Possible ATP trafficking by ATP-shuttles in the olfactory cilia and glucose transfer across the olfactory mucosa.

Odor transduction in the cilia of olfactory sensory neurons involves several ATP-requiring enzymes. ATP is generated by glycolysis in the ciliary lumen, using glucose incorporated from surrounding mucus, and by oxidative phosphorylation in the dendrite. During prolonged stimulation, the cilia maintain ATP levels along their length, by unknown means. We used immunochemistry, RT-PCR, and immunoblotting to explore possible underlying mechanisms. We found the ATP-shuttles, adenylate and creatine kinases, capable of equilibrating ATP. We also investigated how glucose delivered by blood vessels in the olfactory mucosa reaches the mucus. We detected, in sustentacular and Bowman's gland cells, the crucial enzyme in glucose secretion glucose-6-phosphatase, implicating both cell types as putative glucose pathways. We propose a model accounting for both processes.

Embryogenic Competence Acquisition in Sugar Cane Callus Is Associated with Differential H+-Pump Abundance and Activity.

Somatic embryogenesis is an important biological process in several plant species, including sugar cane. Proteomics approaches have shown that H+ pumps are differentially regulated during somatic embryogenesis; however, the relationship between H+ flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H+ flux and somatic embryo maturation in sugar cane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H+-flux and H+-pump (P-H+-ATPase, V-H+-ATPase, and H+-PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H+ pumps. We observed that P-H+-ATPase and H+-PPase were more abundant in embryogenic callus. Compared to non-embryogenic callus, embryogenic callus showed higher H+ influx, especially on maturation day 14, as well as higher H+-pump activity (mainly, P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H+ pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H+-pump protein abundance and, consequently, higher H+ flux and embryogenic competence acquisition in the callus of sugar cane, allowing for the optimization of the somatic embryo conversion process by modulating the activities of these H+ pumps.

Proteomic response of gill microsomes of Crassostrea brasiliana exposed to diesel fuel water-accommodated fraction.

Diesel fuel water-accommodated fraction (diesel-WAF) is a complex mixture of organic compounds that may cause harmful effects to marine invertebrates. Expression of microsomal proteins can be changed by oil exposure, causing functional alterations in endoplasmic reticulum (ER). The aim of this study was to investigate changes in protein expression signatures in microsomes of oysterl Crassostrea brasiliana (=C.gasar) gill after exposure to 10% diesel-WAF for 24 and 72 h. Protein expression signatures of gills of oysters exposed to diesel-WAF were compared to those of unexposed oysters using two-dimensional electrophoresis (2-DE) to identify differentially expressed proteins. A total of 458 protein spots with molecular weights between 30-75 kDa were detected by 2-DE in six replicates of exposed oyster proteomes compared to unexposed ones. Fourteen differentially expressed proteins (six up-regulated and eight down-regulated) were identified. They are: proteins related to xenobiotic biotransformation (cytochrome P450 6 A, NADPH-cytochrome P450 reductase); cytoskeleton (α-tubulin, ß-tubulin, gelsolin); processing and degradation of proteins pathways (thioredoxin domain-containing protein E3 ubiquitin-protein ligase MIB2); involved in the biosynthesis of glycolipids and glycoproteins (beta-1,3-galactosyltransferase 1); associated with stress responses (glutamate receptor 4 and 14-3-3 protein zeta, corticotropin-releasing factor-binding protein); plasmalogen biosynthesis (fatty acyl-CoA reductase 1), and sodium-and chloride-dependent glycine transporter 2 and glyoxylate reductase/hydroxypyruvate reductase. Different patterns of protein responses were observed between 24 and 72 h-exposed groups. Expression pattern of microsomal proteins provided a first insight on the potential diesel-WAF effects at protein level in microsomal fraction of oyster gills and indicated new potential biomarkers of exposure and effect. The present work can be a basis for future ecotoxicological studies in oysters aiming to elucidate the molecular mechanisms behind diesel-WAF toxicity and for environmental monitoring programs.

Assessment of sediment mutagenicity in areas under the influence of a contaminated site undergoing a remediation process.

Soil contamination enters aquatic ecosystems affecting sediment quality. The region studied is the Taquari River, Brazil, close to a site contaminated by wood preservatives, with a runoff route into the river. The first stage of the remediation process (In this article, the terms intervention and remediation have been used with slightly different meanings. We consider intervention to be the first phase of the remediation process, which aims to remove active sources) was an intervention to remove the main active sources. The Salmonella/microsome assay and polycyclic aromatic hydrocarbons (PAHs) were used to assess sediment quality in organic extracts during different intervention phases. The strains used were TA98, TA97a, and TA100 with and without S9mix (±S9). The results indicated the presence of pro-mutagens at site Ta010 (closest to the contaminated site) in all samplings, and the highest result occurred before intervention for TA100 + S9 (1,672 ± 215.9 rev/g). These values decreased during (83 ± 23.6 rev/g) and after this process (403 ± 105.9 rev/g), although the PAHs concentrations increased. Samples from this site presented PAHs with a carcinogenic potential during the assessed periods. After intervention, Ta006 (4 km downstream from Ta010) showed the most significant mutagenesis for TA100 + S9 (764 ± 230.2 rev/g) and, although the total PAHs values were lower, the species considered carcinogenic had higher concentrations. Mutagenesis predicted values of PAHs confirmed that carcinogenic species were predominantly detected by TA100, and the other PAHs by TA97a strains. Marked contaminant release to the river was observed, mainly in Ta010 at different periods. Mutagenicity and PAHs values in an internal stream, upstream from Ta010, showed a dispersion route of these agents. Thus, contamination in Ta010 and possible contribution to Ta006, after intervention, provides a warning regarding environmental quality in the region. Environ. Mol. Mutagen. 59:625-638, 2018. © 2018 Wiley Periodicals, Inc.

Miniaturization of the microsuspension Salmonella/microsome assay in agar microplates.

The Salmonella/microsome assay (Ames test) is the most widely used mutagenicity test for the evaluation of pure chemicals and environmental samples. There are several versions of protocols available in the literature, including those that reduce the amount of sample needed for testing with liquid and agar media. The microsuspension version of the Salmonella/microsome assay is more sensitive than the standard protocol. It is performed using 5-times concentrated bacteria and less sample and S9 mixture, but still uses conventional Petri dishes (90 × 15 mm). It has been extensively used for environmental sample testing, including in effect-directed analysis (EDA). The objective of this study was to miniaturize the microsuspension assay using 12-well microplates instead of the conventional plates. For validation of this miniaturization, thirteen mutagenic compounds were tested using three Salmonella strains that were selected based on their different spontaneous reversion frequencies (low, medium, and high). The conditions of the miniaturized procedure were made as similar as possible to the microsuspension protocol, using the same testing design, metabolic activation, and data interpretation, and the tests were conducted in parallel. The miniaturized plate assay (MPA) and microsuspension procedures provided similar sensitivities although MPA is less laborious and require less sample and reagents, thereby reducing overall costs. We conclude that the MPA is a promising tool and can be particularly suitable for environmental studies such as EDA or monitoring programs. Environ. Mol. Mutagen. 59:488-501, 2018. © 2018 Wiley Periodicals, Inc.

Biotransformation of bromhexine by Cunninghamella elegans, C. echinulata and C. blakesleeana

Abstract Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography–mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard – clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4 min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7 min when RLM were incubated with a CYP3A4 enzyme inhibitor – clarithromycin.

Characterization of ethoxyresorufin O-deethylase activity (EROD) in oyster Crassostrea brasiliana.

Cytochrome P450 family 1 (CYP1) is involved in polycyclic aromatic hydrocarbons (PAHs) biotransformation. PAHs can induce CYP1 protein expression and enzyme activity, the latter being usually quantified as 7-ethoxyresorufin O-deethylase activity (EROD). The aim of this study was to characterize EROD activity in the bivalve mollusk Crassostrea brasiliana. EROD activity was evaluated in cytosolic and microsomal fractions of gills, digestive gland and mantle of C. brasiliana. No EROD activity was detected in mantle, but it was present in microsomal fraction of gills and digestive gland with NADPH as coenzyme. Optima temperature and pH for EROD assay were 30°C and 7.4, respectively. EROD apparent Km (Kmapp) was 4.32µM for gills and 5.56µM for digestive gland. EROD Vmax was 337.3fmol·min-1·mg of protein-1 in gills and 297.7fmol·min-1·mg of protein-1 in digestive gland. Compared to other bivalves, a higher Kmapp and a lower Vmax was found in oyster which may suggest that oyster CYP1-like enzyme has lower affinity for substrate 7-ethoxyresorufin (7-ER) than those species. CYP1 inhibitor ellipticine (ELP) inhibited EROD activity in all tested concentrations in both tissues. The higher ELP concentration, 100µM, inhibited 78% of EROD activity in gills and 47% in digestive gland. The CYP1 inhibitors α-naphthoflavone and furafylline did not inhibited EROD activity in microsomes of both tissues. In conclusion, EROD activity can be used to determine CYP1-like activity in oysters and possibly a CYP1A1/A2-like enzyme is responsible for this catalysis.

Biotransformation of bromhexine by Cunninghamella elegans, C. echinulata and C. blakesleeana.

Fungi is a well-known model used to study drug metabolism and its production in in vitro condition. We aim to screen the most efficient strain of Cunninghamella sp. among C. elegans, C. echinulata and C. blakesleeana for bromhexine metabolites production. We characterized the metabolites produced using various analytical tools and compared them with mammalian metabolites in Rat liver microsomes (RLM). The metabolites were collected by two-stage fermentation of bromhexine with different strains of Cunninghamella sp. followed by extraction. Analysis was done by thin layer chromatography, high performance thin layer chromatography, Fourier transform infrared spectroscopy, high performance liquid chromatography and Liquid chromatography-mass spectrometry. The role of Cytochrome P3A4 (CYP3A4) enzymes in bromhexine metabolism was studied. Fungal incubates were spiked with reference standard - clarithromycin to confirm the role of CYP3A4 enzyme in bromhexine metabolism. Three metabolites appeared at 4.7, 5.5 and 6.4min retention time in HPLC. Metabolites produced by C. elegans and RLM were concluded to be similar based on their retention time, peak area and peak response of 30.05%, 21.06%, 1.34%, and 47.66% of three metabolites and bromhexine in HPLC. The role of CYP3A4 enzyme in metabolism of bromhexine and the presence of these enzymes in Cunninghamella species was confirmed due to absence of peaks at 4.7, 5.4 and 6.7min when RLM were incubated with a CYP3A4 enzyme inhibitor - clarithromycin.

Activation-dependent mitochondrial translocation of Foxp3 in human hepatocytes.

Foxp3 is considered to be the master regulator for the development and function of regulatory T cells (Treg). Recently Foxp3, has been detected in extra lymphoid tissue, and in hepatocytes and has been associated with hepatocellular carcinoma (HCC), although its role has not been defined. Since it is expected that there is a relationship between protein localization, activity and cellular function, the aim of this study was to explore the subcellular localization of Foxp3 in resting and stimulated human hepatocytes. Foxp3 expression was measured by flow cytometry, subcellular fractioning, and immunofluorescence, and this data was used to track the shuttling of Foxp3 in different subcellular compartments in hepatocytes (HepG2 cell line), stimulated by using the PKC activators (PMA), core and preS1/2 antigen from hepatitis B virus (HBV). Our data shows that besides the nuclear location, mitochondrial translocation was detected after stimulation with PMA and at to a lesser extent, with preS1/2. In addition, Foxp3 is localizes at outer mitochondrial membrane. These results suggest a non-canonical role of Foxp3 in the mitochondrial compartment in human hepatocytes, and opens a new field about their role in liver damages during HBV infection.

Cytochrome P450 expression in mouse brain: specific isoenzymes involved in Phase I metabolizing system of porphyrinogenic agents in both microsomes and mitochondria.

Brain cytochrome P450 (CYP) metabolizes a variety of drugs to produce their pharmacological effects within the brain. We have previously observed that porphyrinogenic agents altered CYP levels in brain. The aim of this work was to further study the involvement of mice brain mitochondrial and microsomal Phase I drug metabolizing system when porphyrinogenic agents, such as Enflurane, Isoflurane, allylisopropylacetamide, veronal, ethanol, and Griseofulvin were administered. To this end, CYP2E1, CYP2B1, and CYP3A4 expression were measured. NADPH cytochrome P450 reductase (CPR) expression was also determined. Western Blots were performed in microsomes and mitochondria of whole brain. Some of the drugs studied altered expression mainly in microsomes. Chronic Isoflurane augmented mitochondrial isoform, although this anaesthetic diminished microsomal expression. Ethanol and topical Griseofulvin affected expression in microsomes but not in mitochondria. CYP2E1 mitochondrial activity was induced by acute Enflurane; while the activity of the microsomal protein was enhanced in alcoholised animals. Ethanol also induced CYP2E1 expression in microsomes, although Isoflurane provoked opposite effects in mitochondria and microsomes. Expression of CPR was also induced. Several reports support an emergent role of CYP enzymes in the pathogenesis of neurological disorders, so CYP response in brain could be one of the multiples factors influencing porphyria acute attacks.

Subcellular localization and kinetic characterization of a gill (Na+, K+)-ATPase from the giant freshwater prawn Macrobrachium rosenbergii.

The stimulation by Mg(2+), Na(+), K(+), NH4 (+), and ATP of (Na(+), K(+))-ATPase activity in a gill microsomal fraction from the freshwater prawn Macrobrachium rosenbergii was examined. Immunofluorescence labeling revealed that the (Na(+), K(+))-ATPase α-subunit is distributed predominantly within the intralamellar septum, while Western blotting revealed a single α-subunit isoform of about 108 kDa M r. Under saturating Mg(2+), Na(+), and K(+) concentrations, the enzyme hydrolyzed ATP, obeying cooperative kinetics with V(M) = 115.0 ± 2.3 U mg(-1), K(0.5) = 0.10 ± 0.01 mmol L(-1). Stimulation by Na(+) (V(M) = 110.0 ± 3.3 U mg(-1), K(0.5) = 1.30 ± 0.03 mmol L(-1)), Mg(2+) (V(M) = 115.0 ± 4.6 U mg(-1), K(0.5) = 0.96 ± 0.03 mmol L(-1)), NH4 (+) (V(M) = 141.0 ± 5.6 U mg(-1), K(0.5) = 1.90 ± 0.04 mmol L(-1)), and K(+) (V(M) = 120.0 ± 2.4 U mg(-1), K(M) = 2.74 ± 0.08 mmol L(-1)) followed single saturation curves and, except for K(+), exhibited site-site interaction kinetics. Ouabain inhibited ATPase activity by around 73% with K(I) = 12.4 ± 1.3 mol L(-1). Complementary inhibition studies suggest the presence of F0F1-, Na(+)-, or K(+)-ATPases, but not V(H(+))- or Ca(2+)-ATPases, in the gill microsomal preparation. K(+) and NH4(+) synergistically stimulated enzyme activity (≈25%), suggesting that these ions bind to different sites on the molecule. We propose a mechanism for the stimulation by both NH4(+), and K(+) of the gill enzyme.

Comparative analysis of binding affinities to epidermal growth factor receptor of monoclonal antibodies nimotuzumab and cetuximab using different experimental animal models.

Although pharmaco/toxicological studies have always been conducted in pharmacologically relevant species in which the test material is pharmacologically active, the very specificity of many biopharmaceuticals could present challenges in the identification of a relevant species for pharmaco/toxicological studies. Alternative approaches may improve the predictive value of preclinical assessments of species-specific biopharmaceuticals. This could lead to improved decision-making, reduce the number of experimental animals by eliminating non-relevant studies, and decrease the time and cost involved in the drug development process. As an alternative to utilizing traditional animal models, this study investigated the activity of human EGF and the anti-EGF receptor monoclonal antibodies nimotuzumab and cetuximab using the placenta microsomal fraction of different experimental animals. Ligand-receptor binding curves were obtained from the different experimental animal models, and binding constants were calculated based on the Scatchard plots. The constants for human and monkey EGF receptor expressed on the placental extract showed a K(a)<10(-8)M, while rabbits, mice and rats showed a K(a)>10(-8)M. The K(a) values obtained from animal placentas show that Macaca fascicularis and Cercopitecus aethiops monkeys are relevant species for studying the pharmaco/toxicological properties of nimotuzumab and cetuximab.

Inhibition of rat mammary microsomal oxidation of ethanol to acetaldehyde by plant polyphenols.

We previously reported that the microsomal fraction from rat mammary tissue is able to oxidize ethanol to acetaldehyde, a mutagenic-carcinogenic metabolite, depending on the presence of NADPH and oxygen but not inhibited by carbon monoxide or other cytochrome P450 inhibitors. The process was strongly inhibited by diphenyleneiodonium, a known inhibitor of NADPH oxidase, and by nordihydroguaiaretic acid, an inhibitor of lipoxygenases. This led us to suggest that both enzymes could be involved. With the purpose of identifying natural compounds present in food with the ability to decrease the production of acetaldehyde in mammary tissue, in the present studies, several plant polyphenols having inhibitory effects on lipoxygenases and of antioxidant nature were tested as potential inhibitors of the rat mammary tissue microsomal pathway of ethanol oxidation. We included in the present screening study 32 polyphenols having ready availability and that were also tested against the rat mammary tissue cytosolic metabolism of ethanol to acetaldehyde. Several polyphenols were also able to inhibit the microsomal ethanol oxidation at concentrations as low was 10-50 µM. The results of these screening experiments suggest the potential of several plant polyphenols to prevent in vivo production and accumulation of acetaldehyde in mammary tissue.

Phase 1 and phase 2 metabolic activities along the small intestine in adult male sheep.

Metabolic activities of several xenobiotic metabolizing enzymes were evaluated in both hepatic and enteric subcellular fractions obtained from Corriedale × Merino crossbreed rams by using a biochemical approach. Microsomes obtained from the different segments of sheep small intestinal mucosa displayed cytochrome P450 (CYP)-dependent N-demethylations but not O-deethylase activities apparently occurred. CYP-mediated N-demethylations neither decreased nor increased along the small intestinal mucosa. Percentages of activity for erythromycin N-demethylase in the small intestine were between 29% (duodenum) and 45% (ileum) from that measured in the liver, whereas those determined for triacetyl-oleandomycin N-demethylation ranged between 10% (duodenum) and 15% (jejunum) of the same hepatic activity. Conversely, metabolic rates for aminopyrine and chlorfeniramine N-demethylations in the gut mucosa ranged between 3% and 7% compared to their respective hepatic enzyme activities. Sheep enteric mucosa also displayed metabolic reactions typically mediated by flavin-containing monooxygenases (FMOs), carbonyl reductases (CBRs), carboxylesterases (CES), glutathione S-transferases (GSTs) and uridine diphosphoglucuronyltransferases (UGTs). The FMO-mediated sulfoxidation of methimazole was 2.6-fold higher (P < 0.01) in the ileal compared to the duodenal mucosa. Percentages of activity for the microsomal CBR-dependent biotransformation of menadione were between 12% (ileum) and 19% (duodenum-jejunum) of the total activity measured in the liver; metabolic rates measured in duodenum and jejunum were ∼1.7-fold higher (P < 0.05) than that observed in the ileum. The microsomal CES activity (using p-nitrophenyl acetate as substrate) was around twofold higher in duodenum (P < 0.05) and jejunum (P < 0.01) in comparison to the ileum. Cytosolic GST-dependent activities (toward 1-chloro, 2,4-dinitrobenzene) were similar in the mucosa of duodenum, jejunum and ileum. Microsomal UGT activities (toward 1-naphthol) in duodenum and jejunum were three- and fourfold higher, respectively, compared to that measured in the ileum. The small intestinal mucosa may play a critical defensive role due to its involvement in the detoxification of toxic compounds prior to absorption. In addition, gut metabolic reactions may contribute to the presystemic metabolism of orally administered drugs. These results are a further contribution to the understanding of the relevance of the extra-hepatic metabolism of xenobiotics in ruminant species.

Chronic fluoxetine treatment induces structural plasticity and selective changes in glutamate receptor subunits in the rat cerebral cortex.

It has been postulated that chronic administration of antidepressant drugs induces delayed structural and molecular adaptations at glutamatergic forebrain synapses that might underlie mood improvement. To gain further insight into these changes in the cerebral cortex, rats were treated with fluoxetine (flx) for 4 weeks. These animals showed decreased anxiety and learned helplessness. N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit levels (NR1, NR2A, NR2B, GluR1 and GluR2) were analysed in the forebrain by both western blot of homogenates and immunohistochemistry. Both methods demonstrated an upregulation of NR2A, GluR1 and GluR2 that was especially significant in the retrosplenial granular b cortex (RSGb). However, when analysing subunit content in postsynaptic densities and synaptic membranes, we found increases of NR2A and GluR2 but not GluR1. Instead, GluR1 was augmented in a microsomal fraction containing intracellular membranes. NR1 and GluR2 were co-immunoprecipitated from postsynaptic densities and synaptic membranes. In the immunoprecipitates, NR2A was increased while GluR1 was decreased supporting a change in receptor stoichiometry. The changes of subunit levels were associated with an upregulation of dendritic spine density and of large, mushroom-type spines. These molecular and structural adaptations might be involved in neuronal network stabilization following long-term flx treatment.

Uptake and biochemical response to B[a]P in the sea anemone Anthopleura elegantissima.

In order to evaluate the effects of benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH) in the sea anemone Anthopleura elegantissima at the biochemical level. NADPH cytochrome P450 reductase and cytochrome P450 were assayed in A. elegantissima under toxicant. One toxicity test was performed with 75 organisms distributed in 5 groups. Animals in groups G1, G2 and G3 were given increasing B[a]P. Two groups named GC and GS were used as controls. GC was treated with seawater and GS was treated with acetone. After 72 h of exposure, enzymatic activities were determined. Microsomes were isolated from the columnar tissue and exposed in vitro to the toxicant in order to explore their ability to incorporate B[a]P. Basal activity for this enzyme was 1.69 +/- 0.18 (Mean +/- standard deviation) nmol cyt C red min(-1) mg(-1) and there was no significant effect in GS organisms compared to GC organisms. Significant increases were observed in NADPH cytochrome P450 reductase in G3 organisms. In this group, the enzyme activity was 3.53 +/- 0.40 nmol cyt C red min(-1) mg(-1). For cytochrome P450 content, a gradual increase was observed in organisms in groups G1 to G3. Basal content was 10.25 +/- 0.49 pmol mg(-1) microsomal protein. For G3 animals, P450 content was 27.51 +/- 0.32 pmol mg(-1) microsomal. For the test in vitro, it was found that microsomes isolated from G2 and G3 had the capacity to incorporate this substance when exposed to B[a]P at a level of 4 mu M in the surrounding medium. Spectrum recorded from 350 to 450 nm after a 40-min exposure for these groups showed significant difference from spectra obtained for microsomes in GC, GS and G1. It was concluded that the capacity to increase NADPH cytochrome P450 reductase activity as well as to increase NADPH cytochrome P450 reductase activity as well as to increase P450 content shows the ability of A. elegantissima to induce a mixed function oxidase activity in the presence of B[a]P.

Evaluation of the cytotoxicity, genotoxicity and mutagenicity of diphenyl ditelluride in several biological models.

Diphenyl ditelluride (DPDT) is a potential prototype for the development of novel biologically active molecules. Thus, it is important to evaluate the toxic effects of this compound. In the present study, we evaluated the cytotoxic, genotoxic and mutagenic properties of DPDT in Chinese hamster fibroblast (V79) cells, in strains of the yeast Saccharomyces cerevisiae both proficient and deficient in several DNA repair pathways and in Salmonella typhimurium. DPDT induced frameshift mutations in both S.typhimurium and a haploid wild-type strain of S.cerevisiae. Mutants of S.cerevisiae defective in base excision repair and recombinational repair were more sensitive to DPDT. The results of a lactate dehydrogenase leakage assay suggest that DPDT is cytotoxic to V79 cells. At cytotoxic concentrations, this compound increased thiobarbituric reactive species levels and decreased the glutathione:GSSH ratio in yeast and V79 cells. DPDT generated single- and double-strand DNA breaks in V79 cells, both with and without metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, an induction of oxidative DNA base damage was indicated by a modified comet assay using formamidopyrimidine DNA glycosylase and endonuclease III. Treatment with DPDT also induced micronucleus formation in V79 cells. Pre-incubation with N-acetylcysteine reduced DPDT's oxidative, genotoxic and mutagenic effects in yeast and V79 cells. Our results suggest that the toxic and mutagenic properties of DPDT may stem from its ability to disturb the redox balance of the cell, which leads to oxidative stress and the induction of DNA damage.

Metabolization of nifurtimox and benznidazole in cellular fractions of rat mammary tissue.

Two nitroheterocyclic drugs, nifurtimox (NFX) and benznidazole (BZ), used in the treatment of Chagas' disease have serious side effects attributed to their nitroreduction to reactive metabolites. Here, we report that these drugs reach the mammary tissue and there they could undergo in situ bioactivation. Both were detected in mammary tissue from female Sprague-Dawley rats after their intragastric administration. Only NFX was biotransformed by pure xanthine-oxidoreductase and from tissue cytosol. These activities were purine dependent and were inhibited by allopurinol. Also, only NFX was biotransformed by microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and was inhibited by carbon monoxide and partially by diphenyleneiodonium. NFX treatment produced significant decrease in protein sulfhydryl content after 1, 3 and 6 hours; no increases in protein carbonyl content at any time tested and significantly higher levels of lipid hydroperoxides at 3 and 6 hours; besides, ultrastructural observations after 24 hours showed significant differences in epithelial cells compared to control. These findings indicate that NFX might be more deleterious to mammary tissue than BZ and could correlate with early reports on its ability to promote rat mammary tissue toxicity.

Preparation of drug-loaded polymeric nanoparticles and evaluation of the antioxidant activity against lipid peroxidation.

Antioxidants have been found to be effective as prophylatic and therapeutic agents for different diseases such as diabetes, cancer, and neurodegenerative disorders. However, antioxidant substances can present poor solubility in water, inefficient permeability, gastrointestinal degradation, first-pass effect, and/or instability during storage. These drawbacks can be potentially circumvented by encapsulating the susceptible antioxidants. Polymeric nanoparticles (nanocapsules or nanospheres) have been used to improve the drug efficacy and release. Our group has shown that the in vitro antioxidant effect of melatonin against lipid peroxidation in microsomes and liposomes can be improved by encapsulation of the antioxidant drug in polymeric nanoparticles.

High resistance to lipid peroxidation of bird heart mitochondria and microsomes: Effects of mass and maximum lifespan.

The aim of this investigation was to study the connection between body size, fatty acid composition and sensitivity to lipid peroxidation of heart mitochondria and microsomes isolated from different size bird species: manon (Lonchura striata), quail (Coturnix coturnix var japonica), pigeon (Columba livia), duck (Cairina moschata) and goose (Anser anser), representing a 372-fold range of body mass. Fatty acids of total lipids were determined using gas chromatography and lipid peroxidation was evaluated with a chemiluminescence assay. The fatty acids present in heart organelles of the different bird species analyzed showed a small number of significant allometric trends. In mitochondria, from the individual fatty acid data, palmitoleic acid (C16:1 n7) increased allometrically (r=0.878), while stearic acid (C18:0) was negatively related to body mass (r=-0.903). Interestingly, none of the calculated fatty acid variables, the average fatty acid saturated, monounsaturated, polyunsaturated (PUFA) and the unsaturation index (UI) was established to show significant body size-related variations. In heart microsomes, the content of C18:0 was significantly smaller (r=-0.970) in the birds of greater size. A significant allometric increase in linoleic acid (C18:2 n6) (r=0.986), polyunsaturated (r=0.990) and UI (r=0.904) was observed in the larger birds. The total n6 fatty acids of heart mitochondria did not show significant differences when it was correlated to body mass of the birds. Moreover, positive allometric relationships were shown for microsomes. The total n3 fatty acids of heart mitochondria and microsomes indicated no significant correlations to body mass of birds. The C16:1 n7, C18:0 in mitochondria and C18:0, C18:2 n6, PUFA, UI and PUFA n6 in microsomes showed significant differences when they were correlated to maximum life span (MLSP) of birds. As light emission=chemiluminescence originated from heart organelles was not statistically significant, a lack of correlation between the sensitivity to lipid peroxidation and body size or maximum life span was obtained. These results indicate that the high resistance of bird hearts to the attack by free radicals is body size-independent and would be related to the preservation of cardiac function.