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.

Cytochemical characterization of the endomembranous system during oocyte secondary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The endomembranous system of Serrasalmus spilopleura oocyte secondary growth was analysed using structural and ultrastructural cytochemical techniques. In vitellogenic oocytes, the endoplasmic reticulum components, the nuclear envelope intermembranous space, some Golgi dictiossomes, lysosomes, yolk granules, regions of the egg envelope and sites of the follicle cells react to acid phosphatase detection (AcPase). The cortical alveoli, some heterogeneous cytoplasmic structures, regions of the egg envelope, and sites of the follicle cells are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). The endoplasmic reticulum components, some vesicles, and sites of the follicle cells also react to osmium tetroxide and potassium iodide impregnation (KI). The biosynthetic pathway of lysosomal proteins, such as acid phosphatase, required for vitellogenesis, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes, and, finally, lysosomes. In S. spilopleura oocytes at secondary growth, the endomembranous system takes part in the production of the enzymes needed for vitellogenesis, and in the metabolism of yolk exogenous components (AcPase detection). The endomembranous system compartments also show reduction capacity (KI reaction) and are involved in the metabolism of proteins rich in SH-groups (ZIO reaction).

Endocytic activity in the thrombocytes of the turtle Phrynopys hilarii (freshwater South American species)

The phagocytic process in cells depends on lysosomal enzymes, high-energy metabolism and cellular recognition. In this paper, we investigated the presence of energy and recognition factors in thrombocytes of turtle Phrynopys hilarii (a freshwater South American species). Turtle thrombocytes (P. hilarii) present glycogen - possibly β particles - dispersed in their cytoplasm and glycoproteins in the cell surface, as well as a large number of enzymes involved in the endocytic process (Pellizzon, 1996). The activity of these enzymes depends on high-energy metabolism and on cellular recognition provided by specific glycoconjugates (Alberts et al., 1994). This metabolic characterization is demonstrated by the large amount of glycogen particles observed in the cytoplasm by Thiéry's method. Glycogen labeling was also observed when concanavalin A-peroxidase was used as a marker for thrombocytes and for endocyted charcoal particles. Our results show that these cells have phagocytic ability, suggesting that their function in blood circulation is not limited to aggregation but may also involve a great potential for phagocytosis.

Solid state production of thermostable pectinases from thermophilic Thermoascus aurantiacus

Pectin lyase (Pl) and polygalacturonase (Pg) production by Thermoascus aurantiacus 179-5 was carried out by means of solid-state determination using orange bagasse and wheat bran as a carbon sources. Pg and Pl had optimum activity at pH 5.0 and 10.5 respectively. Maximal activity of the enzymes were determined at 65 °C. Pg was stable in the acidic to neutral pH range and at 60 °C for 1 h. whereas Pl was stable at acidic pH and at 60 °C for 5 h. © 2002 Elsevier Science Ltd. All rights reserved.

Purification and characterization of xylanases from Aspergillus giganteus

A strain of Aspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50°C temperature optimum; optimum pH was 6.0-6.5 for xylanase I and 6.0 for xylanase II. At 50°C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu 2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.

Quantitative HPLC analysis of sesquiterpene lactones and determination of chemotypes in Eremanthus seidelii MacLeish & Schumacher (Asteraceae)

Eremanthus seidelii MacLeish & Schumacher has a restricted occurrence to the Brazilian cerrado surrounding the Furnas (MG) reservoir, in environments that have been seriously damaged by human activity. The present phytochemical investigation reveals that the sesquiterpene lactones (SL) 4β,5-dihydro-2′,3′-dihydroxy-15-desoxy-goyazensolide (1) and 4β,5-dihydro-1′,2′-epoxy-eremantholide-C (2) are the major secondary metabolites in E. seidelii leaves, and an HPLC method was developed for their quantitative analysis. HPLC analysis showed no significant seasonal variation in the concentrations of both SL. No qualitative differences were found in the SL patterns of all individuals sampled. However, there is a different SL quantitative pattern among the plants analyzed, pointing to the existence of three quantitative chemotypes of this species, with differences possibly originating from the activity of the enzymes that cyclize the goyazensolide type SL (1) to a eremantholide type SL (2). ©2005 Sociedade Brasileira de Química.

The hypopharyngeal gland of leaf-cutting ants (Atta sexdens rubropilosa) (Hymenoptera: Formicidae)

The social behavior of ants is controlled by means of dynamic interactions between the environment and the individuals, mainly through pheromones. Among the exocrine glands is the hypopharyngeal gland (HG), located laterally over the pharyngeal plate. The present work aimed to describe and compare the HG in the different castes of the leaf-cutting ant Atta sexdens rubropilosa Forel 1908. The HG of different castes showed similar morphology varying only in relation to the secretory cell number. The secretory intracellular reservoir presented positive reaction to Xylidine Ponceau and P.A.S, indicating the presence of protein and polysaccharides in the secretion. Therefore, we suggest that the function of this gland would be the production of digestive enzymes and/or some sort of mucus, which, together with the secretions produced by the salivary glands of the thorax, would be related to the production of saliva.

Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin or collagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may also be responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Production of pectate lyase by penicillium viridicatum RFC3 in solid-state and submerged fermentation

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500U mL -1 or 300Ug -1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme fromSSF and designated PL I, II, III, IV, V, andVI, while five peaks were obtained fromcrude enzyme fromSmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. Copyright © 2010 Viviani Ferreira et al.

MMP-2 and TIMP-2 in the prostates of male and female mongolian gerbils: Effects of hormonal manipulation

TIMPs in the prostates of male and female gerbils and evaluated the effects of testosterone on the expression of these enzymes. Ventral prostates from male gerbils that were either intact or had been castrated for 7 or 21 days, along with prostates from female gerbils that were either intact or had been treated with testosterone for 7 or 21 days, were submitted to histological, stereological and immunohistochemical analyses. Stereology of prostatic components showed significant alterations of tissue compartments in the ventral male prostate after castration, especially after 21 days, with a significant increase in stroma. Administration of testosterone led to disorganization in the female prostate, with a significant increase in collagen fibers and smooth muscle cells after 21 days, along with the development of epithelial lesions such as PINs. MMP-2 increased after 21 days of castration in males; however, the TIMP-2 immunoreaction for this group was weak or absent. In females, the expression of MMP-2 appeared to decrease after 7 days of treatment with testosterone, but after 21 days, both epithelium and stroma showed a stronger reaction for MMP-2 than the controls. The expression of TIMP-2 in the treated females was similar to its expression in the castrated males. We conclude that the distribution of MMPs and TIMPs in both male and female prostates is altered by androgen manipulation, but the mechanism of stromal regulation appears to be distinct between genders because both the lack of T in castrated males and the excess levels of T in treated females lead to the same effect.

Xylanase and β-Xylosidase from Penicillium janczewskii: Production, Physico-Chemical properties, and application of the crude extract to pulp biobleaching

Extracellular xylanase and β-xylosidase production by a Penicillium janczewskii strain were investigated in liquid cultures with xylan from oat spelts under different physical and chemical conditions. The selected conditions for optimized production of xylanase and β-xylosidase were 7 days, pH 6.5, at 30 °C and 8 days, pH 5.0, at 25 °C, respectively. The xylanase exhibited optimal activity in pH 5.0 at 50 °C and the β- xylosidase in pH 4.0 at 75 °C. The xylanase was more stable at pH 6.0 to 9.5, while the β-xylosidase remained stable at pH ranging from 1.6 to 5.5. The xylanase half-life (T50) at 40, 50, and 60 °C was 183, 15, and 3 min, respectively. β-xylosidase half-life was 144, 8, and 4 min at 50, 65, and 75 °C, respectively. When applied to the biobleaching of Eucalyptus kraft pulp, xylanase dosages of 2 and 4 U/g dried pulp reduced, respectively, kappa number by 3.0 and 3.3 units after 1 h treatment, demonstrating that the use of P. janczewskii xylanases in this process is quite promising. The pulp viscosity was not altered, confirming the absence of cellulolytic enzymes in the fungal extract.

Use of different carbon sources in cultivation of baker's yeast for production of glycerol-3-phosphate dehydrogenase

The physiological state of yeast cells changes during culture growth as a consequence of environmental changes (nutrient limitations, pH and metabolic products). Cultures that grow exponentially are heterogeneous cell populations made up of cells regulated by different metabolic and/or genetic control systems. The strain of baker's yeast selected by plating commercial compressed yeast was used for the production of glycerol-3- phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPD) has been widely used in the enzyme assays with diverse compounds of industrial interest, such as glycerol or glycerol phosphate, as well as a number of important bioanalytical applications. Each cell state determines the level of key enzymes (genetic control), fluxes through metabolic pathways (metabolic control), cell morphology and size. The present study was carried out to determine the effects of environmental conditions and carbon source on GPD production from baker's yeast. Glucose, glycerol, galactose and ethanol were used as carbon sources. Glycerol and ethanol assimilations required agitation, which was dependent on the medium volume in the fermentation flask for the greatest accumulation of intracellular GPD. Enzyme synthesis was also affected by the initial pH of the medium and inoculum size. The fermentation time required for a high level of enzyme formation decreased with the inoculum size. The greatest amount of enzyme (0.45 U/ml) was obtained with an initial pH of 4.5 in the medium containing ethanol or glycerol. The final pH was maintained in YP-ethanol, but in the YP-glycerol the final pH increased to 6.9 during growth.

Characterization of esterase patterns in hepatopancreas of three species of Macrobrachium (Palaemonidae)

The genus Macrobrachium (Bate, 1868) belongs to the Palaemonidae family. These species are commonly found in lakes, floodplains and rivers in tropical and subtropical regions of South America. The Macrobrachium genus encompasses nearly 210 species of ecological and economic importance. In this study, three species of Macrobrachium (M acrobrachium jelskii, M acrobrachium amazonicum and M acrobrachium brasiliense) were studied in order to characterize the esterase patterns in the hepatopancreas, which were still unknown. Esterases are enzymes which catalyze the hydrolysis of esters. In the hepatopancreas, these enzymes play important roles in several metabolic processes involved in some functions of this organ, such as detoxification and digestion. Twelve esterase bands (EST1 to EST12) were detected in these species, and a comparison among them showed no qualitative differences in interspecific bands, or between males and females. Inhibitors were used to classify the esterase bands. The results indicated seven acetylesterases, two carboxylesterases, one arylesterase, and one cholinesterase. The EST11 band was not detected in these procedures because of its lower frequency. Statistical analyses showed no variability among the species, in either interspecific or intraspecific assays. These results support the hypothesis of a high evolutionary conservation of esterases in the hepatopancreas of these crustaceans. The data enabled us to assess the genetic structure of these species through the use of esterasic enzymes. It also contributes to our knowledge about the biology of these poorly studied species. Knowledge on the genetic structure of populations and species are essential when defining priorities for their management and conservation. © 2012 Elsevier Ltd.

Effects of corn replacement by sorghum in broiler diets on performance and intestinal mucosa integrity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Diabetes mellitus triggers oxidative stress in the liver of alloxan-treated rats: A mechanism for diabetic chronic liver disease

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)