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Diethylnitrosamine (DEN) induces hepatocarcinogenesis, increasing mitotic hepatocytes and leading to chronic inflammation. In addition, type 1 diabetes mellitus (T1DM) is also characterized by a proinflammatory state and by requiring insulin exogenous treatment. Given the association of diabetes, insulin treatment, and cell proliferation, our specific goal was to determine whether the liver in the diabetic state presents a greater response to DEN-induced cell cycle alteration, which is essential for the malignant transformation. Male C57BL/6 mice (four-week-old) were divided into 4 groups: C, C + DEN, T1DM, and T1DM + DEN. Mice were euthanized ten weeks after DEN injection. DEN per se produced an increase in liver lipid peroxidation levels. Besides, in T1DM + DEN, we found a greater increase in the proliferation index, in comparison with C + DEN. These results are in agreement with the increased expression observed in cell cycle progression markers: cyclin D1 and E1. In addition, a proapoptotic factor, such as activated caspase-3, evidenced a decrease in T1DM + DEN, while the Vascular Endothelial Growth Factor (VEGF) and the protooncogene p53 showed a higher increase with respect to C + DEN. Overall, the results allow us to highlight a major DEN response in T1DM, which may explain in part the greater predisposition to the development of hepatocarcinoma (HCC) during the diabetic state.

Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP. Our results show that BZL reduces leukocytes in peripheral blood accompanied by an increase in peritoneal macrophages 24h after CLP. In the liver of these septic mice, BZL decreased expression of mRNA and protein for TNF-α and NOS-2 by inhibition of NF-κB and MAPK (p-38 and ERK). The body of evidence suggests that the immunomodulatory effects of BZL could act selectively, as it is able to decrease the systemic inflammatory reaction and the hepatic response but it can increase the number of cells in the site of infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background/aims: Clarification of the role of lipid peroxidation in the onset of liver proliferation has been hampered by the fact that both higher and lower lipid peroxidation have been reported after two-thirds partial hepatectomy. Recently, it has been shown that nitric oxide might be involved in the control of early responses after partial hepatectomy. We analysed the possible involvement of nitric oxide production in lipid peroxidation levels during liver regeneration., Methods: Sham-operated, hepatectomised and sham and hepatectomised rats pretreated with two inhibitors of oxide nitric synthesis (aminoguanidine or N(G)-monomethyl-L-arginine) were used throughout. Animals were killed at 1, 3, 5 and 15 h after surgery. Cytosolic superoxide dismutase and microsomal-lysosomal catalase activities were measured. Lipid peroxidation levels were measured as thiobarbituric acid-reactive substances and conjugated dienes. Cytosolic nitrate (a stable metabolic product of nitric oxide) was enzymatically determined. Inducible-type nitric oxide synthase (iNOS) was analysed in hepatic cytosol by immunoblotting. DNA synthesis 24 and 48 h after surgery was assessed by [3H]thymidine incorporation., Results: Increased lipid peroxidation was found in total homogenate, cytosol and microsomes. The hepatic cytosolic content of nitrates increased, reaching the highest values at 5 h posthepatectomy. Aminoguanidine or N(G)-monomethyl-L-arginine pretreatment blocked the rise of nitric oxide production and lipid peroxidation levels and decreased the DNA synthesis. The increase in hepatic iNOS protein expression at 5 h after partial hepatectomy disappeared with aminoguanidine pretreatment., Conclusions: Our experiments suggest that nitric oxide plays a role in the proliferation mechanism, although it is responsible, at least in part, for the enhanced lipid peroxidation.

The aim of the present study was to measure protein synthesis in regenerating liver and to evaluate the impact of malnutrition in young and old rats. Two groups of male Wistar rats were used: young rats (4 months old) and old rats (18 months old). The rats were allocated to malnutrition or ordinary food intake for 1 week. Half of each group was sham-operated and the other was partially hepatectomized 2 days before the end of diet manipulation. Hepatic protein synthesis was significantly increased in all hepatectomized groups compared with their respective sham group: young well-nourished hepatectomized rats, 44%; young malnourished hepatectomized rats, 55%; old well-nourished hepatectomized rats, 47%; and old malnourished hepatectomized rats only 21%. Hepatic DNA content was unchanged in all groups and liver RNA content was higher in young malnourished hepatectomized rats (21%, P < 0.05). Serum total amino acid concentration did not change in young well-nourished hepatectomized and young malnourished hepatectomized rats. This value did not show significant changes between old well-nourished hepatectomized and old well-nourished sham, but it increased 14% (P < 0.05) in old malnourished hepatectomized. It was concluded that (a) regeneration is not impaired by malnutrition in young rats and may even be better than in rats eating a normal diet, and (b) the deleterious effect of aging is revealed once old animals are exposed to malnutrition. It is manifested in the decreased rate in hepatic protein synthesis observed in old malnourished hepatectomized rats and in the augmentation of total serum amino acid concentration, where the hypercatabolism induced by hepatectomy is significantly greater.

In a previous study we demonstrated that the administration of 20 micrograms/kg b.wt. of glucagon to rats caused a significant diminution of hepatic cytosolic glutathione S-transferase (GST) activity. This inhibition was non-competitive and reversible. We suggested that the effect would be mediated by cytosolic effectors. The present work was performed to characterize the mechanism involved in this inhibition. Liver tissue slices (170 to 200 mg) were incubated during different periods of time (0, 5, 10, 15, 20 and 30 min.) with several concentrations of glucagon (10(-5) M, 10(-8) M and 10(-10) M), dibutiryl cyclic AMP (10(-4) M, 10(-6) M and 10(-9) M), divalent cation ionophore A23187 (10(-4) M, 10(-6) M and 10(-9) M) or vasopressin (10(-7) M, 5 x 10(-7) M and 10(-8) M). The incubation was done with or without calcium in the medium. In all cases the cytosolic GST activity were determined in liver slices. The percentage of inhibition of GST activity was directly related to the increase of concentration of the test substances. An inhibition between 40% to 45% after 10 min. of incubation with the highest concentrations was observed (except vasopressin which caused 10% of inhibition). 10(-10) M glucagon did not produce a decrease of GST activity. The inhibition disappeared in calcium-free incubated slices, but direct relationship between plasma-membrane calcium influx and inhibition of GST activity (r = 0.950, P < 0.001, n = 24) could be obtained. By using calmodulin antagonists, we conclude that the inhibition process of the enzyme was mediated by calmodulin. In summary, we propose that plasma-membrane calcium influx induced by high concentrations of glucagon activates calmodulin, which promotes a modification (actually a methylation, according to other authors) on GST, thereby causing a decrease in its activity.

The intravenous administration of insulin plus glucose in anesthetized rats caused, within 30 min, an increase of about 56% in hepatic cytosolic glutathione S-transferase (GST) activity, but it did not affect the microsomal enzyme. The injection of glucagon resulted, at the same time, in a 43% drop in the hepatic cytosolic GST, without affecting the microsomal GST. The insulin-dependent increase in cytosolic GST activity was abolished by the pretreatment of the animals with an inhibitor of protein synthesis (cycloheximide). A kinetic analysis revealed a non-competitive inhibition caused by glucagon upon the cytosolic enzyme. In addition, the presence of insulin did not interfere with the effectiveness of glucagon, and vice versa. We propose that: (1) the effect of insulin on hepatic cytosolic GST activity requires protein synthesis; (2) glucagon produces an inhibition of hepatic cytosolic GST, which could be mediated by cytosolic effectors such as adenosine 3'-5'-cyclic monophosphate (cAMP); (3) the effects of glucagon and insulin were not mutually exclusive; (4) hepatic microsomal GST is regulated by different mechanism(s).

The present study was designed to investigate the effect of partial (two-thirds) hepatectomy (PH) on hepatic and intestinal glutathione S-transferases (GSTs) activities. A significant decrease of cytosolic hepatic GSTs activity was observed after the PH. The lowest value of hepatic GSTs was obtained 48 h after the surgery. On the other hand, intestinal GSTs activities increased after PH, reaching the highest values 48 h after the hepatic lobes resection. The hepatic GSTs activities diminution was attributed, in part, to the high accumulation of bile acids in the liver tissue of hepatectomized rats, also demonstrated by a higher retention of [14C] taurocholate. The kinetic analysis performed with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate showed two sets of parameters, indicating the presence of isozymes of high and low affinities. Vmax1 and Vmax2 were lower in PH rats suggesting a non competitive inhibition mechanism. The inhibitory effect of bile acids decreased during liver regeneration process of hepatectomized rats disappearing at 7 days after PH. Conversely, in non regenerating rats (GABA treated) the inhibitory mechanism was still observed at 7 days after the surgery. The increase of intestinal GSTs activities (isozymes of high and low affinities) was attributed to the presence of polyamines, mainly putrescine, produced during the hepatic regeneration process. In this regard, it was showed that GABA treatment, which inhibits polyamine synthesis, completely abolished the increase on intestinal GSTs activities. Finally, the treatment with exogenous putrescine showed that in hepatectomized and sham-operated rats, the polyamine induced GSTs activities in both tissues. In PH rats, the putrescine dependent increase of hepatic GSTs was masked by the inhibitory effect of bile acids. In addition, a summation effect of endogenous and exogenous putrescine was probably the reason of the induction of intestinal GSTs after PH. The GSH/GSSG ratio did not change during the treatments, as well as the microsomal GST activity of both tissues. The work points out the hypothetical detoxification power of the intestine during the hepatocellular insufficiency which follows a two-thirds hepatectomy.

Choleresis induced by dehydrocholate (DHC) stimulates the discharge into bile of lysosomes, which are implicated in the biliary excretion of proteins. Contrary to taurocholate-induced choleresis, DHC choleresis is not affected by microtubule (mt) inhibition. Therefore, the role of mt's in the biliary protein excretion during bile salt choleresis was analyzed in this study. Normal rats and rats treated with the mt poisons colchicine or vinblastine or with the acidotropic agent chloroquine (Cq) were used. The analysis of the protein component in bile was made on SDS-polyacrylamide gel, and the individual polypeptides were quantitated by densitometry. The excretion of bile polypeptides were compared with that of lysosomal acid phosphatase. Bile flow and bile salt output did not show changes on account of treatments. The biliary excretion of acid phosphatase was stimulated by DHC, and it was not affected by mt inhibitors but was markedly diminished by Cq. DHC choleresis produced different effects on the bile polypeptides. The biliary excretion of polypeptide of high molecular mass (84-140 kDa) was stimulated by DHC. Cq treatment increased their basal biliary excretions, whereas DHC-induced secretion was qualitatively and quantitatively similar to that of controls. The 69-kDa polypeptide (albumin) also increased during DHC-induced choleresis, but it showed a different excretory pattern. Cq treatment inhibited such an increase but no correlation with the excretory pattern of the lysosomal marker was found. The biliary excretion of polypeptides of low molecular mass (down to 14 kDa) suffered a transitory decrease and then a subsequent increase over basal values during the DHC choleresis.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of aflatoxin B1 (AFB1) on the glutathione S-transferase activity (GST) and on non-protein thiol levels of different tissues was studied in adult male Wistar rats. Animals received a single dose of the toxin (100 or 500 micrograms/kg body wt., p.o.), and were studied 6 or 24 h after administration. GST was determined in liver, renal cortex, duodenum, jejunum-ileum and distal ileum, using 3 substrates: 1-chloro-2,4-dinitrobenzene (CDNB), trans-4-phenyl-3-buten-2-one (PBO) and 1,2-epoxyethylbenzene (STOX). The non-protein thiol content of all tissues tested increased with the lowest dose at 6 h, returning to normal values at 24 h, while the higher dose produced a significant decrease in reduced thiol levels at 6 h, returning to normal values at 24 h. AFB1 administration induced, independently of dose and tissue, total GST (CDNB) and epoxide-transferase activity (STOX) while A--C-type transferases (PBO) were inhibited. Almost all activities returned to normal values at 24 h. In cases of enzyme induction there was in general an increase in Vmax and a decrease in apparent Km. The opposite was seen in cases of inhibition. In conclusion, the results provide evidence that extrahepatic GST could be important in the overall process of detoxification of AFB1. The behavior seen in hepatic and extrahepatic tissues revealed the functions of catalysis (B-type transferases) and covalent bond formation, as well as inactivation by probable AFB1 metabolites (A--C-type transferases).

The effect of streptozotocin (SZ) on hepatobiliary function was studied in rats on the 1st, 7th and 15th days of treatment. Serum glucose increased significantly on the 1st day, and then remained high. Bile flow, bile acids output and BSP biliary excretion were significantly decreased on the 1st day of treatment, whereas serum sorbitol dehydrogenase was increased. All the parameters tested apart from serum glucose tended to normalize with time. The results suggested a transient toxic effect of SZ on the hepatocyte.

The formation of bilirubin monoglucuronide (BMG) and diglucuronide (BDG) was studied in isolated rat hepatocytes with appropriate viability. Isolated cells were obtained from normal rats and from rats pretreated with spironolactone (SP). A fixed number of cells (4.8 X 10(6)) was incubated in a medium containing uridine diphosphoglucuronic acid (UDPGA, 3.4 mM) and bilirubin (11.3 microM, 29 microM, 50 microM and 81 microM) for different time intervals (from 0 to 25 min). The pellet of cells and the supernatant fraction were subjected to alkaline methanolysis, and the proportions of BMG and BDG were estimated by thin-layer chromatography. No conjugates were detected at time O or in the absence of UDPGA in the incubation system. BMG and BDG were detected after 2 min of incubation and then they increased up to 15 min of incubation. Both conjugates were mostly found in the supernatant fraction, and a predominance of BMG was apparent. Normal cells also synthesized increasing amounts of BMG and BDG with the increase of bilirubin substrate concentration up to 50 microM. When hepatocytes from SP-treated rats were used, a more rapid rate of glucuronidation, that was mainly produced at the expense of BMG found in the supernatant fraction, was clear. The results probably indicate that enzymic conversion of BMG to BDG may be rate limiting in isolated hepatocytes although other possible mechanisms were not excluded.