to 99% by dithizone, 93% by activated charcoal, 64% by furosemide, 82% by Prussian Blue and 92% by combining furosemide and Prussian Blue. In view of the inherent toxicity of dithizone, it is suggested that treatment with a combination of Prussian Blue-furosemide or with activated charcoal should be adequate.(ABSTRACT TRUNCATED AT 250 WORDS)

Thallium replaces potassium in activation of the (Na+,K+)-ATPase of rat liver plasma membranes.

The capacity of thallium to substitute for K+ in activation of (Na+,K+)-ATPase of liver plasma membranes was studied. Our results indicate that T1+ can replace K+ in the activation of the (Na+,K+)-ATPase of liver plasma membranes. In the presence of Na+, similar activation is obtained with T1+ concentrations only 1/10 of those of K+. In all other aspects, the (Na+,K+)- and (Na+,T1+)-ATPases were found to be identical.

Parameters for the adsorption of thallium ions by activated charcoal and Prussian blue.

Activated charcoal and Prussian blue are effective antidotes in acute thallium (T1+) intoxication in rats. They act by trapping any metal present in or secreted into the gut by the gastro-intestinal epithelium. It was therefore of interest to determine the parameters of the Langmuir adsorption isotherms of T1+ ions for these two adsorbents. The data from equilibration experiments were analyzed by direct least-squares fitting to a hyperbola and with Langmuir's equation to give the following results: Activated charcoal: K1(-1) = 192 micrograms ml-1, K2 = 124 mg g-1 Prussian Blue: K1(-1) = 130 micrograms ml-1, K2 = 72 mg g-1. These high values provide in vitro confirmation of their in vivo antidotal efficacy and show that activated charcoal can replace Prussian blue when this latter drug is unobtainable.