Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.     

Cytotoxic and Genotoxic Effects of <Emphasis Type="Italic">cis</Emphasis>-Tetraammine(oxalato)Ruthenium(III) Dithionate on the Root Meristem Cells of <Emphasis Type="Italic">Allium cepa</Emphasis>

Ruthenium complexes have attracted much attention as possible building blocks for new transition-metal-based antitumor agents. The present study examines the mitotoxic and clastogenic effects induced in the root tips of Allium cepa by cis-tetraammine(oxalato)ruthenium(III) dithionate {cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆)} at different exposure durations and concentrations. Correlation tests were performed to determine the effects of the time of exposure and concentration of ruthenium complex on mitotic index (MI) and mitotic aberration index. A comparison of MI results of cis-[Ru(C₂O₂)(NH₃)₄]₂(S₂O₆) to those of lead nitrate reveals that the ruthenium complex demonstrates an average mitotic inhibition eightfold higher than lead, with the frequency of cellular abnormalities almost fourfold lower and mitotic aberration threefold lower. A. cepa root cells exposed to a range of ruthenium complex concentrations did not display significant clastogenic effects. Cis-tetraammine(oxalato)ruthenium(III) dithionate therefore exhibits a remarkable capacity to inhibit mitosis, perhaps by inhibiting DNA synthesis or blocking the cell cycle in the G2 phase. Further investigation of the mechanisms of action of this ruthenium complex will be important to define its clinical potential and to contribute to a novel and rational approach to developing a new metal-based drug with antitumor properties complementary to those exhibited by the drugs already in clinical use.     

Ovarian and Uterine Ultrasonography in <Emphasis Type="Italic">Aotus azarai infulatus</Emphasis>

We evaluated the uterus and ovaries of owl monkeys (Aotus azarai infulatus) via gynecological ultrasound examination. We evaluated the subjects in 2 different time periods. The first period (P1) was characterized by the absence of mating, with daily examinations, during 4 mo (n = 10). At the end of P1, we paired the subjects for 30 d, but without ultrasonographic evaluation. The second period (P2) was characterized by the presence of mating, with examinations once a week, during 7 consecutive months (n = 9). We evaluated the uterus and ovaries in sagittal and transverse scans, using a 5–12 MHz linear array probe. The uterine volume (UV) was directly proportional to the number of previous parturitions. The right ovary volume (RtOV) is greater than the left (LtOV) in P1 and P2. There is a positive correlation (p < 0.05) between the females’ mass, RtOV (r = 0.28) and LtOV (r = 0.16).     

Antioxidant effects of different extracts from Melissa officinalis, Matricaria recutita and Cymbopogon citratus

Considering the important role of oxidative stress in the pathogenesis of several neurological diseases, and the growing evidence of the presence of compounds with antioxidant properties in the plant extracts, the aim of the present study was to investigate the antioxidant capacity of three plants used in Brazil to treat neurological disorders: Melissa officinalis, Matricaria recutita and Cymbopogon citratus. The antioxidant effect of phenolic compounds commonly found in plant extracts, namely, quercetin, gallic acid, quercitrin and rutin was also examined for comparative purposes. Cerebral lipid peroxidation (assessed by TBARS) was induced by iron sulfate (10 μM), sodium nitroprusside (5 μM) or 3-nitropropionic acid (2 mM). Free radical scavenger properties and the chemical composition of plant extracts were assessed by 1′-1′ Diphenyl-2′ picrylhydrazyl (DPPH) method and by Thin Layer Chromatography (TLC), respectively. M. officinalis aqueous extract caused the highest decrease in TBARS production induced by all tested pro-oxidants. In the DPPH assay, M. officinalis presented also the best antioxidant effect, but, in this case, the antioxidant potencies were similar for the aqueous, methanolic and ethanolic extracts. Among the purified compounds, quercetin had the highest antioxidant activity followed by gallic acid, quercitrin and rutin. In this work, we have demonstrated that the plant extracts could protect against oxidative damage induced by various pro-oxidant agents that induce lipid peroxidation by different process. Thus, plant extracts could inhibit the generation of early chemical reactive species that subsequently initiate lipid peroxidation or, alternatively, they could block a common final pathway in the process of polyunsaturated fatty acids peroxidation. Our study indicates that M. officinalis could be considered an effective agent in the prevention of various neurological diseases associated with oxidative stress.     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Lead chloride affects sperm motility and acrosome reaction in mice

Lead is highly toxic and persistent in the environment and, thus, a major concern for public health. In this study, the effects of lead chloride (PbCl₂) on mouse epididymal sperm were evaluated. Male mice were subcutaneously injected with 74 and 100 mg PbCl₂/kg body weight for four consecutive days. Sperm was collected from the epididymis and several parameters of sperm function, such as sperm density, motility, viability, mitochondrial function, acrosome integrity and morphology, were evaluated. Furthermore, DNA fragmentation was assessed by the terminal deoxylnucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assay and chromatin integrity was evaluated by sperm chromatin structure assay (SCSA). In order to assess direct effects on existing sperm population, we sacrificed one group for each condition at day 5. The effects of lead upon one entire spermatogenic cycle were evaluated on day 35. Both lead concentrations used in this work affected sperm motility, although no significant differences were observed in sperm viability, mitochondrial function and DNA/chromatin integrity. However, a decrease in the percentage of intact acrosomes was also observed, mirroring a lead-induced premature acrosome reaction. Thus, the results obtained indicate that, together with impaired motility, the effect of lead toxicity on acrosome integrity, leading to premature reaction, may compromise the ability of sperm to fertilize the oocyte.     

Variation of heartwood and sapwood in 18-year-old Eucalyptus globulus trees grown with different spacings

Heartwood and sapwood development was studied in 18-year-old Eucalyptus globulus trees from pulpwood plantations with different spacings (3 × 2, 3 × 3, 4 × 3, 4 × 4 and 4 × 5 m), on cross-sectional discs taken at breast height. The trees possessed a large proportion of heartwood, on average 60% of the wood cross-sectional surface. Spacing was a statistically significant source of variation of heartwood area, which ranged between 99 and 206 cm² for the closer (3 × 2) and wider (4 × 5) spacings, respectively. There was a positive and high statistical significant correlation between heartwood diameter and tree diameter (heartwood diameter = −0.272 + 0.616 dbh; r ² = 0.77; P < 0.001), and larger trees contained more heartwood regardless of spacing. Heartwood proportion in cross-section remained practically constant between spacings but increased with tree diameter class: 55.1, 62.2, 65.0 and 69.5% for diameter at breast height classes <15, 15–20, 20–25 and >25 cm, respectively. The sapwood width did not depend on tree diameter growth and remained practically constant at an average of 18 mm (range 15–21 mm), but sapwood area showed a good linear regression with tree diameter. Therefore, tree growth enhancement factors, such as wide spacings, will induce formation of larger heartwoods that can negatively impact raw-material quality for pulping. The increase in heartwood in relation with tree dimensions should therefore be taken into account when designing forest management guidelines.     

Photosynthetic changes and oxidative stress caused by iron ore dust deposition in the tropical CAM tree Clusia hilariana

The effect of iron solid particulate matter (SPM_Fe) deposited onto soil and leaves on photosynthesis and oxidative stress was evaluated in Clusia hilariana, a CAM tropical tree of high occurrence in Brazilian restingas. Significant increases in iron content were found in plants exposed to SPM_Fe applied onto leaf and soil surfaces. However, only the application of SPM_Fe on leaves of C. hilariana caused significant reductions in some evaluated characteristics such as photosynthetic rate, stomatal conductance, transpiration, organic acid accumulation, potential quantum yield of PSII, and changes in daily CAM photosynthesis pattern. Increase in relative membrane permeability and reduction in catalase and superoxide dismutase activities in the leaves of plants exposed to SPM_Fe also were observed; however, lipid peroxidation did not change. These responses seem to be due to the combination of physical effects such as increase of leaf temperature, reduction in light absorption, obstruction of stomatal pores, and biochemical effects triggered by oxidative stress.     

Long-term effects of overfeeding during lactation on insulin secretion--the role of GLUT-2

Overnutrition during critical developmental periods is believed to be a risk factor for the emergence of metabolic disorders in adulthood. The present study investigated the effects of pups overfeeding during lactation on offspring's insulin secretion. To study the consequences of overnutrition early in life in rats, litter size reduction has been shown to be an appropriate experimental model. To induce early postnatal overnutrition, litter size was reduced to three pups per litter at the third day following birth [overfed group (OG)]. In the control group (CG), the litter size was adjusted to 10 pups per litter. Metabolic parameters and glucose-stimulated insulin secretion were assessed. OG pups ingested more milk at 10 and 21 days and had an augmented food intake at 1 year compared to the CG. Consistently, body weight, body fat, and fasting plasma levels of insulin were higher in 1-year-old OG rats. In addition, OG rats exhibited enhanced insulin secretion, accompanied by elevated content of GLUT-2 in pancreatic islets compared to CG. These findings indicate that early postnatal overnutrition during a critical developmental period in life may program permanent alterations in glucose-stimulated insulin secretion.     

The potential of Metarhizium anisopliae and Beauveria bassiana isolates for the control of Aedes aegypti (Diptera: Culicidae) larvae

Fungal isolates were screened against Aedes aegypti larvae. Exposure of larvae to conidial suspensions resulted in 6–90% mortality. An inoculum persistence assay using one of the most virulent isolates showed an approximate half-life of 10 days in water containers. Not all larvae surviving to form pupae resulted in adult emergence.