Characterization of new polymorphic functional markers for sugarcane

Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[02/01167-1]

An integrated molecular map of yellow passion fruit based on simultaneous maximum-likelihood estimation of linkage and linkage phases

The development of genetic maps for auto-incompatible species, such as the yellow passion fruit (Passiflora edulis Sims f.flavicarpa Deg.) is restricted due to the unfeasibility of obtaining traditional mapping populations based on inbred lines. For this reason, yellow passion fruit linkage maps were generally constructed using a strategy known as two-way pseudo-testeross, based on monoparental dominant markers segregating in a 1:1 fashion. Due to the lack of information from these markers in one of the parents, two individual (parental) maps were obtained. However, integration of these maps is essential, and biparental markers can be used for such an operation. The objective of our study was to construct an integrated molecular map for a full-sib population of yellow passion fruit combining different loci configuration generated from amplified fragment length polymorphisms (AFLPs) and microsatellite markers and using a novel approach based on simultaneous maximum-likelihood estimation of linkage and linkage phases, specially designed for outcrossing species. Of the total number of loci, approximate to 76%, 21%, 0.7%, and 2.3% did segregate in 1:1, 3:1, 1:2:1, and 1:1:1:1 ratios, respectively. Ten linkage groups (LGs) were established with a logarithm of the odds (LOD) score >= 5.0 assuming a recombination fraction : <= 0.35. On average, 24 markers were assigned per LG, representing a total map length of 1687 cM, with a marker density of 6.9 cM. No markers were placed as accessories on the map as was done with previously constructed individual maps.

Development and characterization of microsatellite markers for turmeric (Curcuma longa)

P>Curcuma longa L. is a sterile, triploid, vegetatively-propagated crop cultivated mainly in Southeast Asia. When dried rhizomes are ground, the resulting yellow powder is used by the food industry as a natural food dye. Moreover, many pharmacological compounds have broadened the commercial application of the crop. However, conventional breeding is difficult and hence, improvement has been limited to germplasm selection. To better utilize the germplasm collections and facilitate genotype selection, a total of 17 polymorphic microsatellite loci were developed using a CT/GT/CTT enriched genomic library. All microsatellites resulted in amplified PCR products, showing a banding pattern of 2-11 polymorphic bands per locus, enabling genotype discrimination. These results can be used in further studies aimed at characterizing C. longa genetic resource collections and also to improve breeding strategies.

Associations between glutathione peroxidase-1 Pro198Leu polymorphism, selenium status, and DNA damage levels in obese women after consumption of Brazil nuts

Objective: Alterations in selenium (Se) status may result in suboptimal amounts of selenoproteins, which have been associated with increased oxidative stress levels. The Pro198Leu polymorphism at the glutathione peroxidase-1 (GPx1) gene is supposed to be functional. The response of Se status, GPx activity, and levels of DNA damage to a Se supplementation trial between the genotypes related to that polymorphism was investigated. Methods: A randomized trial was conducted with 37 morbidly obese women. Participants consumed one Brazil nut, which provided approximately 290 mu g of Se a day, for 8 wk. Blood Se concentrations, erythrocyte GPx activity, and DNA damage levels were measured at baseline and at 8 wk. The results were compared by genotypes. Results: The genotype frequencies were 0.487, 0.378, and 0.135 for Pro/Pro (the wild-type genotype), Pro/Leu, and Leu/Leu, respectively. At baseline, 100% of the subjects were Se deficient, and after the supplementation, there was an improvement in plasma Se (P < 0.001 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), erythrocyte Se (P = 0.00 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), and GPx activity (P = 0.00 for Pro/Pro, P < 0.00001 for Pro/Leu, P < 0.001 for Leu/Leu). In addition, the Pro/Pro group showed a decrease in DNA damage after Brazil nut consumption compared with baseline (P < 0.005), and those levels were higher in Leu/Leu subjects compared with those with the wild-type genotype (P < 0.05). Conclusion: Consumption of one unit of Brazil nuts daily effectively increases Se status and increases GPx activity in obese women, regardless of GPx1 Pro198Leu polymorphism. However, the evaluated biomarkers showed distinct results in response to the supplementation when the polymorphism was considered. (c) 2011 Elsevier Inc. All rights reserved.

Analysis of chemokines and reactive oxygen species formation by rat and human neutrophils induced by microcystin-LA, -YR and -LR

Microcystins (MC), a family of heptapeptide toxins produced by some genera of Cyanobacteria, have potent hepatotoxicity and tumor-promoting activity. Leukocyte infiltration in the liver was observed in MC-induced acute intoxication. Although the mechanisms of hepatotoxicity are still unclear, neutrophil infiltration in the liver may play an important role in triggering toxic injury and tumor development. The present study reports the effects of MC-LA, MC-YR and MC-LR (1 and 1000 nM) on human and rat neutrophils functions in vitro. Cell viability, DNA fragmentation, mitochondrial membrane depolarization and intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Extracellular ROS content was measured by lucigenin-amplified chemiluminescence, and cytokines were determined by ELISA. We found that these MC increased interleukin-8 (IL-8), cytokine-induced neutrophil chemoattractant-2 alpha beta (CINC-2 alpha beta) and extracellular ROS levels in human and rat neutrophils. Apart from neutrophil presence during the inflammatory process of MC-induced injury, our results suggest that hepatic neutrophil accumulation is further increased by MC-induced neutrophil-derived chemokine. (c) 2008 Elsevier Ltd. All rights reserved.

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA ""alleles"" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Direct Detection of Mycobacterium bovis in Bovine Lymph Nodes by PCR

P>Thirty-five lymph node samples were taken from animals with macroscopic lesions consistent with Mycobacterium bovis infection. The animals were identified by postmortem examination in an abattoir in the northwestern region of state of Parana, Brazil. Twenty-two of the animals had previously been found to be tuberculin skin test positive. Tissue samples were decontaminated by Petroff`s method and processed for acid-fast bacilli staining, culture in Stonebrink and Lowenstein-Jensen media and DNA extraction. Lymph node DNA samples were amplified by PCR in the absence and presence (inhibitor controls) of DNA extracted from M. bovis culture. Mycobacterium bovis was identified in 14 (42.4%) lymph node samples by both PCR and by culture. The frequency of PCR-positive results (54.5%) was similar to that of culture-positive results (51.5%, P > 0.05). The percentage of PCR-positive lymph nodes increased from 39.4% (13/33) to 54.5% (18/33) when samples that were initially PCR-negative were reanalysed using 2.5 mu l DNA (two samples) and 1 : 2 diluted DNA (three samples). PCR sensitivity was affected by inhibitors and by the amount of DNA in the clinical samples. Our results indicate that direct detection of M. bovis in lymph nodes by PCR may be a fast and useful tool for bovine tuberculosis epidemic management in the region.

Mass Spectrometric Analysis of a Cyclic 7,8-Butanoguanine Adduct of N-Nitrosopyrrolidine: Comparison to Other N-Nitrosopyrrolidine Adducts in Rat Hepatic DNA

The well established rat hepatocarcinogen N-nitrosopytrolidine (NPYR, 1) requires metabolic activation to DNA adducts to express its carcinogenic activity. Among the NPYR-DNA adducts that have been identified, the cyclic 7,8-butanoguanine adduct 2-amino-6,7,8,9-tetrahydro-9-hydroxypyrido[2,1-f]purine-4(3H)-one (6) has been quantified using moderately sensitive methods, but its levels have never been compared to those of other DNA adducts of NPYR in rat hepatic DNA. Therefore, in this study, we developed a sensitive new LC-ESI-MS/MS-SRM method for the quantitation of adduct 6 and compared its levels to those of several other NPYR-DNA adducts formed by different mechanisms. The new method was shown to be accurate and precise, with good recoveries and low fmol detection limits. Rats were treated with NPYR by gavage at doses of 46, 92, or 184 mg/kg body weight and sacrificed 16 h later. Hepatic DNA was isolated and analyzed for NPYR-DNA adducts. Adduct 6 was by far the most prevalent, with levels ranging from about 900-3000 mu mol/mol Gua and responsive to dose. Levels of adducts formed from crotonaldehyde, a metabolite of NPYR, were about 0.2-0.9 mu mol/mol dGuo, while those of adducts resulting from reaction with DNA of tetrahydrofuranyl-like intermediates were in the range of 0.01-4 mu mol/mol deoxyribonucleoside. The results of this study demonstrate that, among typical NPYR-DNA adducts, adduct 6 is easily the most abundant in hepatic DNA. Since previous studies have shown that it can be detected in the urine of NPYR-treated rats, the results suggest that it is a potential candidate as a biomarker for assessing human exposure to and metabolic activation of NPYR.

Mate attenuates DNA damage and carcinogenesis induced by diethylnitrosamine and thermal injury in rat esophagus

Drinking hot mate has been associated with risk for esophageal cancer in South America. Thus. the aims of this study were to evaluate the modifying effects of mate intake on DNA damage and esophageal carcinogenesis induced by diethylnitrosamine (DEN) and thermal injury (TI) in male Wistar rats. At the initiation phase of carcinogenesis, rats were treated with DEN (8 x 80 mg/kg) and submitted to TI (water at 65 degrees C, 1 ml/rat, instilled into the esophagus). Concomitantly, the animals received mate (2.0% w/v) for 8 weeks. Samples of peripheral blood were collected 4 h after the last DEN application for DNA damage analysis. At weeks 8 and 20, samples from esophagus and liver were also collected for histological and immunohistochemical analysis. Mate significantly decreased DNA damage in leukocytes, cell proliferation rates in both esophagus and liver and the number of preneoplastic liver lesions from DEN/TI-treated animals at week 8. A significant lower incidence of esophageal papillomas and liver adenomas and tumor multiplicity was observed in the animals previously treated with mate at week 20. Thus, mate presented protective effects against DNA damage and esophageal and liver carcinogenesis induced by DEN. (C) 2009 Elsevier Ltd. All rights reserved.

Functional characterization of the putative Aspergillus nidulans DNA damage binding protein homologue DdbA

Nucleotide excision repair (NER) eliminates helix-distorting DNA base lesions. Seven XP-deficient genetic complementation groups (XPA to XPG) have already been identified in mammals, and their corresponding genes have been cloned. Hereditary defects in NER are associated with several diseases, including xeroderma pigmentosum (XP). UV-DDB (XPE) is formed by two associated subunits, DDB1 and DDB2. UV-DDB was identified biochemically as a protein factor that exhibits very strong and specific binding to ultraviolet (UV)-treated DNA. As a preliminary step to characterize the components of the NER in the filamentous fungus Aspergillus nidulans, here we identified a putative DDB1 homologue, DdbA. Deletion and expression analysis indicated that A. nidulans ddbA gene is involved in the DNA damage response, more specifically in the UV light response and 4-nitroquinoline oxide (4-NQO) sensitivity. Furthermore, the Delta ddbA strain cannot self-cross and expression analysis showed that ddbA can be induced by oxidative stress and is developmentally regulated in both asexual and sexual processes. The Delta ddbA mutation can genetically interact with uvsB(ATR), atmA(ATM), nkuA(KU70), H2AX-S129A (a replacement of the conserved serine in the C-terminal of H2AX with alanine), and cshB (a mutation in CSB Cockayne`s syndrome protein involved in the transcription-coupled repair subpathway of NER) mutations. Finally, to determine the DdbA cellular localization, we constructed a GFP:DdbA strain. In the presence and absence of DNA damage, DdbA was mostly detected in the nuclei, indicating that DdbA localizes to nuclei and its cellular localization is not affected by the cellular response to DNA damage induced by 4-NQO and UV light.

Protective properties of quercetin against DNA damage and oxidative stress induced by methylmercury in rats

Aim of the study was to find out whether consumption of quercetin (QC), an abundant flavonoid in the human diet, protects against DNA damage caused by exposure to organic mercury. Therefore, rats were treated orally with methylmercury (MeHg) and the flavonoid with doses that reflect the human exposure. The animals received MeHg (30 mu g/kg/bw/day), QC (0.5-50 mg/kg/bw/day), or combinations of both over 45 days. Subsequently, the glutathione levels (GSH) and the activities of glutathione peroxidase (GPx) and catalase (CAT) were determined, and DNA damage was measured in hepatocytes and peripheral leukocytes in single cell gel electrophoresis assays. MeHg decreased the concentration of GSH and the activity of GPx by 17 and 12%, respectively and caused DNA damage to liver and blood cells, while with QC no such effects were seen. When the flavonoid was given in combination with MeHg, the intermediate and the highest concentrations (5.0 and 50.0 mg/kg/bw/day) were found to cause DNA protection; DNA migration was reduced by 54 and 65% in the hepatocytes and by 27 and 36% in the leukocytes; furthermore, the reduction in GSH and GPx levels caused by MeHg treatment was restored. In summary, our results indicate that consumption of QC-rich foods may protect Hg-exposed humans against the adverse health effects of the metal.

Lutein improves antioxidant defense in vivo and protects against DNA damage and chromosome instability induced by cisplatin

Lutein (LT) is the second most prevalent carotenoid in human serum, and it is abundantly present in dark, leafy green vegetables. The objectives of this study were to evaluate the genotoxicity and mutagenicity of LT, and its protective effects in vivo against DNA damage and chromosome instability induced by cisplatin (cDDP). For this purpose, we used the comet assay and micronucleus (MN) test, and we evaluated the antioxidant effects of LT by determination of enzymatic (catalase-CAT) and non-enzymatic (reduced glutathione-GSH) activity. Mice were divided into six groups: cDDP, mineral oil (OM), LT groups and LT + cDDP groups. To perform the MN test on peripheral blood (PB) cells, blood samples were collected before the first treatment (T0), and 36 h (T1) and 14 days (T2) after the first treatment. To perform the comet assay, blood samples were collected 4 h after the first and the last treatment. Oxidative capacity was analyzed in total blood that was collected 24 h after the last treatment, when bone marrow (BM) sample was also collected for the MN test. No genotoxic or mutagenic effects of LT were observed for the doses evaluated. We did find that this carotenoid was able to reduce the formation of crosslinks and chromosome instability induced by cDDP. No differences were observed in CAT levels, and LT treatment increased GSH levels compared with a negative control group, reinforcing the role of this carotenoid as an antioxidant.

Evaluation of curcumin and cisplatin-induced DNA damage in PC12 cells by the alkaline comet assay

A very appropriate method for antigenotoxicity evaluation of antioxidants is the comet assay, since this analytical method detects initial DNA lesions that are still subject to repair; in other words, lesions that are very associated to damages resulting from the generation and subsequent action of reactive species. However, a solid evaluation should be developed in order to avoid inexact interpretations. In our study, besides the association of curcumin with cisplatin, curcumin and cisplatin agents were also tested separately. Classical genotoxic compounds, when tested by the comet assay, present an increase in the nucleoid tail; however, the cisplatin treatment has resulted in a decrease of DNA migration. This was an expected effect, as the cross-links between cisplatin and DNA decrease the DNA electrophoretic mobility. A similar effect was observed with the curcumin treatment, which decreased the nucleoid tail. Such effect was not expected and reinforced the necessity of including in the study, separate treatment groups with potentially antigenotoxic substances. The comet assay results have been analyzed using specific software for image analysis, as well as the classical visual analysis, and we have observed that the effect of decrease in DNA electrophoretic mobility was more easily observed when the data were analyzed by the software.

The azo dye Disperse Orange 1 induces DNA damage and cytotoxic effects but does not cause ecotoxic effects in Daphnia similis and Vibrio fischeri

Azo dyes constitute the largest group of colorants used in industry and can pass through municipal waste water plants nearly unchanged due to their resistance to aerobic treatment, which potentially exposes humans and local biota to adverse effects. Unfortunately, little is known about their environmental fate. Under anaerobic conditions, some azo dyes are cleaved by microorganisms forming potentially carcinogenic aromatic amines. In the present study, the azo dye Disperse Orange 1, widely used in textile dyeing, was tested using the comet, Salmonella/microsome mutagenicity, cell viability, Daphnia similis and Microtox (R) assays. The human hepatoma cell line (HepG2) was used in the comet assay and for cell viability. In the mutagenicity assay. Salmonella typhimurium strains with different levels of nitroreductase and o-acetyltransferase were used. The dye showed genotoxic effects with respect to HepG2 cells at concentrations of 0.2, 0.4, 1.0, 2.0 and 4.0 mu g/mL. In the mutagenicity assay, greater responses were obtained with the strains TA98 and YG1041, suggesting that this compound mainly induces frameshift mutations. Moreover, the mutagenicity was greatly enhanced with the strains overproducing nitroreductase and o-acetyltransferase, showing the importance of these enzymes in the mutagenicity of this dye. In addition, the compound induced apoptosis after 72 h in contact with the HepG2 cells. No toxic effects were observed for either D. similis or Vibrio fischeri. (C) 2011 Elsevier B.V. All rights reserved.

Bixin and lycopene modulation of free radical generation induced by cisplatin-DNA interaction

This work evaluated the Modulation of reactive oxygen species (ROS) produced by the cisplatin-human DNA interaction in a cell-free experimental model by the carotenoids bixin and lycopene extracted from, natural dietary Sources and purified through luminol- and Cypridina luciferin methoxy-analogue (MCLA)- enhanced chemiluminescence assays. The results showed that the ROS generation by DNA-cisplatin interaction was inhibited by both lycopene and bixin in a concentration-dependent manner. At a concentration of 100 mu M, lycopene and bixin inhibited Superoxide anion (O center dot(2)) generation at 90% and 82%, respectively, and the total ROS generation at 44% and 42%, respectively. The formation of significant amounts of isomers or degradation products of both carotenoids was not observed after ROS scavenging, as evaluated by high-performance liquid chromatography. Taken together, these results Suggest that carotenoids can be helpful to Modulate the oxidative stress found in cancer therapy with cisplatin. (c) 2008 Elsevier Ltd. All rights reserved.