Iuri Marques de Oliveira

DNA damage in organs of mice treated acutely with patulin, a known mycotoxin

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0-3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3-138.4) and kidneys (31.5-99); decreased levels of GSH (hippocampus--from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus--from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus--DI from 127.5 to 39.8) and lipid peroxidation (hippocampus--20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.

Protective effects of three extracts from Antarctic plants against ultraviolet radiation in several biological models

The photoprotective effect of the methanolic extracts of three Antarctic plant species - Deschampsia antarctica Desv., Colobanthus quitensis (Kunth) Bartl., and Polytrichum juniperinum Hedw. against UV-induced DNA damage was investigated in hamster lung fibroblasts (V79 cells) and in a biomonitor organism Helix aspersas, using comet assay. The protective, mutagenic, and antimutagenic profiles of these extracts were also evaluated using haploid strains of the simple eukaryote Saccharomyces cerevisiae, and antioxidant activity were investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assay, as well as the hypoxanthine/xanthine oxidase assay. At the concentration range employed, the extracts were not cytotoxic or mutagenic to S. cerevisiae. In addition, the treatment with these extracts enhanced survival, and decreased induced reverse, frameshift, and forward mutations in a dose-response manner in all UVC doses employed. The plants extracts did not generate DNA strand breaks in V79 cells, and the treatment significantly decreased DNA damage induced by UVC. Extracts significantly decreased UVC-induced lipid peroxidation in V79 cells, showing a clear antioxidant property. Moreover, results of comet assay in V79 cells, employing Fpg, Endo III, and Endo V enzymes, demonstrated significant reduction of UVC-induced DNA damage after pre-incubation with these extracts. The treatment with all tested extracts were much less efficient against UVC-induced cytotoxicity in the yeast strain defective in photolyase as compared to the wild type strain, suggesting that this DNA repair pathway is stimulated by substances present in the extracts. All extracts showed a significant inhibitory effect in the hypoxanthine/xanthine oxidase assay, and they had the ability to scavenge DPPH. In H. aspersas, the treatment was able to protect against UVC-induced damage. In conclusion, D. antarctica, C. quitensis, and P. juniperinum extracts present photoprotective properties, which can be attributed to molecules, such as flavonoids and carotenoids, which act as UV-absorbing molecules and as antioxidants, as well as stimulate DNA-repair processes.

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 DPDTs 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.

NAP prevents acute cerebral oxidative stress and protects against long-term brain injury and cognitive impairment in a model of neonatal hypoxia–ischemia

Hypoxia-ischemia (HI) is a common cause of neonatal brain damage with lifelong morbidities in which current therapies are limited. In this study, we investigated the effect of neuropeptide NAP (NAPVSIPQ) on early cerebral oxidative stress, long-term neurological function and brain injury after neonatal HI. Seven-day-old rat pups were subjected to an HI model by applying a unilateral carotid artery occlusion and systemic hypoxia. The animals were randomly assigned to groups receiving an intraperitoneal injection of NAP (3 μg/g) or vehicle immediately (0 h) and 24 h after HI. Brain DNA damage, lipid peroxidation and reduced glutathione (GSH) content were determined 24 h after the last NAP injection. Cognitive impairment was assessed on postnatal day 60 using the spatial version of the Morris water maze learning task. Next, the animals were euthanized to assess the cerebral hemispheric volume using the Cavalieri principle associated with the counting point method. We observed that NAP prevented the acute HI-induced DNA and lipid membrane damage and also recovered the GSH levels in the injured hemisphere of the HI rat pups. Further, NAP was able to prevent impairments in learning and long-term spatial memory and to significantly reduce brain damage up to 7 weeks following the neonatal HI injury. Our findings demonstrate that NAP confers potent neuroprotection from acute brain oxidative stress, long-term cognitive impairment and brain lesions induced by neonatal HI through, at least in part, the modulation of the glutathione-mediated antioxidant system.

The role of two putative nitroreductases, Frm2p and Hbn1p, in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H2O2 and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright

NAP prevents hippocampal oxidative damage in neonatal rats subjected to hypoxia-induced seizures

Neonatal seizures in which hypoxic-ischemic encephalopathy is the main triggering etiology have a challenging diagnosis and limited efficacy of treatment. NAP (NAPVSIPQ) has shown extensive neuroprotective and antioxidant capacity in vitro and in vivo. To evaluate its neuroprotective role in the context of seizures associated with perinatal hypoxia, we assessed the integrity of DNA and lipid membranes as well as the redox status in the hippocampus of 10-day-old rats exposed to hypoxia-induced seizures (HS) with and without NAP treatment. Rats were exposed to transient global hypoxia (12 min exposure to 5-7% O2 was able to induce electrographic seizures) or room air with subsequent intraperitoneal NAP (0.03, 0.3 or 3 microg/g) or vehicle administration. Results showed elevated DNA damage immediately after the insult until 72 h post-HS, while oxidized bases were only detected 3, 6 and 24 h later. In addition, thiobarbituric acid reactive species peaked at 6 h in parallel with decreased levels of reduced glutathione between 3 and 72 h post-HS insult. Our findings expand on the knowledge about the time course of HS-induced oxidative damage and demonstrate for the first time that a single NAP injection dose-dependently prevents HS-induced oxidative damage to DNA and lipid membranes, in correlation with modulation of the glutathione system. Hence, NAP may represent a promising therapeutic strategy for avoiding HS-induced oxidative damage.

MutY-glycosylase: An overview on mutagenesis and activities beyond the GO system

MutY is a glycosylase known for its role in DNA base excision repair (BER). It is critically important in the prevention of DNA mutations derived from 7,8-dihydro-8-oxoguanine (8-oxoG), which are the major lesions resulting from guanine oxidation. MutY has been described as a DNA repair enzyme in the GO system responsible for removing adenine residues misincorporated in 8-oxoG:A mispairs, avoiding G:C to T:A mutations. Further studies have shown that this enzyme binds to other mispairs, interacts with several enzymes, avoids different transversions/transitions in DNA, and is involved in different repair pathways. Additional activities have been reported for MutY, such as the repair of replication errors in newly synthesized DNA strands through its glycosylase activity. Moreover, MutY is a highly conserved enzyme present in several prokaryotic and eukaryotic organisms. MutY defects are associated with a hereditary colorectal cancer syndrome termed MUTYH-associated polyposis (MAP). Here, we have reviewed the roles of MutY in the repair of mispaired bases in DNA as well as its activities beyond the GO system.

Genetic toxicity of three symmetrical diselenides in yeast

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organoselenium (OS) compounds. The aim of the present study was to investigate the mutagenic effects of three symmetrical derivatives from the prototype DPDS (p-chloride-phenyl-diselenide, p-methyl-phenyl-diselenide and p-methoxy-phenyl-diselenide) in yeast. In summary, the cellular effects of the three OS compounds studied in this work appear to be variable according to the substituent group in the aromatic ring and are concentration-dependent. The presence of methoxyl group in the aromatic ring of DPDS structure results in elevation of the cytotoxic and mutagenic potential while the introduction of a methyl group increases the cytotoxic effect.

Transplantation of bone marrow mononuclear cells prolongs survival, delays disease onset and progression and mitigates neuronal loss in pre-symptomatic, but not symptomatic ALS mice

Cell-based therapy provides a novel strategy to restore lost neurons or modulate the degenerating microenvironment in amyotrophic lateral sclerosis (ALS). This study verified the therapeutic potential of bone marrow mononuclear cells (BMMCs) in SOD1G93A mice. BMMCs were obtained from enhanced green fluorescent protein (EGFP) transgenic C57BL/6 mice (EGFPBMMCs) or from SOD1G93A transgenic mice (mSOD1BMMCs) and given to mice at the pre-symptomatic or late symptomatic stage. Survival, body weight and motor performance data were recorded. DNA integrity was evaluated using the alkaline comet assay. The spinal cords were collected to assess motoneuron preservation and cell migration. EGFPBMMCs and mSOD1BMMCs transplantation to pre-symptomatic SOD1G93A mice prolonged survival and delayed disease progression. The effects were more significant for the EGFPBMMC-transplanted mice. In late symptomatic mice, EGFPBMMCs promoted a discrete increase in survival, without other clinical improvements. DNA from EGFPBMMCs and mSOD1BMMCs was found in the spinal cords of transplanted animals. DNA damage was not modified by BMMCs in any of the studied groups. Despite positive behavioral effects observed in our study, the limited results we observed for late transplanted mice call for caution before clinical application of BMMCs in ALS.

Antigenotoxic and antimutagenic effects of diphenyl ditelluride against several known mutagens in Chinese hamster lung fibroblasts

The present study evaluates antigenotoxic and antimutagenic properties of diphenyl ditelluride (DPDT) against several known mutagens in Chinese hamster lung fibroblasts (V79 cells). DPDT was not cytotoxic and genotoxic at concentrations ranging from 0.01 to 0.1 μM. The pre-treatment for 2h with this organotellurium compound at non-cytotoxic dose range (0.01, 0.05 and 0.1 μM) increased cell survival after challenge with hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BOOH), methylmethanesulphonate (MMS) or ultraviolet (UV)C radiation. In addition, the pre-treatment with DPDT decreased the DNA damage and Formamidopyrimidine DNA-glycosylase (Fpg)- and Endonuclease III (Endo III) sensitive sites induction by the studied genotoxic agents, as verified by comet assay and modified comet assay, respectively. The pre-treatment also reduced micronucleus frequency, revealing the protector effect of DPDT against MMS and UVC-induced mutagenesis. Our results demonstrate that DPDT-treated cells at concentration range of 0.01-0.1 μM do not change thiobarbituric acid reactive species (TBARS) levels and ROS generation. Moreover, DPDT pre-treatment at this concentration range decreases the ROS induction by H2O2 and t-BOOH treatment indicating antioxidant potential. On the other hand, concentrations higher than 0.1 μM increase TBARS formation and inhibited superoxide dismutase (SOD) activity, suggesting pro-oxidative effect of this compound at high concentrations. Our results suggest that DPDT presents antigenotoxic and antimutagenic properties at concentration range of 0.01-0.1 μM. The protection effect could be attributed to antioxidant capacity of DPDT at this concentration range in V79 cells.