Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Purported Interactions of Amyloid-ß and Glucocorticoids in Cytotoxicity and Genotoxicity: Implications in Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized by the presence of aggregates of the amyloid-ß peptide (Aß) that are believed to be neurotoxic. One of the purposed damaging mechanisms of Aß is oxidative insult, which eventually could damage the cellular genome. Stress and associated increases in glucocorticoids (GCs) have been described as a risk factor for the development of AD, although the purported genotoxic effects of GCs have not been fully characterized. Therefore, it is possible to speculate about purported synergistic effects of GCs on the Aß-driven genotoxic damage. This in vitro study addresses the single and combined cyto/genotoxic effects of Aß and GCs in SH-SY5Y cells. Cytotoxicity was determined by the MTT assay, and the genotoxic effects were studied using the comet assay. A comet assay derivation allows for measuring the presence of the FPG-sensitive sites (mainly 8-oxoguanines) in the DNA, apart from the DNA strand breaks. Treatment with Aß (10 µM, 72 h) induced cytotoxicity (35% decrease in cell viability) and DNA strand breaks, but had no significant effect on oxidative DNA damage (FPG sites). Corticosterone showed no effect on cell viability, genotoxicity, or reparation processes. Corticosterone was unable to neither reverse nor potentiate Aß driven effects. The present results suggest the existence of alternative mechanisms for the Aß driven damage, not involving oxidative damage of DNA. In addition, could be suggested that the interaction between Aß and GCs in AD does not seem to involve DNA damage.

Validation of an antiviral assay method for quantifying IFN-α5 activity in macaque and human serum.

BACKGROUND: IFN-α5 has been demonstrated to induce stronger signaling and higher expression of antiviral genes than IFN-α2, which is the current treatment in chronic viral hepatitis. However, there is no specific and validated quantification method in order to conduct kinetic studies as part of the preclinical and clinical evaluation for regulatory purposes. RESULTS: A novel integration of an antiviral assay against the cytopathic effect of the encephalomyocarditis virus in HeLa cells with a very sensitive method for assay processing - the Vialight(®) Plus assay - is presented for IFN-α5 activity quantification. The bioassay has been validated in macaque and human serum and it has been demonstrated to be selective, precise and accurate. CONCLUSION: The validated bioassay meets suitable acceptance criteria for these types of biological assays.

Toxicity studies of poly(anhydride) nanoparticles as carriers for oral drug delivery.

PURPOSE: To evaluate the acute and subacute toxicity of poly(anhydride) nanoparticles as carriers for oral drug/antigen delivery. METHODS: Three types of poly(anhydride) nanoparticles were assayed: conventional (NP), nanoparticles containing 2-hydroxypropyl-ß-cyclodextrin (NP-HPCD) and nanoparticles coated with poly(ethylene glycol) 6000 (PEG-NP). Nanoparticles were prepared by a desolvation method and characterized in terms of size, zeta potential and morphology. For in vivo oral studies, acute and sub-acute toxicity studies were performed in rats in accordance to the OECD 425 and 407 guidelines respectively. Finally, biodistribution studies were carried out after radiolabelling nanoparticles with (99m)technetium. RESULTS: Nanoparticle formulations displayed a homogeneous size of about 180 nm and a negative zeta potential. The LD(50) for all the nanoparticles tested was established to be higher than 2000 mg/kg bw. In the sub-chronic oral toxicity studies at two different doses (30 and 300 mg/kg bw), no evident signs of toxicity were found. Lastly, biodistribution studies demonstrated that these carriers remained in the gut with no evidences of particle translocation or distribution to other organs. CONCLUSIONS: Poly(anhydride) nanoparticles (either conventional or modified with HPCD or PEG6000) showed no toxic effects, indicating that these carriers might be a safe strategy for oral delivery of therapeutics.

Kidney and liver distribution of ochratoxin A in male and female F344 rats.

The impact of age and gender on Ochratoxin A (OTA) distribution in kidney and liver were studied. OTA was quantified in kidney and liver of young and mature rats of both sexes. Data was fit simultaneously using the population approach with NONMEM program. Fed and fasted mature males showed a 30% decrease and an 11% increase in relative bioavailability, respectively, in comparison with the rest of the groups. The OTA concentrations reached in kidney and liver were very similar between both organs. The models that best fit to data were the ones that considered that distribution of OTA to kidney and liver occurs from the central compartment and that elimination occurs mainly from the liver compartment. The kinetic analysis revealed that both, the apparent volume of distribution of the central compartment (V/F) and the apparent volume of distribution of the liver and kidney compartments (V(L,K)/F) increased significantly with body weight. Thus, the sex differences observed in organs distribution are a reflection of the differences in relative bioavailability observed in adult males, as a consequence of the fed and fasted conditions and to the significant higher body weight of mature males which directly affected the V/F and V(L,K)/F.

DNA strand cleaving properties and hypoxia-selective cytotoxicity of 7-chloro-2-thienylcarbonyl-3-trifluoromethylquinoxaline 1,4-dioxide.

The heterocyclic N-oxide, 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine, 1), shows promising antitumor activity in preclinical studies, but there is a continuing need to explore new compounds in this general structural category. In the work described here, we examined the properties of 7-chloro-2-thienylcarbonyl-3-trifluoromethylquinoxaline 1,4-dioxide (9h). We find that 9h causes redox-activated, hypoxia-selective DNA cleavage that mirrors the lead compound, tirapazamine, in both mechanism and potency. Furthermore, we find that 9h displays hypoxia-selective cytotoxicity against human cancer cell lines.

Occurrence of ochratoxin A in Southern Spanish generous wines under the denomination of Origin "Jerez-Xérès-Sherry and 'manzanilla' Sanlúcar de Barrameda".

The mycotoxin ochratoxin A (OTA) has toxic effects in animals; the most relevant of them is nephrotoxicity. OTA has also been classified as a possible carcinogen for humans (group 2B) by the International Agency for Research on Cancer (IARC). Therefore, exposure to OTA through contaminated food can represent health impairment to humans. The maximum permitted level for this mycotoxin in wine is 2.0 µg/L. The presence of OTA in Spanish wines produced using the traditional methods under the Denomination of Origin "Jerez-Xérès-Sherry andmanzanilla Sanlúcar de Barrameda" was evaluated by a High performance Liquid Chromatography method with fluorescence detection and immunoaffinity column purification. A recovery of 95.4% and a limit of detection and quantification of 0.009 µg/L and 0.02 µg/L respectively, were achieved. In manzanilla, fino, amontillado and oloroso wine, the mean OTA values were 0.042, 0.044, 0.144, and 0.319 µg/L, respectively. These levels are not different from other data given in the reference literature on white wines, although fino and manzanilla wines have very low OTA levels.

Antiproliferative effect of flavomannin-6,6'-dimethylether from Tricholoma equestre on Caco-2 cells.

Several studies have been performed reporting antitumoral activity of different mushroom extracts. The current study reports the antiproliferative activity of flavomannin-6,6'-dimethylether obtained from a very common edible mushroom: Tricholoma equestre(L.)P.Kumm, and the characterization of its effects at molecular level. Concentrations causing 50% and 80% growth inhibition on human adenocarcinoma colorectal Caco-2 cells were determined (in microg/mL: IC(50)=96+/-3 after 24 h and 78+/-7 after 48 h, IC(80)=112+/-4 after 24 h and 90+/-3 after 48 h) by using MTT method. It was demonstrated that flavomannin-6,6'-dimethylether induced an arrest in G0/G1 phase of the cell cycle by flow cytometry analysis and an increase of p27 protein level by Western blot. Furthermore, this compound did not induce apoptosis by flow cytometry or DNA fragmentation by gel electrophoresis. Thus, it could be a promising agent due to its cytostatic effect against Caco-2 tumoral cells, and the absence of a genotoxic effect.

Cytotoxic, mutagenic and genotoxic effects of new anti-T. cruzi 5-phenylethenylbenzofuroxans. Contribution of phase I metabolites on the mutagenicity induction.

5-Phenylethenylbenzofuroxans have displayed in vitro and in vivo activity against Trypanosoma cruzi, the etiologic agent of American Trypanosomiasis. On the basis of benzofuroxans pre-clinical studies we evaluated the potential of six 5-phenylethenyl derivatives to induce cytotoxicity, mutagenicity and genotoxicity using different in vitro models. Cytotoxic effects were evaluated using a set of cells, mammal pre-monocytic macrophages, V-79 lung fibroblast from Chinese hamster, and colorectal adenocarcinoma Caco-2 cells, in the MTT viability assay. Mutagenicity was tested in the Ames assay using Salmonella typhimurium TA98 strain with and without metabolic activation by S9-rat liver homogenate. The genotoxic potentials were evaluated with the alkaline single cell gel electrophoresis (comet assay) in V-79 cells. In view of the Ames test results we study whether the main mammals' phase I metabolites, the corresponding o-nitroanilines, are involved in the mechanism of mutagenicity. These metabolites are produced by NADPH-dependent enzymes in cytosol and by xanthine oxidase and cytochrome P450 in microsomes from rat liver. Among them, the electronic property of phenyl substituent seems to be responsible for this effect. It could be pointed out that the equimolecular mixture of compounds 1 and 2 (5E- and 5Z-(2-phenylethenyl)benzofuroxan, respectively) could be used in further clinical studies as anti-T. cruzi drug.

New copper-based complexes with quinoxaline N1,N4-dioxide derivatives, potential antitumoral agents.

Taking into account our previous studies on cytotoxic metal compounds, new copper complexes with 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives as ligands were synthesized and characterized by different spectroscopic methods. The hypoxic selective cytotoxicity towards V79 cells and the superoxide dismutase-like activity of the complexes were determined and related to physicochemical properties of the compounds. In particular, the copper(II) complex with 3-amino-6-chloro-7-fluoroquinoxaline-2-carbonitrile N1,N4-dioxide showed cytotoxic selectivity in hypoxia being the most lipophilic compound of the series. On the contrary, the complex with 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide was cytotoxic but not selective and that with 3-amino-7-chloro-6-methoxy-quinoxaline-2-carbonitrile N1,N4-dioxide was not cytotoxic towards V79 cells neither in oxia nor in hypoxia in the assayed conditions. The sigmam Hammett substituent electronic descriptor was related to the effect in hypoxic conditions and the SOD-like activity was correlated to the effect in normoxia.

Comparative acute systemic toxicity of several quinoxaline 1,4-di-N-oxides in Wistar rats.

The acute toxicity of six quinoxaline 1,4-di-N-oxides has been evaluated in an attempt to determine: a) the feasibility of testing systemic toxicity of these compounds in a very preliminary phase without an adequate formulation for in vivo administration, b) the LD50 range and the toxic target organ of these compounds in order to have an approximation of the structure-activity relationship. Quinoxaline 1,4-di-N-oxides have shown a great variety of biological activities with potential therapeutic application in cancer, malaria, etc. Problems of toxicity hinder the progression of these compounds to clinical phases. The compounds dissolved in DMSO at their solubility limit were administered i.v. to female Wistar rats (8 weeks, 160 g), using an infusion pump (300 microL; 20 microl/min). Animals were observed for a period of 14 days. This dose of the vehicle (1.7 ml/kg) was well tolerated by the animals. The LD50 could not be determined, but a marked hypoactivity was induced by the treatment. The same compounds were also injected intraperitoneally, suspended in 0.01% Tween 80/0.09 % saline, and the animals that did not die were observed for a period of 14 days. The LD50 could be estimated to be in a range between 30 and 120 mg/kg, except for one of the compounds. A decrease in the evolution of body weight and hypoactivity were the principal symptoms induced by the treatment. In both assays, histopathologic study of heart, liver, kidney, lung, spleen and ovaries indicated that the target organs may be heart and spleen. In conclusion, the i.v. route is not adequate for estimating the LD50 of these compounds due to solubility problems; by i.p. route, the LD50 interval is between 30 and 120 mg/kg. The data did not permit the deduction of any specific structure-activity relationship.

Synthetic chalcones, flavanones, and flavones as antitumoral agents: biological evaluation and structure-activity relationships.

A series of synthetic chalcones, flavanones, and flavones has been synthesized and evaluated for antitumor activity against the human kidney carcinoma cells TK-10, human mammary adenocarcinoma cells MCF-7 (estrogen receptor-positive), and human colon adenocarcinoma cells HT-29. The most active series is the chalcone ones with the best results against TK-10 and HT-29 cells. Fourteen out of 53 analyzed compounds resulted very active against at least two of the studied tumoral cells. Alkaline single cell gel electrophoresis, comet assay, was performed as a study of the chromosomal aberrations promoted by the compounds on normal cells. Four active and two inactive chalcones were studied in the comet assay against normal human kidney cells (HK-2). A structure-activity relationship analysis of these compounds was performed and for 4- and 3,4-disubstituted derivatives a quantitative correlation was obtained in the case of anti-HT-29 activity.

Selective hypoxia-cytotoxins based on vanadyl complexes with 3-aminoquinoxaline-2-carbonitrile-N1,N4-dioxide derivatives.

A new vanadyl complex with the formula VO(L1)2, where L1=3-amino-6(7)-chloroquinoxaline-2-carbonitrile N(1), N(4)-dioxide, has been synthesized and characterized by elemental analyses, conductometry, fast atom bombardment mass spectroscopy (FAB-MS) and electronic, Fourier transform infrared (FTIR), Raman, nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopies. Results were compared with those previously reported for analogous vanadium complexes with other 3-aminoquinoxaline-2-carbonitrile N1,N4-dioxide derivatives as ligands. As an effort to develop novel metal-based selective hypoxia-cytotoxins and to improve bioavailability and pharmacological and toxicological properties of aminoquinoxaline carbonitrile N-dioxides bioreductive prodrugs, the new complex and VO(L)2 complexes, with L=3-amino-6(7)-bromoquinoxaline-2-carbonitrile N1,N4-dioxide (L2) and 3-amino-6(7)-methylquinoxaline-2-carbonitrile N1,N4-dioxide (L3), were subjected to cytotoxic evaluation in V79 cells in hypoxic and aerobic conditions. The complexes resulted in vitro more potent cytotoxins than the free ligands (i.e. potencies P(VO(L1)2)=3.0, P(L1)=9.0 microM) and Tirapazamine (P=30.0 microM) and showed excellent selective cytotoxicity in hypoxia, being no cytotoxic in oxia. In addition, the solubility in hydrophilic solvents resulted significantly higher for the vanadyl complexes than for the free ligands. These results could be indicative that complexation of the quinoxaline-2-carbonitrile N1,N4-dioxide derivatives with vanadium could improve their bioavailability. In addition, a new aspect of the series has been investigated. A detailed comparison of the electrochemical behavior of the free ligands and the complexes has been performed searching for a correlation between reduction potentials of the complexes and their activities and hypoxia selectivities.

Indazole N-oxide derivatives as antiprotozoal agents: synthesis, biological evaluation and mechanism of action studies.

A series of indazole N-oxide derivatives have been synthesized and their antichagasic and leishmanocidal properties studied. 3-Cyano-2-(4-iodophenyl)-2H-indazole N1-oxide exhibited interesting antichagasic activity on the two parasitic strains and the two parasitic stages evaluated. Furthermore, besides its trypanocidal activity, 3-cyano-2-(4-nitrophenyl)-2H-indazole N1-oxide showed leishmanocidal activity in the three parasitic strains evaluated. To gain insight into the mechanism of action, electrochemical behaviour, ESR experiment, inhibition of parasitic respiration and QSAR were performed.

Pediatric meningosarcoma: clinical evolution and genetic instability.

This report presents a female diagnosed with a frontoparietal interhemispheric meningosarcoma who, parallel to the clinical worsening, revealed an increase in the genetic instability (in bleomycin cultures) and the complexity of the karyotypes, with the acquisition of a clonal deletion of 17p13 (the locus for the TP53 tumor suppressor gene). The genetic findings of this patient suggest that the increased genetic instability could contribute to tumor progression as well as to treatment resistance, possibly in the background of the clonal deletion of TP53.

1, 2, 4-Triazine N-oxide derivatives: studies as potential hypoxic cytotoxins. Part II.

New 1, 2, 4-Triazine N-oxide and N, N'-dioxide derivatives were synthesized in order to obtain compounds as selective hypoxic cell cytotoxins. The starting heterocycles have been prepared using a standard microwave oven in a clean and good-yielded process. The reactivity of methyl-1, 2, 4-triazine N(4)-oxide and N(1), N(4)-dioxide with different electrophilic agents has been studied. The desired products were obtained only when iminium electrophiles were employed. The regioselectivity of this process has been studied by means of experimental and theoretical (at ab initio level) procedures. Theoretically was expected that the most stable intermediates where the benzylic-like anion from position 5. A fact which agreed with the experimental observed regioselectivity. The new compounds were tested for their cytotoxicity in oxia and hypoxia. Some of them proved to be less active in hypoxic conditions than tirapazamine, 3-amino-benzo[1, 2-e]1, 2, 4-triazine N(1), N(4)-dioxide. Derivative 19, 6-methyl-5-[2-(5-nitrothienyl)ethenyl)-1, 2, 4-triazine N(4)-oxide, was the most cytotoxic compound, but it was non-selective.

1, 2, 4-Triazine N-oxide derivatives: studies as potential hypoxic cytotoxins. Part III.

New 5-(2-arylethenyl)-1, 2, 4-triazine N-oxide and N, N'-dioxide derivatives were synthesized in order to obtain compounds as selective hypoxic cell cytotoxins. The desired products were obtained when the 5-methyl heterocycle reacted with the corresponding iminium electrophiles. The new compounds were tested for their cytotoxicity in oxia and hypoxia. Some of them proved to be less active in hypoxic conditions than Tirapazamine, 3-aminobenzo[1, 2-e]1, 2, 4-triazine N(1), N(4)-dioxide. Derivative 11, 6-methyl-5-[2-(5-nitrofuryl)ethenyl)-1, 2, 4-triazine N(4)-oxide, was the most cytotoxic compound, but it was non-selective. Some derivatives were studied as DNA-binding agents in oxic conditions showing poor affinity for this biomolecule. This result showed that the cytotoxic activity in oxia is DNA damage not dependent. Electrochemical and ESR spectroscopy studies were performed in order to determine the ability of compounds to produce radicals and the relation of these in the mechanism of cytotoxicity.