The carotenoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in human cells.

The role of dietary antioxidants in human health remains controversial. Fruits and vegetables in the diet are associated with lower rates of chronic disease, and this is often attributed to their content of antioxidants, and a resulting protection against oxidative stress. However, large-scale human trials with antioxidant supplements have shown, if anything, an increase in mortality. We have investigated the biological properties of beta-cryptoxanthin, a common carotenoid, in cell culture model systems, using the comet assay to measure DNA damage. At low concentrations, close to those found in plasma, beta-cryptoxanthin does not itself cause damage, but protects transformed human cells (HeLa and Caco-2) from damage induced by H(2)O(2) or by visible light in the presence of a photosensitizer. In addition, it has a striking effect on DNA repair, measured in different ways. Incubation of H(2)O(2)-treated cells with beta-cryptoxanthin led to a doubling of the rate of rejoining of strand breaks and had a similar effect on the rate of removal of oxidized purines by base excision repair. The latter effect was confirmed with an in vitro assay: cells were incubated with or without beta-cryptoxanthin before preparing an extract, which was then incubated with substrate DNA containing 8-oxo-7,8-dihydroguanine; incision was more rapid with the extract prepared from carotenoid-preincubated cells. No significant increases were seen in protein content of human 8-oxoguanine DNA glycosylase 1 or apurinic endonuclease 1. The apparent cancer-preventive effects of dietary carotenoids may depend on the enhancement of DNA repair as well as antioxidant protection against damage.

Antitumoral effect of phenazine N5,N10-dioxide derivatives on Caco-2 cells.

We studied the in vitro antitumoral effect of a series of phenazine di- N-oxide derivatives, named 2-chloroacetylamino-7(8)-nitrophenazine N(5), N(10)-dioxide (1), 2-amino-7(8)-(1,3-dioxol-2-yl)phenazine N(5), N(10)-dioxide (2), 2-chloroacetylamino-7(8)-(1,3-dioxol-2-yl)phenazine N(5), N(10)-dioxide (3), and 2-amino-7(8)-methoxyphenazine N(5), N(10)-dioxide (4), on Caco-2 cells. These phenazine N(5), N(10)-dioxide derivatives belong to our in-house chemical library. The products were selected according to their stereoelectronic characteristics and taking into account their differential cytotoxicity against V79 cells. Human colorectal adenocarcinoma cell line Caco-2 was used to study the cell growth inhibition capacity of these compounds, their capacity of altering the cell cycle and possible induction of apoptosis, DNA fragmentation, and genotoxic damage. The IC 50 after 24 h of incubation was lower for 1, 2, and 3 (4.8, 46.8, and 8.2 microM, respectively) than for 4 (474.7 microM). Compound 1 induced arrest in the G2/M phase at 24 and 48 h of treatment and apoptosis at the highest doses at 24 h of treatment. These facts were corroborated with caspase 3, caspase 9, and cytochrome c activation and DNA fragmentation at 24 h of treatment. The derivatives studied induced neither significant single strand breaks nor oxidative damage at the different studied times. We concluded that among the series of N(5), N(10)-dioxide phenazine derivatives analyzed, 1, which contains a nitro moiety and a chloroacetamide group, is the most promising as an antitumoral compound.

Cytotoxic activity of fucoxanthin, alone and in combination with the cancer drugs imatinib and doxorubicin, in CML cell lines.

In the present study, we evaluate the in vitro cytotoxicity of fucoxanthin (Fx) on two human leukemia cell lines, K562 and TK6, alone and in combination with the conventional anticancer drugs imatinib (Imat) and doxorubicin (Dox). For the purpose, we assessed the cytotoxic and proliferation effects by cell count, induction of DNA damage by comet assay, and cell death by nuclear condensation, annexin V staining, coupled with propidium iodide uptake, and protein expression of Bax, caspase-3, and Bcl-2 (western blot). Our results show that Imat increased cytotoxicity in TK6 cells and inhibited proliferation in K562 cells, while Dox decreased cell viability and proliferation in both cell lines. Fx per se increased cytotoxicity against K562 cells and decreased cell proliferation of K562 and TK6 cells. The effects were confirmed by phase contrast microscopy. However, the antiproliferative effects are not explained by induction of DNA damage or cell death. In co-incubation, Fx increased antiproliferative effects of both drugs in the cell lines tested, however no differences where observed relative to Fx alone. This study unveiled in vitro cytotoxicity of Fx by inhibition of cell proliferation in both cell lines. Further studies are needed to elucidate the signal transduction pathways and molecular targets involved in that effect.

A quinoxaline 1,4-di-N-oxide derivative induces DNA oxidative damage not attenuated by vitamin C and E treatment.

Some anticancer compounds are pro-drugs which give rise to toxic species through enzymatic reduction. The quinoxaline-di-N-oxide derivative Q-85 HCl (7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride) is a bioreductive compound selectively toxic in hypoxia. Due to the possibility of secondary tumors the study of the genotoxic capability of antitumoral drugs is very important. The aim of this study was to assess the ability of Q-85 HCl to produce reactive oxygen species (ROS) and oxidative DNA damage in Caco-2 cells, both in hypoxia and in well-oxygenated conditions. Secondly, we attempted to evaluate the effect of vitamins C and E under hypoxic and normoxic conditions, in order to determine if these antioxidant substances modify Q-85 HCl effect in hypoxic cells or possibly exert a protective action in normal cells. Caco-2 cells were treated with Q-85 HCl for 2h, at high concentrations in normoxia (0.1-5 microM) and at low concentrations in hypoxia (0.002-0.1 microM). In normoxia, a dose-related significant increase in intracellular ROS level was evident; in hypoxia all the concentrations produced very high level of ROS. Just after the treatment and 24h later, oxidative DNA damage was evaluated by the modified comet assay after post-digestion of the cells with formamidopyrimidine-DNA glycosylase (FPG) and endonuclease III (Endo III). Q-85 HCl treatment evoked a significant dose-dependent increase in the total comet score of the cells both in hypoxia and normoxia, indicating that this compound or some metabolite is able to oxidize purine and pyrimidine bases. After 24h DNA damage caused by the compound was completely repaired with only one exception: cells treated with the highest concentration of Q-85 HCl in hypoxia and post-digested with FPG. Vitamin C (5-100 microM) and vitamin E (500-400 microM) did not have a pro-oxidant effect in Caco-2 cells. Treatment of cells with vitamin C (10 microM) or vitamin E (100 microM) did not significantly reduce oxidative DNA damage in hypoxia and normoxia. In conclusion, the use of these vitamins would not hinder toxicity against hypoxic cells, but a protective effect in normoxic cells was not evident.

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.

Phenazine 5,10-dioxide derivatives as hypoxic selective cytotoxins.

The synthesis and evaluation as hypoxic selective cytotoxins of 2-amino- or 2-hydroxyphenazine 5,10-dioxide derivatives and reduced analogues are reported. In vitro cytotoxicities on V79 cells under hypoxic and aerobic conditions were determined. Some derivatives, such as 7(8)-bromo-2-hydroxyphenazine 5,10-dioxide, showed selective toxicity toward hypoxic cells and along with derivatives 7(8)-bromo-2-aminophenazine 5,10-dioxide and 7(8)-chloro-2-aminophenazine 5,10-dioxide behave as hypoxic trigger cytotoxins. These compounds represent interesting leads for further chemical modifications and biological studies.

Vanadium(V) complexes with salicylaldehyde semicarbazone derivatives bearing in vitro anti-tumor activity toward kidney tumor cells (TK-10): crystal structure of [VVO2(5-bromosalicylaldehyde semicarbazone)].

As a contribution to the development of novel vanadium complexes with pharmacologically interesting moieties, new dioxovanadium(V) semicarbazone complexes with the formula cis-VO(2)L, where L=5-bromosalicylaldehyde semicarbazone and 2-hydroxynaphtalen-1-carboxaldehyde semicarbazone have been synthesized and characterized by (1)H and (13)C NMR, Raman and FTIR spectroscopies. Results were compared with those previously reported for other three analogous complexes of this series. The five complexes were tested in three different human tumor cell lines for bioactivity as potential anti-tumor agents, showing selective cytotoxicity on TK-10 cell line. Results showed that structural modifications on the semicarbazone moiety could have a significant effect on the anti-tumor activity of the vanadium complexes. In addition, the electrochemical behavior of all the complexes was studied. No apparent correlation could be demonstrated between reduction potentials of the complexes and their anti-tumor activities. The molecular structure of the novel [V(V)O(2)(5-bromosalicylaldehyde semicarbazone)] complex was solved by X-ray diffraction methods. The vanadium atom shows a distorted square pyramidal coordination sphere. The (VO(2))(+) cation is coordinated to a nearly planar (L)(-) anion acting as a tridentate ligand through both oxygen and one nitrogen atoms.

Ruthenium (II) nitrofurylsemicarbazone complexes: new DNA binding agents.

Complexes of the type [Ru(II)Cl(2)(DMSO)(2)L], where L are 5-nitrofurylsemicarbazone derivatives, were prepared in an effort to combine the potential anti-tumor activity of the metal and the free ligands. The new complexes are excellent DNA binding agents for calf thymus DNA. So, their in vitro anti-tumor activity was tested in cellular models and the complexes were found to be non-cytotoxic on the tumor cell lines assayed, neither in aerobic conditions nor in the bio-reductive assay performed. Redox behavior, lipophilicity and stability were studied in order to explain the lack of cellular cytotoxic effects. The complexes resulted 10-100 times more hydrophilic than the parent ligands thus the bio-activity of these compounds would be compromised by their inadequate lipophilic properties.

DNA damage in lens epithelium of cataract patients in vivo and ex vivo.

PURPOSE: DNA damage has been described in the human cataractous lens epithelium, and oxidative stress generated by UV radiation and endogenous metabolic processes has been suggested to play a significant role in the pathogenesis of cataract. In this study, the aim was to explore the quality and relative quantity of DNA damage in lens epithelium of cataract patients in vivo and after incubation in a cell culture system. METHODS: Capsulotomy specimens were analysed, before and after 1 week of ex vivo cultivation, using the comet assay to measure DNA strand breaks, oxidized purine and pyrimidine bases and UV-induced cyclobutane pyrimidine dimers. RESULTS: DNA strand breaks were barely detectable, oxidized pyrimidines and pyrimidine dimers were present at low levels, whereas there was a relatively high level of oxidized purines, which further increased after cultivation. CONCLUSION: The observed levels of oxidized purines in cataractous lens epithelium may support a theory consistent with light damage and oxidative stress as mediators of molecular damage to the human lens epithelium. Damage commonly associated with UV-B irradiation was relatively low. The levels of oxidized purines increased further in a commonly used culture system. This is of interest considering the importance and versatility of ex vivo systems in studies exploring the pathogenesis of cataract.

Novel quinoxaline 1,4-di-N-oxide derivatives as new potential antichagasic agents.

As a continuation of our research and with the aim of obtaining new agents against Chagas disease, an extremely neglected disease which threatens 100 million people, eighteen new quinoxaline 1,4-di-N-oxide derivatives have been synthesized following the Beirut reaction. The synthesis of the new derivatives was optimized through the use of a new and more efficient microwave-assisted organic synthetic method. The new derivatives showed excellent in vitro biological activity against Trypanosoma cruzi. Compound 17, which was substituted with fluoro groups at the 6- and 7-positions of the quinoxaline ring, was the most active and selective in the cytotoxicity assay. The electrochemical study showed that the most active compounds, which were substituted by electron-withdrawing groups, possessed a greater ease of reduction of the N-oxide groups.

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.

Oxidative DNA damage induced by Ochratoxin A in the HK-2 human kidney cell line: evidence of the relationship with cytotoxicity.

Ochratoxin A (OTA) is a mycotoxin produced by species of the genera Aspergillus and Penicillium. The kidneys are the target organ of this mycotoxin and it is considered a potent renal carcinogen in male rats. The mechanisms of its genotoxicity and carcinogenicity have been studied thoroughly, but controversial results have been published. The aim of this study was to evaluate the ability of OTA to produce single-strand DNA breaks and oxidative DNA damage in the human renal proximal tubular epithelial cell line (HK-2), due to the fact that there is no study on human kidney cells as the toxic target. In addition, we attempted to determine if biotransformation processes mediate OTA genotoxicity. Therefore, single-cell gel electrophoresis assay (comet assay) was performed after 3h- and 6h-treatments using different OTA concentrations, both cytotoxic and non-cytotoxic, in order to be able to distinguish a genotoxic effect of the mycotoxin from an indirect effect derived from its general cellular toxicity. No effect was shown where no cytotoxicity was found, both in the presence and in the absence of metabolic activation (10% rat liver S9-mix). However, oxidative DNA damage was shown at cytotoxic concentrations when formamidopyrimidine DNA glycosylase (FPG) and endonucleaseIII (EndoIII) were introduced in the assay with or without metabolic activation. Furthermore, at these concentrations, an elevation of reactive oxygen species was measured and pre-incubation with N-acetyl-L-cysteine was able to produce a slight protective effect on OTA-induced oxidative DNA damage as well as cytotoxicity. These data suggest that OTA is not acting as a direct genotoxic carcinogen and that oxidative stress is implicated in the genotoxicity and cytotoxicity observed in these human renal cells.

In vitro gene expression data supporting a DNA non-reactive genotoxic mechanism for ochratoxin A.

Ochratoxin A (OTA) is a mycotoxin often found in cereals and agricultural products. There is unequivocal evidence of renal carcinogenicity of OTA in male rats, although the mechanism of action is unknown. At present, available data support an epigenetic mechanism (DNA non-reactive) resulting from oxidative stress and cytotoxicity, because a direct OTA interaction with DNA has not been demonstrated. Genotoxic mechanism (DNA-reactive vs. DNA non-reactive) may have implications on human risk assessment. Therefore, the aim of the present work was to identify biological pathways modulated by OTA in vitro in a human renal cell line (HK-2) to contribute to the elucidation of the mechanism of OTA toxicity. For that purpose, cells were exposed to 50 microM OTA during 6 and 24 h, and gene expression profiles were analyzed using Affymetrix Human Genome U133 A 2.0 Gene Chips. Under the same experimental conditions, genotoxicity was evaluated by the modified comet assay using FPG and Endo III to detect oxidative DNA damage, and intracellular ROS level by the H(2)DCF assay. After 6 h, with slight cytotoxicity (83% survival), genes involved in mitochondrial electron transport chain were up-regulated; and after 24 h, with a more pronounced cytotoxicity (51% survival), genes implicated in oxidative stress response were also up-regulated. Increase in intracellular ROS level and oxidative DNA damage was evident at both exposure times being more pronounced with high cytotoxicity. On the contrary, up-regulation of genes implicated in DNA damage response, as cell cycle control or apoptosis, was not detected at any exposure time. In conclusion, these results support a DNA non-reactive mechanism of OTA genotoxicity.

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.

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.

DNA damage induced by a quinoxaline 1,4-di-N-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay.

The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 microM in hypoxia; 20, 40 microM in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24-168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 +/- 13, 343 +/- 30 and 399 +/- 1; control comet score: 42 +/- 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 +/- 14 and 312 +/- 9; control comet score: 27 +/- 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time- and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.

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.