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.

Low level and sub-chronic exposure to methylmercury induces hypertension in rats: nitric oxide depletion and oxidative damage as possible mechanisms

Increased risk of hypertension after methylmercury (MeHg) exposure has been suggested. However, the underlying mechanisms are not well explored. In this paper, we have analyzed whether sub-chronic exposure to MeHg increases systolic blood pressure even at very low levels. In addition, we analyzed if the methylmercury-induced hypertension is associated with a decreased plasmatic nitric oxide levels and with a dysregulation of the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), as well as the levels of MDA and glutathione. For this study, Wistar rats were treated with methylmercury chloride (100 mu g/kg per day) or vehicle. Total treatment time was 100 days. Malondialdehyde (MDA) and circulating NOx levels and superoxide dismutase (SOD) and catalase (CAT) activities were determined in plasma, whereas glutathione levels were determined in erythrocytes. Our results show that long-term treatment at a low level of MeHg affected systolic blood pressure, increasing and reducing the levels of plasmatic MDA and NOx, respectively. However, the activity of SOD did not decrease in the MeHg exposed group when compared to the control. We found a negative correlation between plasmatic nitrite/nitrate (NOx) levels and systolic blood pressure (r = -0.67; P = 0.001), and a positive correlation between MDA and systolic blood pressure (r = 0.61; P = 0.03), thus suggesting increased inhibition of NO formation with the increase of hypertension. In conclusion, long-term exposure to a low dose of MeHg increases the systolic pressure and is associated, at least in part, with increased production of ROS as judged by increased production of malondialdehyde and depressed NO availability.

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.

Evaluation of protective effects of fish oil against oxidative damage in rats exposed to methylmercury

The present study evaluates a possible protective effect of fish oil against oxidative damage promoted by methylmercury (MeHg) in sub-chronically exposed rats. Reduced glutathione peroxidase and catalase enzyme activity and reduced glutathione levels were observed in MeHg-exposed animals compared to controls. Methylmercury exposure was also associated with DNA damage. Administration of fish oil to the methylmercury-exposed animals did not ameliorate enzyme activity or glutathione levels. On the other hand, a significant DNA protective effect (about 30%) was observed with fish oil treatment. There were no differences in the total mercury concentration in rat liver, kidney, heart or brain after MeHg administration with or without fish oil co-administration. Histopathological analyses showed a significant leukocyte infiltration in rat tissues after MeHg exposure, but this effect was significantly reduced after co-administration of fish oil. Taken together, our findings demonstrate oxidative damage even after low-level MeHg exposure and the protective effect of fish oil. This protection seems not to be related to antioxidant defenses or mercury re-distribution in rat tissues. It is probably due to the anti-inflammatory effects of fish oil. (C) 2010 Elsevier Inc. All rights reserved.

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.

A second Candida albicans resistance gene (Carg2) regulates tissue damage, but not fungal clearance, in sub-lethal murine systemic infection

The severity of systemic infection with the yeast Candida albicans has been shown to be under complex genetic control. C57/L mice carry an allele that is associated with an increase in tissue destruction when compared with C57BI/6 mice; however, the gene affects only the severity of tissue lesions, and does not influence the magnitude of the fungal burden in either kidney or brain. Studies in [C57/L x C57BI/6]F1 hybrid mice, and [C57/L x C57BI/6]F1 x C57/L backcross mice, demonstrated that the gene behaves as a simple Mendelian co-dominant. (C) 1998 Academic Press.

Anticlastogenic and antigenotoxic effects of selenomethionine on doxorubicin-induced damage in vitro in human lymphocytes

The use of antioxidants during chemotherapy has been shown to reduce or prevent the undesirable effects experienced by healthy cells. Micronutrient selenium is well known for its antioxidant properties; however, selenium exhibits a bimodal nature in that both its beneficial and toxic properties lie within a limited and narrow dose range. The present study investigated the possible protective effects of selenomethionine (SM) on the cytotoxicity, genotoxicity and clastogenicity of the chemotherapic doxorubicin (DXR), a key chemotherapic used in cancer treatment. Human peripheral lymphocytes were treated in vitro with varying concentrations of SM (0.25 mu M, 0.5 mu M, 1.0 mu M and 2.0 mu M), tested in combination with DXR (0.15 mu g/mL). SM alone was not cytotoxic and when combined with DXR treatment, reduced the DNA damage index significantly, the frequency of chromosomal aberrations, the number of aberrant metaphases and the frequency of apoptotic cells. The mechanism of chemoprotection of SM may be related to its antioxidant properties as well as its ability to interfere with DNA repair pathways. Therefore this study showed that SM is effective in reducing the genetic damage induced by the antitumoral agent DXR. (C) 2007 Elsevier Ltd. All rights reserved.

Genetic interactions of the Ashergillus nidulans atmA(ATM) homolog with different components of the DNA damage response pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for Delta atmA, Delta uvsB, Delta chkA, and Delta chkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APCI), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

Photodynamic therapy with aluminum-chloro-phtalocyanine induces necrosis and vascular damage in mice tongue tumors

In this paper we describe the efficacy of the liposomal-AlClPc (aluminum-chloro-phthalocyanine) formulation in PDT study against Ehrlich tumor cells proliferation in immunocompetent swiss mice tongue. Experiments were conduced in sixteen tumor induced mice that were divided in three control groups: (1) tumor without treatment; (2) tumor with 100 J/cm(2) laser (670 nm) irradiation; and (3) tumor with AlClPc peritumoral injection; and a PDT experimental group when tumors received AlClPc injection followed by tumor irradiation. Control groups present similar macroscopically and histological patterns after treatments, while PDT treatment induced 90% of Ehrlich tumor necrosis after 24 h of one single showing the efficacy of liposome-AlClPc (aluminum-chloro-phthalocyanine) mediated PDT application, on the treatment of oral cancer. (C) 2008 Elsevier B.V. All rights reserved.

Prolonged recruitment manoeuvre improves lung function with less ultrastructural damage in experimental mild acute lung injury

The effects of prolonged recruitment manoeuvre (PRM) were compared with sustained inflation (SI) in paraquat-induced mild acute lung injury (ALI) in rats. Twenty-four hours after ALI induction, rats were anesthetized and mechanically ventilated with VT = 6 ml/kg and positive end-expiratory pressure (PEEP) = 5 cmH(2)O for 1 h. SI was performed with an instantaneous pressure increase of 40 cmH(2)O that was sustained for 40 s, while PRM was done by a step-wise increase in positive inspiratory pressure (PIP) of 15-20-25 cmH(2)O above a PEEP of 15 cm H(2)O (maximal PIP = 40 cmH(2)O), with interposed periods of PIP = 10 cmH(2)O above a PEEP = 15 cmH(2)O. Lung static elastance and the amount of alveolar collapse were more reduced with PRM than SI, yielding improved oxygenation. Additionally, tumour necrosis factor-alpha, interleukin-6, interferon-gamma, and type III procollagen mRNA expressions in lung tissue and lung epithelial cell apoptosis decreased more in PRM. In conclusion, PRM improved lung function, with less damage to alveolar epithelium, resulting in reduced pulmonary injury. (C) 2009 Elsevier BLV. All rights reserved.

Attentional modulation of visual cortex activity following right parietal damage in humans

Pathological inattention following parietal damage causes perceptual impairments for visual stimuli in the contralesional hemifield. Here we used functional magnetic resonance imaging (fMRI) to examine visual cortex activity in parietal patients as they performed a spatial attention task. Righthemisphere patients and healthy controls viewed counterphasing checkerboards in which coloured targets appeared briefly within the contralesional and ipsilesional hemifields. In separate fMRI runs participants focused their attention covertiy on the left or right hemifield, or on both hemifields concurrentiy. They were required to detect coloured targets that appeared briefly within the attended hemifield(s), and to withhold responses to distractor stimuli. Neural activit}' was significantly attenuated in early visual areas within the damaged hemisphere. Crucially, although attention significantiy modulated early visual activit}' within the intact (left) hemisphere, there was relatively littie modulation of activity within the affected hemisphere. Our findings suggest that parietal lesions alter early cortical responses to contralesional visual inputs.

Urinary glycosaminoglycans as biomarker for urothelial injury: Is it possible to discriminate damage from recovery?

OBJECTIVES The glycosaminoglycan (GAG) layer is referred to as a bladder protective factor. We reproduced an experimental model of urothelial damage to assess GAG metabolism in the process of injury and recovery of the urothelium. METHODS Wistar female rats were bladder catheterized and instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24-hour urine samples were obtained. The urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycan were determined in all groups and in nonmanipulated rats (day 0). Additionally, sulfated-GAG synthesis was assessed by the incorporation of [S-35]-inorganic sulfate. The bladders were analyzed by histochemical staining for HA and immunofluorescence for heparin sulfate and syndecan-4. RESULTS Urinary HA and sulfated-GAG were elevated after PS injection (P <0.05). A greater concentration of [S-35] -labeled GAG in the PS group animals on the fifth day and, especially, on the seventh day represented increased GAG synthesis at these periods (P <0.05). Bladder sections from the PS group animals on day 1 showed a greater amount of HA in the urothelium. PS instillation damaged the urothelium layer of heparin sulfate and syndecan-4 seen in the control animals. On day 5, patchy areas of a restored layer were seen, and, on day 7, this layer had completely regenerated. CONCLUSIONS Urinary GAG cannot differentiate urothelial damage from recovery. Elevated levels of urinary GAG can result from either desquamation of the surface cell GAG layer or increased GAG synthesis to regenerate the damaged urothelium.