A Synthetic Snake-Venom-Based Tripeptide Protects PC12 Cells from the Neurotoxicity of Acrolein by Improving Axonal Plasticity and Bioenergetics.

The synthetic peptide p-BTX-I is based on the native peptide (formed by glutamic acid, valine and tryptophan) isolated from Bothrops atrox venom. We have previously demonstrated its neuroprotective and neurotrophic properties in PC12 cells treated with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Now, we have investigated the neuroprotective effects and mechanisms of p-BTX-I against the toxicity of acrolein in PC12 cells. Studies have demonstrated that acrolein might play an important role in the etiology of Alzheimer's disease (AD), which is characterized by neuronal and synaptic loss. Our results showed that not only acrolein reduced cell differentiation and cell viability, but also altered the expression of markers of synaptic communication (synapsin I), energy metabolism (AMPK-α, Sirt I and glucose uptake), and cytoskeleton (ß-III-tubulin). Treatment with p-BTX-I increased the percentage of differentiation in cells treated with acrolein and significantly attenuated cell viability loss, besides counteracting the negative effects of acrolein on synapsin I, AMPK-α, Sirt I, glucose uptake, and ß-III-tubulin. Additionally, p-BTX-I alone increased the expression of apolipoprotein E (apoE) gene, associated with the proteolytic degradation of ß-amyloid peptide aggregates, a hallmark of AD. Taken together, these findings demonstrate that p-BTX-I protects against acrolein-induced neurotoxicity and might be a tool for the development of novel drugs for the treatment of neurodegenerative diseases.

Gold-Coated Superparamagnetic Iron Oxide Nanoparticles Attenuate Collagen-Induced Arthritis after Magnetic Targeting.

The aim of the study was to evaluate if gold-coated superparamagnetic iron oxide nanoparticles (AuSPION) magnetic-targeted to the arthritic articulation of collagen induced arthritis (CIA) rats are able to ameliorate rheumatoid arthritis without producing significant biological adverse effects in comparison to colloidal Au nanoparticles (AuC) and metotrexate (MTX). Male Wistar rats were divided into control; arthritic; AuSPION (150 µg kg-1); AuC (150 µg kg-1) and MTX (2.5 µg kg-1). Treatments were administered thrice every other day by the intraperitoneal route 15 min after all groups had a neodymium magnet coupled to the right ankle joint (kept for 1 h). Paw edema and body weight were measured weekly. Joint sections were evaluated by Haematoxylin & Eosin and immunohistochemistry (TNF-α, IL-1ß). Biomarkers of oxidative stress were used to evaluate toxicity. Among the evaluated treatments, AuSPION led to significant clinical improvements (decreased edema and infiltration by leukocytes as well as less positively immunostained cells for both TNF-α and IL-1ß in synovium) accompanied by a lack of toxicity as indicated by redox state and genotoxicity assays. Our results clearly indicate that the magnetic targeting of AuSPION suppresses joint edema and inflammation, cytokine expression as well as the redox imbalance, thereby contributing to an amelioration of arthritis severity in CIA rats. The results demonstrate for the first time the potentiality of AuSPION administration under a magnetic field as an attractive alternative for future treatments of rheumatic diseases.

Immunomodulatory actions and epigenetic alterations induced by proteases from Bothrops snake venoms in human immune cells.

The aim of this study was to evaluate the immunomodulatory effects of two toxins from Bothrops snake venoms (the P-I metalloprotease Batroxase and the thrombin-like serine protease Moojase) on human peripheral blood mononuclear cells (PBMC), also investigating changes in the expression of genes related to epigenetic alterations and their immunotherapeutic potential. After 24 h of PBMC stimulation, Batroxase (2 µg/mL) and Moojase (4 µg/mL) increased some cytokine levels (including IL-6 and IL-10), but did not promote cell death processes (apoptosis/necrosis) or alterations in the global DNA methylation levels. Gene expression experiments (RT-qPCR) showed that most of the genes with altered transcript levels encode enzymes that act on histones, such as acetyltransferases (HAT1), deacetylases (HDACs), methyltransferases (DOT1L) or demethylases (KDM5B), indicating that these toxins may alter gene regulation through epigenetic changes mainly related to histones and to methyl-CpG binding proteins (MECP2). Subsequently, the immunotherapeutic potential of these toxins was evaluated using in vitro cytotoxicity assays with NK cells and K562 leukemic cells. Both toxins were able to potentiate the NK cell cytotoxic effects against K562 tumor cells, and the effect of Batroxase was dependent on the concomitant stimulus with IL-2, whereas Moojase increased the NK cytotoxicity independently of IL-2. Thus, Batroxase and Moojase presented interesting immunomodulatory effects that could be explored for the development of new strategies in anticancer immunotherapies.

CR-LAAO causes genotoxic damage in HepG2 tumor cells by oxidative stress.

Snake venom L-amino acid oxidases (SV-LAAOs) are enzymes of great interest in research due to their many biological effects with therapeutic potential. CR-LAAO, an L-amino acid oxidase from Calloselasma rhodostoma snake venom, is a well described SV-LAAO with immunomodulatory, antiparasitic, microbicidal, and antitumor effects. In this study, we evaluated the genotoxic potential of this enzyme in human peripheral blood mononuclear cells (PBMC) and HepG2 tumor cells, as well as its interaction with these cells, its impact on the expression of DNA repair and antioxidant pathway genes, and reactive oxygen species (ROS)-induced intracellular production. Flow cytometry analysis of FITC-labelled CR-LAAO showed higher specificity of interaction with HepG2 cells than PBMC. Moreover, CR-LAAO significantly increased intracellular levels of ROS only in HepG2 tumor cells, as assessed by fluorescence. CR-LAAO also induced genotoxicity in HepG2 cells and PBMC after 4 h of stimulus, with DNA damages persisting in HepG2 cells after 24 h. To investigate the molecular basis underlying the genotoxicity attributed to CR-LAAO, we analyzed the expression profile (mRNA levels) of 44 genes involved in DNA repair and antioxidant pathways in HepG2 cells by RT2 Profiler polymerase chain reaction array. CR-LAAO altered the tumor cell expression of DNA repair genes, with two downregulated (XRCC4 and TOPBP1) and three upregulated (ERCC6, RAD52 and CDKN1) genes. In addition, two genes of the antioxidant pathway were upregulated (GPX3 and MPO), probably in an attempt to protect tumor cells from oxidative damage. In conclusion, our data suggest that CR-LAAO possesses higher binding affinity to HepG2 tumor cells than to PBMC, its genotoxic mechanism is possibly caused by the oxidative stress related to the production of H2O2, and is also capable of modulating genes related to the DNA repair system and antioxidant pathways.

Cocoplum (Chrysobalanus icaco L.) anthocyanins exert anti-inflammatory activity in human colon cancer and non-malignant colon cells.

Cocoplum (Chrysobalanus icaco L.) (CP) is an anthocyanin-rich fruit found in tropical areas around the globe. CP polyphenols are associated with beneficial effects on health, including reduction of inflammation and oxidative stress. Due to its functional properties, the consumption of this fruit may be beneficial in the promotion of human health and reduce the risk for chronic diseases. The objective of this study was to assess the anti-inflammatory and anti-proliferative activities of anthocyanins extracted from CP (1.0 to 20.0 µg ml-1 gallic acid equivalents [GAE]) in CCD-18Co non-malignant colonic fibroblasts and HT-29 colorectal adenocarcinoma cells. Tumor necrosis factor alpha (TNF-α, 10 ng mL-1) was used to induce inflammation in CCD-18Co cells. CP anthocyanins were identified and quantified using HPLC-ESI-MSn. The chemical analysis of CP extract identified delphinidin, cyanidin, petunidin and peonidin derivatives as major components. Cell proliferation was suppressed in HT-29 cells at 10.0 and 20.0 µg ml-1 GAE and this was accompanied by increased intracellular ROS production as well as decreased TNF-α, IL-1ß, IL-6, and NF-κB1 expressions at 20.0 µg ml-1 GAE. Within the same concentration range, there was no cytotoxic effect of CP anthocyanins in CCD-18Co cells and TNF-α-induced intracellular ROS-production was decreased by 17.3%. IL-1ß, IL-6 and TNF-α protein expressions were also reduced in TNF-α-treated CCD-18Co cells by CP anthocyanins at 20.0 µg ml-1 GAE. These results suggest that cocoplum anthocyanins possess cancer-cytotoxic and anti-inflammatory activities in both inflamed colon and colon cancer cells.

Low levels of methylmercury induce DNA damage in rats: protective effects of selenium.

In this study we examined the possible antigenotoxic effect of selenium (Se) in rats chronically exposed to low levels of methylmercury (MeHg) and the association between glutathione peroxidase (GSH-Px) activity and DNA lesions (via comet assay) in the same exposed animals. Rats were divided into six groups as follows: (Group I) received water; (Group II) received MeHg (100 mug/day); (Group III) received Se (2 mg/L drinking water); (Group IV) received Se (6 mg/L drinking water); (Group V) received MeHg (100 mug/day) and Se (2 mg/L drinking water); (Group VI) received MeHg (100 mug/day) and Se (6 mg/L drinking water). Total treatment time was 100 days. GSH-Px activity was determined spectrophotometrically and DNA damage was determined by comet assay. Mean GSH-Px activity in groups I, II, III, IV, V and VI were, respectively: 40.19 +/- 17.21; 23.63 +/- 6.04; 42.64 +/- 5.70; 38.50 +/- 7.15; 34.54 +/- 6.18 and 41.39 +/- 11.67 nmolNADPH/min/gHb. DNA damage was represented by a mean score from 0 to 300; the results for groups I, II, III, IV, V and VI were, respectively: 6.87 +/- 3.27; 124.12 +/- 13.74; 10.62 +/- 3.81; 13.25 +/- 1.76; 86.87 +/- 11.95 and 76.25 +/- 7.48. There was a significant inhibition of GSH-Px activity in group II compared with group I (P < 0.05). Groups V and VI did not show a difference in enzyme activity compared with groups III and IV, showing the possible protective action of Se. Comet assay presented a significant difference in DNA migration between group II and group I (P < 0.0001). Groups V and VI showed a significant reduction in MeHg-induced genotoxicity (P < 0.001) when compared with group II. A negative correlation (r = -0.559, P < 0.05) was found between GSH-Px activity and DNA lesion, showing that the greater the DNA damage, the lower the GSH-Px activity. Our findings demonstrated the oxidative and genotoxic properties of MeHg, even at low doses. Moreover, Se co-administration reestablished GSH-Px activity and reduced DNA damage.

Protection by Panax ginseng C.A. Meyer against the genotoxicity of doxorubicin in somatic cells of Drosophila melanogaster

Panax ginseng is one of the most widely prescribed herbal medicines for the treatment of cancer, diabetes, chronic inflammation, and neurodegenerative and cardiovascular diseases. Since the use of alternative medicines in combination with conventional therapy may increase the risk of unwanted interactions, we investigated the possible genotoxicity of a water-soluble form of the dry root of P. ginseng (2.5, 5.0 or 10.0 mg/mL) and its ability to protect against the genotoxicity of doxorubicin (DOX; 0.125 mg/mL) by using the Drosophila melanogaster wing somatic mutation and recombination test (SMART) with standard and high-bioactivation crosses of flies. Panax ginseng was not genotoxic at the concentrations tested, whereas DOX-induced genotoxicity in marker-heterozygous flies resulted mainly from mitotic recombination. At low concentrations, P. ginseng had antirecombinogenic activity that was independent of the concentration of extract used. Recombination events may promote cancer, but little is known about the ability of P. ginseng to inhibit such recombination or modulate DNA repair mechanisms.

Genotoxic studies in hypertensive and normotensive rats treated with amiodarone.

Amiodarone, a benzofuran derivative, is a very effective antiarrhythmic medication, but has potential to cause side effects. Although its cytotoxicity potential is very well-known, there are few reports about its genotoxicity effects. Since amiodarone has not been investigated in genotoxicity studies, and the spontaneously hypertensive rat (SHR) is a well-characterized model for hypertension, the aim of the present study was to perform cytogenetic analysis on chromosome aberrations in bone marrow cells of SHRs and normotensive Wistar-Kyoto rats (WKYs) that received oral amiodarone treatment for 4 weeks. Amiodarone activity was also monitored using electrocardiograms. The presence of bradycardia in amiodarone-treated rats confirmed that this drug was really active. Metaphase analysis on bone marrow cells showed that there were significant differences in total chromosomal damage and percentage abnormal metaphase between WKY and SHR negative controls. In the SHR negative control, the frequencies of basal chromosomal aberrations and abnormal metaphases were significantly higher (p<0.05). There were high numbers of chromosomal aberrations in all amiodarone-treated groups, compared with negative controls. In amiodarone-treated groups, the most frequent chromosomal aberration was chromatid breaks. More chromosomal aberrations were found in WKYs that received amiodarone, with a statistically significant difference in comparison with negative controls (p<0.05). However, in SHR rats there was no significant difference between the amiodarone and negative groups regarding chromosomal damage induction. These results showed that treatment with amiodarone was genotoxic in WKYs, but not in SHRs. Further studies are needed to confirm whether amiodarone is genotoxic or efficient and harmless, among humans undergoing therapy.

Cytogenetic analysis in lymphocytes from workers occupationally exposed to low levels of ionizing radiation.

The aim of the present study was to perform a cytogenetic analysis in peripheral lymphocytes of 36 individuals occupationally exposed to low levels of ionizing radiation, and compare the results with 36 controls, using the chromosomal aberrations test (CA), sensitivity to bleomycin and cytokinesis-blocked micronucleus assay (MN). The frequencies of CA/100 cells observed for the exposed workers were not significantly higher than in controls (P>0.05). The mean break/cell (b/c) for the controls and exposed workers was 0.59±0.39 and 0.57±0.29, respectively (P>0.01). The MN frequencies were significantly increased (P<0.01) in exposed workers (6.13±3.18) in comparison with controls (5.11±3.85). The mean MN was also statistically higher in the non-smoker exposed when compared with non-smoker controls, 5.80±3.09 and 5.15±4.08, respectively (P<0.01). The cytogenetic analysis of MN proved to be the most sensitive biological marker to assess the cellular response to low levels of irradiation.

Comparative study of multiple dosage of quercetin against cisplatin-induced nephrotoxicity and oxidative stress in rat kidneys.

Quercetin, a typical bioflavonoid ubiquitously present in fruits and vegetables, is considered to be helpful for human health. Cisplatin (cDDP) is one of the most active cytotoxic agents in the treatment of a wide range of solid tumors. The aim of this study was to investigate the possible effect of quercetin, a bioflavonoid with antioxidant potential, on cisplatin-induced nephrotoxicity and lipid peroxidation in rats. Gavage administrations of water, propylene glycol and quercetin (50 mg/kg) were made 24 and 1 h before saline or cDDP (5 mg/kg) ip injections and were repeated daily for 2, 5 or 20 subsequent days. Rats were killed 2, 5 and 20 days after ip injections, and blood and urine samples were collected to determine plasma creatinine, urine volume and osmolality. The kidneys were removed to determine the levels of thiobarbituric acid-reactive substances (TBARS) and for histological studies. Cisplatin increased lipid peroxidation, urine volume and plasma creatinine levels and decreased urine osmolality. Treatment with quercetin attenuated these alterations. These results demonstrate the role of oxidative stress and suggest a protective effect of quercetin on cisplatin-induced nephrotoxicity in adult Wistar rats.

Cytogenetic effects of mebendazole on in vivo and in vitro mammalian systems

Mebendazole (MBZ), (methyl-5 benzoyl benzimidazole-2-carbamate), a potent antihelmintic agent, was tested for clastogenicity in Wistar rat bone marrow cells (l3OO, 1750, 3500 and 7000 mg/kg b.w.) and for both clastogenicity and antimitotic potential in human peripheral blood lymphocytes in culture (5, 10 and 20 mug/ml culture medium). One-hundred metaphases/treatment were analyzed for induction of chromosome aberrations and 2000 cells/treatment were counted to determine the mitotic index. MBZ did not induce an increase in the frequency of chromosome aberrations, however it was effective in blocking the cell cycle at metaphase.