Adrenergic and serotonergic function in dna synthesis during rat liver regeneration and in hepatocyte cultures

The adult mammalian liver is predominantly in a quiescent state with respect to cell division. This quiescent state changes dramatically, however, if the liver is injured by toxic, infectious or mechanic agents (Ponder, 1996). Partial hepatectomy (PH) which consists of surgical removal of two-thirds of the liver, has been used to stimulate hepatocyte proliferation (Higgins & Anderson 1931). This experimental model of liver regeneration has been the target of many studies to probe the mechanisms responsible for liver cell growth control (Michalopoulos, 1990; Taub, 1996). After PH most of the remaining cells in the renmant liver respond with co-ordinated waves of DNA synthesis and divide in a process called compensatory hyperplasia. Hence, liver regeneration is a model of relatively synchronous cell cycle progression in vivo. In contrast to hepatomas, cell division is terminated under some intrinsic control when the original cellular mass has been regained. This has made liver regeneration a useful model to dissect the biochemical and molecular mechanisms of cell division regulation. The liver is thus, one of the few adult organs that demonstrates a physiological growth rewonse (Fausto & Mead, 1989; Fausto & Webber, 1994). The regulation of liver cell proliferation involves circulating or intrahepatic factors that are involved in either the priming of hepatocytes to enter the cell cycle (Go to G1) or progression through the cell cycle. In order to understand the basis of liver regeneration it is mandatory to define the mechanisms which (a) trigger division, (b) allow the liver to concurrently grow and maintain dilferentiated fimction and (c) terminate cell proliferation once the liver has reached the appropriate mass. Studies on these aspects of liver regeneration will provide basic insight of cell growth and dilferentiation, liver diseases like viral hepatitis, toxic damage and liver transplant where regeneration of the liver is essential. In the present study, Go/G1/S transition of hepatocytes re-entering the cell cycle after PH was studied with special emphasis on the involvement of neurotransmitters, their receptors and second messenger function in the control of cell division during liver regeneration

The cytoprotective effect of alpha-tocopherol and daidzein against d-galactosamine-induced oxidative damage in the rat liver

We hypothesized that the hepatotoxicity that develops after the induction of oxidative stress (induced by d-galactosamine [GalN]) can be ameliorated by alpha-tocopherol (ATC) and the soy isoflavone daidzein. To test this, we ranked and assigned male Wistar rats into 6 groups, which involved pretreatment (ATC or daidzein) for 1 hour followed by treatment (GalN) for 23 hours. Histopathologic analysis showed that GalN administration induced marked necrosis (P < .001), steatosis (P < .001), both lobular and portal inflammations (P < .001), overall histopathologic score (P < .001), and activation of caspase-3 in the liver (P < .001). Immunohistochemical staining of malondialdehyde-protein adducts, a measure of oxidative stress, was increased in response to GalN (P < .001). Paradoxically, there were increases in total (P < .05) and cytosolic superoxide dismutase (P < .001) activities after GalN administration, indicative of an up-regulation of antioxidant defenses. The concentration of total protein (P < .001), albumin (P < .01), and globulin fractions (P < .001) in the plasma, as well as the activity of aspartate aminotransferase (P < .001), was significantly perturbed after GalN treatment, reflective of overall acute hepatic injury. Administration of daidzein showed a significant amelioration of the Ga1N-induced increase in malondialdehyde-protein adducts (P < .01) and cytosolic superoxide dismutase activities (P < .01) in the liver. However, all other variables were not significantly altered in response to daidzein. In response to ATC pretreatment, the total histopathologic score (P < .05), degree of necrosis (P < .05), and both lobular (P < .05) and portal (P = .05) inflammations were significantly ameliorated. To conclude, both daidzein and ATC protect the liver against oxidative damage possibly via different pathways.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K(+) channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.

Antioxidant activity of isocoumarins isolated from Paepalanthus bromelioides on mitochondria

The isocoumarins (1-50 mu M) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8 '-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH*) and superoxide (O-2(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H2O2 accumulation and Fe2+ -citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O-2(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H2O2. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: Uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca2+ at different conditions, a mitochondrial permeability transition (MPT) linked to protein,thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 mu M on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 mu M Ca2+; inhibition of these processes was assessed in non-energized organelles in the presence of 300 mu M t-butyl hydroperoxide plus 500 mu M Ca2+. Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca2+. Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin. (c) 2006 Published by Elsevier B.V..

Absence of carcinogenic and anticarcinogenic effects of annatto in the rat liver medium-term assay

Annatto (Bixa orellana L.) is a natural food colorant extensively used in many processed foods, especially dairy products. The lower cost of production and the low toxicity, make annatto a very attractive and convenient pigment in substitution to the many synthetic colorants. In the present study we investigate the carcinogenic and anticarcinogenic effects of dietary annatto in Wistar rat liver using the preneoplastic glutathione S-transferase (GST-P) foci and DNA damage biomarkers. Annatto, containing 5% bixin, was administered in the diet at concentrations of 20, 200, and 1000 ppm (0.07; 0.80 and 4.23 bixin/kg body wt/day, respectively), continuously during 2 weeks before, or 8 weeks after DEN treatment (200 mg/kg body wt, i.p.), to evaluate its effect on the liver-carcinogenesis medium-term bioassay. The comet assay was used to investigate the modifying potential of annatto on DEN (20 mg/kg body wt)-induced DNA damage. The results showed that annatto was neither genotoxic nor carcinogenic at the highest concentration tested (1000 ppm). No protective effects were also observed in both GST-P foci development and comet assays. In conclusion, in such experimental conditions, annatto shows no hepatocarcinogenic effect or modifying potential against DEN-induced DNA damage and preneoplastic foci in the rat liver. (C) 2004 Elsevier Ltd. All rights reserved.

Influence of aqueous extract of Agaricus blazei on rat liver toxicity induced by different doses of diethylnitrosamine

The modifying potential of prior administration of an aqueous extract of the mushroom Agaricus blazei Murrill (Agaricaceae) (Ab) on hepatotoxicity induced by different doses of diethylnitrosamine (DEN) in male Wistar rats was evaluated. During 2 weeks, animals of groups G3 (Ab+DEN50), G5 (Ab+DEN100), G7 (Ab+DEN200), and G8 (Ab-treated) were treated with the A. blazei through drinking water. After this period, groups G2 (DEN50), G3 (Ab+DEN50), G4 (DEN100) G5 (Ab+DEN100), G6 (DEN200), and G7 (Ab+DEN200) were given a single i.p. injection of 50, 100 and 200 mg/kg of DEN, respectively, while groups G1 (nontreated) and G8 (Ab-treated) were treated with 0.9% NaCl only. All animals were killed 48 h after DEN or NaCl treatments. The hepatocyte replication rate was estimated by the index of the proliferating cell nuclear antigen (PCNA) positive hepatocytes and the appearance of putative preneoplastic hepatocytes through expression of the enzyme glutathione S-transferase placental form (GSTP). After DEN-treatment, ALT levels, PCNA labeling index, and the number of GST-P positive hepatocytes were lower in rats that received A. blazei treatment and were exposed to 100 mg/kg of DEN. Our findings suggest that previous treatment with A. blazei exerts a hepatoprotective effect on both liver toxicity and hepatocarcinogenesis process induced by a moderately toxic dose of DEN. (C) 2002 Elsevier B.V. Ireland Ltd. All rights reserved.

Protective effects of Ginkgo biloba against rat liver carcinogenesis

Ginkgo biloba (EGb) has been proposed as a promising candidate for cancer chemoprevention and has shown protective effects on the liver against chemically induced oxidative injury and fibrosis. The potential beneficial effects of EGb were investigated in two rat liver carcinogenesis bioassays induced by diethylnitrosamine (DEN). In a short-term study for anti-initiating screening, male Wistar rats were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb and initiated 14 days later with a single dose of DEN (100 mg/kg i.p.). The respective groups were killed 24 h or 2 weeks after DEN-initiation. Liver samples were collected for the analysis of proliferating cell nuclear antigen (PCNA), transforming growth factor alpha (TGF-alpha), p53, apoptosis and induction of single hepatocytes and minifoci positive for the enzyme glutathione S-transferase P-form (GST-P). In a medium-term study for anti-promoting screening, the animals received a single dose of DEN (200 mg/kg i.p.) and, 2 weeks later, were fed a basal diet or supplemented diet with 500 or 1000 ppm EGb for 6 weeks. All animals underwent 70% partial hepatectomy (PH) at week 3 and killed at week 8. Liver samples were colleted to analyze development of preneoplastic foci of altered hepatocytes (FAH) expressing GST-P. In the short-term study, pretreatment of rats with 1000 ppm EGb significantly reduced the rates of cell proliferation, apoptosis and p53, TGF-a immunoreactivity and the number of GST-P-positive hepatocytes. In the medium-term study, EGb treatment during the post-initiation stage failed to reduce the development of DEN-induced GST-P-positive foci. Thus, EGb presented inhibitory actions during initiation but not promotion of rat liver carcinogenesis induced by DEN. (C) 2008 Elsevier B.V. All rights reserved.

Metalloproteinases 2 and-9 activity during promotion and progression stages of rat liver carcinogenesis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Absence of chemopreventive influence of propolis on the rat liver altered foci development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Iron chelating-mediated antioxidant activity of Plectranthus barbatus extract on mitochondria

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diuron lacks promoting potential in a rat liver bioassay

The promoting activity of the herbicide Diuron was evaluated in a medium-term rat liver carcinogenesis bioassay that uses as endpoint immunohistochemically identified glutathione S-transferase positive (GST-P+) foci. Male Wistar rats were allocated to the following groups: G1 to G6 were initiated for liver carcinogenesis by a single dose of diethylnitrosamine (DEN, 200 mg/kg) while groups G7 and G8 received only 0.9% NaCl (DEN vehicle). From the 2nd week animals were fed a basal diet (G1 and G7) or a diet added with Diuron at 125, 500, 1250, 2500 and 2500 ppm (G2 to G5 and G8, respectively) or 200 ppm Hexaclorobenzene (HCB; G6). The animals were submitted to 70% partial hepatectomy at the 3rd week and sacrificed at the 8th week. The herbicide did not alter ALT or creatinine serum levels. No conspicuous GST-P+ foci development was registered in non-initiated rats fed Diuron at 2500 ppm. While DEN-initiated animals fed Diuron at 1250 or 2500 ppm developed mild centrilobular hypertrophy, DEN-initiated HCB-fed animals showed severe liver centrilobular hypertrophy and significant GST-P+ foci development. These findings indicate that the medium-term assay adopted in this study does not reveal any liver carcinogenesis initiating or promoting potential of Diuron in the rat.

Mechanism for the uncoupling of oxidative phosphorylation by juliprosopine on rat brain mitochondria

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Early cytotoxic and genotoxic effects of atrazine on Wistar rat liver: A morphological, immunohistochemical, biochemical, and molecular study

Risk assessments suggest that intermediate and long-term exposure to triazine herbicides and its metabolites through water can cause severe damage to human health. The objective of this study was to investigate the possible effects of atrazine on Wistar rats submitted to subacute treatment. For this purpose, the activity of catalase and alanine aminotransferase was quantified, and the effect of the herbicide on cell membranes was examined based on the measurement of lipid peroxidation and consequent formation of malondialdehyde and on the mRNA expression of antioxidant enzymes (Mn-superoxide dismutase [SOD] and GSTM1) and connexins. In addition, we evaluated histopathological alterations in the liver, cellular expression of SOD and glutathione (GST), activation of heat shock proteins (HSPs) by immunohistochemistry, and the induction of apoptosis. The genotoxic potential of the herbicide was investigated by the micronucleus test in bone marrow smears. Adult male Wistar rats were treated with an aqueous solution of atrazine at a concentration of 400 mg/kg/day, by gavage, for 14 consecutive days. Control groups were also included. The results showed an increase of catalase levels and maintenance of the expression of antioxidant enzymes (SOD and GST). In addition, lipid peroxidation, hepatic tissue degeneration, activation of HSP90, increased levels of connexin mRNA, and genotoxicity were observed. In conclusion, atrazine induced early hepatic oxidative stress that triggered defense mechanisms to maintain the morphophysiological integrity of the liver. Further studies are needed to better understand the effects of this herbicide on human health. (C) 2011 Elsevier B.V. All rights reserved.

Cytotoxicity of monocrotaline in isolated rat hepatocytes: Effects of dithiothreitol and fructose

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)