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

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

The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes

Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poisoning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and FoF1-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracellular Ca2+ homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca2+ homeostasis followed by necrosis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death. © 2012 Elsevier Ltd.

Cissus quadrangularis Linn exerts dose-dependent biphasic effects: osteogenic and anti-proliferative, through modulating ROS, cell cycle and Runx2 gene expression in primary rat osteoblasts

Objectives: This report highlights phytoconstituents present in Cissus quadrangularis (CQ) extract and examines biphasic (proliferative and anti-proliferative) effects of its extract on bone cell proliferation, differentiation, mineralization, ROS generation, cell cycle progression and Runx2 gene expression in primary rat osteoblasts. Materials and methods: Phytoconstituents were identified using gas chromatography-mass spectroscopy (GC-MS). Osteoblasts were exposed to different concentrations (10-100g/ml) of CQ extract and cell proliferation and cell differentiation were investigated at different periods of time. Subsequently, intracellular ROS intensity, apoptosis and matrix mineralization of osteoblasts were evaluated. We performed flow cytometry for DNA content and real-time PCR for Runx2 gene expression analysis.Results: CQ extract's approximately 40 bioactive compounds of fatty acids, hydrocarbons, vitamins and steroidal derivatives were identified. Osteoblasts exposed to varying concentrations of extract exhibited biphasic variation in cell proliferation and differentiation as a function of dose and time. Moreover, lower concentrations (10-50g/ml) of extract slightly reduced ROS intensity, although they enhanced matrix mineralization, DNA content in S phase of the cell cycle, and levels of Runx2 expression. However, higher concentrations (75-100g/ml) considerably induced the ROS intensity and nuclear condensation in osteoblasts, while it reduced mineralization level, proportion of cells in S phase and Runx2 level of the osteogenic gene.Conclusions: These findings suggest that CQ extract revealed concentration-dependent biphasic effects, which would contribute notably to future assessment of pre-clinical efficacy and safety studies.

Antiulcerogenic effect and cytotoxic activity of semi-synthetic crotonin obtained from Croton cajucara Benth.

Trans-dehydrocrotonin, the major diterpene isolated from the bark of Croton cajucara, has good antiulcerogenic activity which, however, is accompanied by toxic effects. on the basis of these results, a semi-synthetic crotonin, named 4SRC, was prepared to determine whether this substance has similar antiulcerogenic activity with lower or no toxicity. The natural crotonin was also isolated from the bark of C. cajucara but was not used due to the small amount obtained. The cytotoxic effect of semi-synthetic crotonin, expressed as cell viability, was assessed in (a) lung fibroblast cell line (V79) derived from Chinese hamsters, a system commonly used for cytotoxicity studies, and (b) rat hepatocytes isolated from male Wistar rats. After treatment, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (MTT reduction), total acid content and neutral red uptake assays. To evaluate V79 cell viability, different concentrations of semi-synthetic crotonin were incubated with the cells. To evaluate the antiulcerogenic effects of semi-synthetic crotonin (50, 100 and 200 mg/kg), we used the models of gastric ulcer induced by ethanol/HCl, stress, indomethacin/bethanechol, and ethanol in male Swiss mice and male Wistar rats. The substance had an IC50 = 500 muM in the neutral red uptake and MTT reduction tests and an IC50 = 200 muM in the nucleic acid content test. With regard to hepatocyte viability after treatment with semi-synthetic crotonin at different concentrations, semi-synthetic crotonin had an IC50 = 10-500 muM in the nucleic acid content and MTT reduction tests and an IC50 = 120 muM in the neutral red uptake test. In another experiment, V79 cells were incubated with the metabolites produced by hepatocytes treated with different concentrations of semi-synthetic crotonin. After a 4-h incubation, semi-synthetic crotonin had an IC50 = 500 muM in the MTT reduction and neutral red uptake tests and an IC50 = 370 muM in nucleic acid content test. The substance had significant antiulcerogenic activity in all models studied, suggesting the presence of a possible antisecretory effect combined with a cytoprotective effect. For this reason, the effect of semi-synthetic crotonin was also evaluated on biochemical parameters of gastric juice and gastric wall mucus, both obtained from pylorus-ligated mice. No significant differences were observed in these parameters between semi-synthetic crotonin-treated and control animals. The results obtained with semi-synthetic crotonin are promising, with a significant preventive effect against gastric ulcer induced by different agents. Our data also show that semi-synthetic crotonin was less toxic than dehydrocrotonin and that the cytotoxic effects decreases with the time that isolated hepatocytes were in culture. (C) 2003 Elsevier B.V. B.V. All rights reserved.

Papel da mitocôndria na citoxicidade induzida pela abamectina em hepatócitos isolados de rato

Pós-graduação em Ciência Animal - FMVA

Mecanismos de toxicidade do fipronil em hepatócitos isolados de rato

Pós-graduação em Ciência e Tecnologia Animal - FEIS

Farmacocinética e farmacodinâmica de derivado ftalimídico planejado para o tratamento da anemia falciforme

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

Glucose- and insulin-induced phosphorylation of the insulin receptor and its primary substrates IRS-1 and IRS-2 in rat pancreatic islets

The presence of tyrosine-phosphorylated proteins was studied in cultured rat pancreatic islets, Immunoblotting performed with total extracts of islets cultured in the presence of 1.8 or 5.6 mM glucose revealed at least three distinct tyrosine-phosphorylated bands (25 kDa, 95 kDa and 165-185 kDa). After 12 h incubation in medium containing 1.8 mM glucose, a pulse exposition to 11 or 22 mM glucose or to 10(-7) M insulin led to a substantial increase in the phosphorylation of all three bands, with no appearance of novel bands. Immunoprecipitation with specific antibodies demonstrated that the signal detected at 95 kDa corresponds to the beta subunit of the insulin receptor (IR) while the band at 165-185 kDa corresponds to the early substrates of the insulin receptor, IRS-1 and IRS-2. Immunoprecipitation with IRS-I or IRS-2 antisera detected their association with the lipid metabolizing enzyme phosphatidylinositol 3-kinase (PI 3-kinase), Thus, this is the first demonstration that elements involved in the insulin-signalling pathway of traditional target tissues are also present in pancreatic islets and are potentially involved in auto- and paracrine-signalling in this organ.

Effects of fasting and intermittent fasting on rat hepatocarcinogenesis induced by diethylnitrosamine

The influences of fasting on DEN-initiation and of intermittent fasting (IF) on the rat liver chemical carcinogenesis process were evaluated in a 52-week long assay. Three groups of adult male Wistar rats were used: Groups I to 3 were treated with a single i.p. injection of 200 mg/kg of diethylnitrosamine (DEN). Group 2 was submitted to 48 h fasting prior to DEN treatment. After the 4th week, Group 3 was submitted to IF, established as 48 h weekly fasting during 48 weeks, while Groups I and 2 were fed ad libitum until the 52nd week. All animals were submitted to 70% partial hepatectomy and sacrificed at the 3rd and 52nd weeks, respectively. Fasting prior to DEN-initiation did not influence the development of altered foci of hepatocytes (AFHs) and of hepatic nodules (Group 2 vs. Group G1). IF inhibited the development of preneoplastic lesions, since this dietary regimen decreased the number and the size of glutathione S-transferase (GST-P) positive foci and the number and size of liver nodules (Group G3 vs. Group G1), the inhibitory effect of IF was also reflected in the development of clear and basophilic cell foci. These results indicate that long-term IF regimen exerts an anti-promoting effect on rat hepatocarcinogenesis induced by DEN. (C) 2002 Wiley-Liss, Inc.

Chemoprevention of preneoplastic liver foci development by dietary mushroom Agaricus blazei Murrill in the rat

The chemopreventive potential of an Agaricus blazei (Ab) Murrill mushroom meal was investigated in a medium-term rat liver carcinogenesis assay. Male Wistar rats initiated for hepatocarcinogenesis with diethylnitrosamine (DEN, 200 mg/kg i.p.) were fed during a 6-week period with the dry powdered mushroom strains Ab 29 or 26, each one with opened (OB) or closed basidiocarp (CB), mixed at 10% level in a basal diet. All experimental animals and controls were subjected to partial hepatectomy at week 3 and killed at week 8. Chemopreventive activity of the mushroom meal was observed for the Ab 29 (OB and CB) and Ab 26 (CB) strains in terms of the number of putative preneoplastic altered foci of hepatocytes which express either the enzyme glutathione S-transferase, placental form (GST-P+) or the transforming growth factor-alpha, and for the Ab 29 (OB) and Ab 26 (CB) strains on the size of GST-P-divided by foci. This was associated with inhibition of foci cell proliferation in the animals fed the Ab 29 (013) and Ab 26 (CB) strains. The results suggest that the protective influence of the Ab meal against the DEN potential for rat liver carcinogenicity depends on both the strain and period of mushroom harvest. (C) 2003 Elsevier Ltd. All rights reserved.

Chlorhexidine induces DNA damage in rat peripheral leukocytes and oral mucosal cells

Objective: Chlorhexidine digluconate is widely used in dental practice for decreasing plaque control, controlling gingivitis and disinfecting root canals. However, the undesirable effects of chlorhexidine digluconate regarding its genotoxicity are conflicting in the literature. Thus, the aim of this study was to investigate the genotoxicity of chlorhexidine digluconate in rat peripheral blood and oral mucosal cells by the single cell gel (comet) assay and micronucleus assay.Methods: Thirty male Wistar rats were distributed into three groups: negative control; experimental group orally treated with 0.5 ml of 0.12% chlorhexidine digluconate, twice daily, during 8 days; and positive control, which received 4-nitroquinoline 1-oxide at 0.5 g/l by drinking water.Results: A statistically significant increase of DNA damage was observed in leukocytes and oral mucosal cells of the chlorhexidine digluconate treated group, as assessed by the comet assay. However, no increase of micronucleated cells was detected in reticulocytes from peripheral blood cells.Conclusions: Taken together, the data indicate that chlorhexidine digluconate is able to induce primary DNA damage in leukocytes and in oral mucosal cells, but no chromosome breakage or loss in erythrocytes.

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.

Tomato oleoresin inhibits DNA damage but not diethylnitrosamine-induced rat hepatocarcinogenesis

Various studies have shown that lycopene, a non-provitamin A carotenoid, exerts antioxidant, antimutagenic and anticarcinogenic activities in different in vitro and in vivo systems. However, the results concerning its chemopreventive potential on rat hepatocarcinogenesis are ambiguous. The aim of the present study was to investigate the antigenotoxic and anticarcinogenic effects of dietary tomato oleoresin adjusted to lycopene concentration at 30, 100 or 300ppm (administered 2 weeks before and during or 8 weeks after carcinogen exposure) on liver of male Wistar rats treated with a single intraperitoneal dose of 20 or 100 mg/kg of diethylnitrosamine (DEN), respectively. The level of DNA damage in liver cells and the development of putative preneoplastic single hepatocytes, minifoci and foci of altered hepatocytes (FHA) positive for glutathione S-transferase (GST-P) were used as endpoints. Significant reduction of DNA damage was detected when the highest lycopene concentration was administered before and during the DEN exposure (20 mg/kg). However, the results also showed that lycopene consumption did not reduce cell proliferation in normal hepatocytes or the growth of initiated hepatocytes into minifoci positive for GST-P during early regenerative response after 70% partial hepatectomy, or the number and area of GST-P positive FHA induced by DEN (100 mg/ kg) at the end of week 10. Taken together, the data suggest a chemopreventive effect of tomato oleoresin against DNA damage induced by DEN but no clear effectiveness in initiating or promoting phases of rat hepatocarcinogenesis. (c) 2008 Elsevier GmbH. 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)