Genotoxicity evaluation of fried meat: A comprehensive review.

Some years ago, the IARC published the carcinogenic potential of processed and red meat. It is known that frying meat can produce genotoxic substances. A systematic review of the literature was conducted to evaluate in vitro and in vivo genotoxicity of fried meat. A total of 31 scientific articles were retrieved and analyzed. The meat extraction methods have been grouped into 6 types based on their similarity to an initially described method or on the general methodology used (solid-liquid extraction or others). The in vitro mutagenic results have been summarised by type of meat studied (beef, pork, others), cooking conditions (method, time and temperature), extraction method, and test used, with or without S9. Most articles assessed the mutagenicity of the extracts using the Ames test. Meat extracts were consistently positive in strains TA98/TA1538 with metabolic activation. In the in vitro studies with meat from restaurants, positive results were always found with variations in the number of His+ revertants between samples or between restaurants. The few in vivo studies retrieved show evidence of induced DNA damage in colon cells and chromosome aberrations in bone marrow cells after daily treatment with fried red meat for 4 weeks or longer.

Applying the comet assay to fresh vs frozen animal solid tissues: A technical approach.

The in vivo comet assay is usually performed in fresh tissues by processing cells immediately after collection, an approach that is not always possible from a logistical point of view. Although the comet assay has been applied to frozen rodent tissue samples on several occasions, there is currently no agreement on the best way to freeze and thaw them. We have tested two different thawing procedures and compared the levels of DNA strand breaks (SBs) and Fpg-sensitive sites in fresh and frozen (for up to year) liver, kidney and lung tissue samples, from untreated and methyl methanosulfonate treated rats. Tissues were snap frozen, stored at -80 °C and processed in such a way that the tissue remained frozen until the cells were in suspension. Our results showed that comparable levels of DNA SBs were detected in fresh and frozen liver and lung samples stored at -80 °C for up to 1 year and 3 months, respectively. In kidney, similar levels of SBs were detected either in fresh or in frozen tissues stored for up to 1 year. However, more studies are needed to control the variability observed in the Fpg-sensitive site levels in this tissue at the different freezing periods.

A simple chemical method reduces ochratoxin A in contaminated cocoa shells.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species, which contaminates cocoa among other food commodities. It has been previously demonstrated that the toxin is concentrated in cocoa shells. The aim of this study was to assay a simple chemical method for ochratoxin A reduction from naturally contaminated cocoa shells. In order to determine the efficiency of the method, a high-performance liquid chromatography method with fluorescence detection was set up beforehand and validated. Ochratoxin A was extracted from cocoa shells with methanol-3% sodium bicarbonate solution and then purified with immunoaffinity columns. The recovery attained was 88.7% (relative standard deviation = 6.36%) and the limits of detection and quantification were 0.06 and 0.2 kg/kg, respectively. For decontamination experiments, the solvent extractor ASE 200 was used. First, aqueous solutions of 2% sodium bicarbonate and potassium carbonate were compared under the same conditions (1,500 lb/in2 at 40 degrees C for 10 min). Higher ochratoxin A reduction was obtained with potassium carbonate (83 versus 27%). Then, this salt was used under different conditions of pressure, temperature, and time. The greatest ochratoxin A reduction was achieved with an aqueous potassium carbonate solution (2%), at 1,000 lb/in2 at 90 degrees C for 10 min. This method could probably be applicable to the cocoa industry because it is fast and relatively economic. From the point of view of human health, the use of potassium carbonate, partially eliminated by rinsing the sample with water, does not likely represent a risk for human health.

Minimizing creatine kinase variability in rats for neuromuscular research purposes.

Rat serum or plasma creatine kinase (CK) activity is widely used to evaluate myopathic processes, to test the myotoxicity of different drugs, or to analyse the benefits of emerging gene therapies in some neuromuscular disorders. However, great variability is found in this determination. The aim of this study has been to control some factors of variation in order to reduce variability and increase the reproducibility of analytical data. 8-10-week-old Wistar-Han rats were used. The study consisted of four sequential phases. Phase I aimed to analyse the effect of ether and isoflurane as anaesthetic drugs. The objective of Phase II was to evaluate bleeding rats via retro-orbital sinus vs. tail vein. Phases III and IV were designed as two separate, repeated measure experiments on two factors: habituation to laboratory handling procedures in Phase III and gender in Phase IV. The repeated factor was the storage temperature of blood sample prior to centrifugation. Ether did not significantly increased the CK value. Using isoflurane, getting rats accustomed to laboratory handling procedures and whole blood refrigeration prior to centrifugation and serum separation resulted in statistically significant reduction in CK value and variability. Male rats showed significantly higher values than female rats. In the light of our findings, CK value and variability in rats may be minimized by choosing tail vein as site of bleeding, getting rats accustomed to laboratory handling procedures and maintaining whole blood refrigerated until centrifugation and serum separation.

Genotoxic evaluation of poly(anhydride) nanoparticles in the gastrointestinal tract of mice.

Gantrez® AN 119-based NPs have been developed as oral drug carriers due to their strong bioadhesive interaction with components of the gastrointestinal mucosa and to their adaptable surface. The use of mannosamine to coat Gantrez® AN 119-based NPs results in a high mucus-permeable carrier, able to reach the gastrointestinal epithelium. Although their efficacy to transport a therapeutic agent has been demonstrated, their safety has not yet been thoroughly studied. They have proved to be non-cytotoxic, non-genotoxic and non-mutagenic in vitro; however, the in vivo toxicity profile has not yet been determined. In this study, the in vivo genotoxic potential of Gantrez® AN 119 NPs coated with mannosamine (GN-MA-NP) has been assessed using the in vivo comet assay in combination with the enzyme formamidopyrimidine DNA glycosylase in mice, following the OECD test guideline 489. To determine the relevant organs to analyse and the sampling times, an in vivo biodistribution study was also carried out. Results showed a statistically significant induction of DNA strand breaks and oxidized bases in the duodenum of animals exposed to 2000 mg/kg bw. However, this effect was not observed at lower doses (i.e. 500 and 1000 mg/kg which are closer to the potential therapeutic doses) or in other organs. In conclusion, GN-MA-NP are promising nanocarriers as oral drug delivery systems.

In vitro evaluation of the genotoxicity of poly(anhydride) nanoparticles designed for oral drug delivery.

In the last years, the development of nanomaterials has significantly increased due to the immense variety of potential applications in technological sectors, such as medicine, pharmacy and food safety. Focusing on the nanodevices for oral drug delivery, poly(anhydride) nanoparticles have received extensive attention due to their unique properties, such as their capability to develop intense adhesive interactions within the gut mucosa, their modifiable surface and their biodegradable and easy-to-produce profile. However, current knowledge of the possible adverse health effects as well as, toxicological information, is still exceedingly limited. Thus, we investigated the capacity of two poly(anhydride) nanoparticles, Gantrez® AN 119-NP (GN-NP) and Gantrez® AN 119 covered with mannosamine (GN-MA-NP), and their main bulk material (Gantrez® AN 119-Polymer), to induce DNA damage and thymidine kinase (TK+/-) mutations in L5178Y TK+/- mouse lymphoma cells after 24h of exposure. The results showed that GN-NP, GN-MA-NP and their polymer did not induce DNA strand breaks or oxidative damage at concentrations ranging from 7.4 to 600µg/mL. Besides, the mutagenic potential of these nanoparticles and their polymer revealed no significant or biologically relevant gene mutation induction at concentrations up to 600µg/mL under our experimental settings. Considering the non-genotoxic effects of GN-NP and GN-MA-NP, as well as their exceptional properties, these nanoparticles are promising nanocarriers for oral medical administrations.

Evaluation of the cytotoxicity, genotoxicity and mucus permeation capacity of several surface modified poly(anhydride) nanoparticles designed for oral drug delivery.

The main concerns with drugs designed for oral administration are their inactivation or degradation in the harsh conditions of the gastrointestinal tract, their poor solubility through the gastrointestinal mucus gel layer, the poor intestinal epithelium permeability that limits their absorption, and their toxicity. In this context, poly(anhydride) nanoparticles are capable of protecting the drug from the harsh environment, reduce the drug's toxicity and, by virtue of surface modification, to enhance or reduce their mucus permeability and the bioadhesion to specific target cells. The copolymer between methyl vinyl ether and maleic anhydride (commercialized as Gantrez® AN 119) are part of the poly(anhydride) nanoparticles. These biocompatible and biodegradable nanoparticles (NPs) can be modified by using different ligands. Their usefulness as drug carriers and their bioadhesion with components of the intestinal mucosa have been described. However, their toxicity, genotoxicity and mucus permeation capacity has not been thoroughly studied. The aim of this work was to evaluate and compare the in vitro toxicity, cell viability and in vitro genotoxicity of the bioadhesive empty Gantrez® AN 119 NPs modified with dextran, aminodextran, 2-hydroxypropyl-ß-cyclodextrin, mannosamine and poly-ethylene glycol of different molecular weights. Results showed that, in general, coated NPs exhibit better mucus permeability than the bare ones, those coated with mannosamine being the most permeable ones. The NPs studied did not affect cell metabolism, membrane integrity or viability of Caco-2 cells at the different conditions tested. Moreover, they did not induce a relevant level of DNA strand breaks and FPG-sensitive sites (as detected with the comet assay).

Design and evaluation of "3 + 1" mixed ligand oxorhenium and oxotechnetium complexes bearing a nitroaromatic group with potential application in nuclear medicine oncology.

The synthesis and evaluation of a series of oxotechnetium and oxorhenium complexes containing a nitroaromatic moiety as potential radiopharmaceuticals for targeting tumour hypoxia is presented. 99mTc labelling was performed in high yield (>85%) and radiochemical purity (>90%). Their structure was corroborated by means of the rhenium complexes. Reduction potentials were in the range for bioreducible compounds. 99mTc complexes III-VI were selected for "in vivo" experiments in view of the results of cytotoxicity studies. Biodistribution in normal animals was characterized by high initial blood, lung and liver uptake, fast blood and soft tissue depuration and preferential excretion via the hepatobiliary system. Initial tumour uptake was moderate but tumour/muscle ratios for complexes III and IV, were favourable at all time points. Although the results are encouraging further development is still necessary in order to achieve higher tumour uptake and lower gastrointestinal activity.

Toxicity evaluation of nanocarriers for the oral delivery of macromolecular drugs.

Oral administration is the most commonly used and accepted route for drug administration. However, two of the main concerns are the poor intestinal epithelium permeability and rapid degradation, which limit absorption of drugs. In this context, nanocarriers have shown great potential for oral drug delivery. Nevertheless, special importance should be given to the possible toxic effect of these nanocarriers, such as their bioaccumulation in different tissues of the body, as well as, the different physicochemical parameters influencing their properties and so their potential toxic effect. This review describes first some aspects related to the behavior of nanosystems within the gastrointestinal tract and then some aspects of nanotoxicology and its evaluation, including the most popular techniques and approaches used for in vitro and in vivo toxicity studies. It also reviews the physicochemical characteristics of polymeric nanoparticles that may influence the development of toxicological effects, and finally it summarizes the toxicity results that have been published regarding polymeric nanocarriers.

Carbonyl reductase and NADPH cytochrome P450 reductase activities in human tumoral versus normal tissues.

The use of bioreductive agents in enzyme-directed bioreductive therapy has been proposed to take advantage not only of hypoxia in tumours, but also of the presence of reductases that metabolise such compounds. In this study, we studied the activities of NADPH cytochrome P450 reductase (P450R) and carbonyl reductase (CR) in 17 human lung tumours and 18 human breast tumours, together with the corresponding normal tissues. For lung cancer but not for breast cancer there was a significant difference in the CR activity between normal and tumour tissue. CR activity was increased with respect to the normal tissue between 2-fold and 40-fold indicating heterogeneity in tumour samples. No relationship was found between CR activity and the histological type, tumoral grade or TNM stage of the tumours. Although some variation in P450R activity in tumoral versus normal tissues was found in the majority of the samples studied, no significant differences could be demonstrated.

Hypoxia-selective agents derived from 2-quinoxalinecarbonitrile 1,4-di-N-oxides. 2.

Hypoxic cells are an important target for antitumor therapy because tumors are typically characterized by such cells. Virtually all tumors which are present as solid masses contain hypoxic cells, while normal cells generally have an adequate supply of oxygen. Accordingly, antitumor agents can be made selective for tumors by virtue of high activity under hypoxic conditions. The initial purpose of this work was to determine the influence of different groups in position 3. Thus, the synthesis of some 3-NH-substituted derivatives (2a, 3a, 4a) starting from 3-amino-2-quinoxalinecarbonitrile 1,4-di-N-oxide (1a) is described. Reductive deamination of compounds 1a-k provides the 2-quinoxalinecarbonitriles 5a-k, which are more potent, while selectivity is maintained or increased in some derivatives. The compound 7-(4-nitrophenyl)-2-quinoxalinecarbonitrile 1,4-di-N-oxide (5k) is 150-fold more potent than tirapazamine (3-amino-1,2,4- benzotriazine 1,4-di-N-oxide), which has been used as a standard. Three derivatives (5g,i,k) show a hypoxic cytotoxicity ratio (HCR) > or = 200, better than that of tirapazamine (HCR = 75) in V79 cells. Replacement of the 3-amino group by chlorine affords the potent but nonselective 3-chloro derivatives 6a-k showing similar toxicities under both aerobic and hypoxic conditions. These compounds were used as intermediates for the synthesis of a new series of water-soluble compounds derived from 3-[[(N,N- dialkylamino)alkyl[amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxides 10a-i and 11a-i. The 7-chloro and the 7-trifluoromethyl derivatives 10b,f have demonstrated high potency (0.4 and 0.3 microM) and excellent selectivity (HCR = 250 and 340). Several 7-chloro analogues, 12b, 13b.1,b.2, and 14b, and the dimer 16b have been prepared and evaluated in order to determine the optimum lateral chain in position 3, which appears to be the [(N,N-dimethylamino)propyl]amino moiety.

Hypoxia-selective agents derived from quinoxaline 1,4-di-N-oxides.

Hypoxic cells, which are a common feature of solid tumors, but not normal tissues, are resistant to both anticancer drugs and radiation therapy. Thus the identification of drugs with selective toxicity toward hypoxic cells is an important objective in anticancer chemotherapy. The benzotriazine di-N-oxide (SR 4233, Tirapazamine) has been shown to be an efficient and selective cytotoxin for hypoxic cells. Since the bioreductive activation of Tirapazamine is thought to be due to the presence of the 1,4-di-N-oxide moiety, a series of 3-aminoquinoxaline-2-carbonitrile 1,4-di-N-oxides with a range of electron-donating and -withdrawing substitutents in the 6- and/or 7- positions has been synthesized and evaluated for toxicity to hypoxic cells. Electrochemical studies of the quinoxaline di-N-oxides and Tirapazamine showed that as the electron-withdrawing nature of the 6(7)-substituent increases, the reduction potential becomes more positive and the compound is more readily reduced. Apart from the unsubstituted 6a and the 6,7-dimethyl derivative 6c, the quinoxaline di-N-oxides have reduction potentials significantly more positive than Tirapazamine (Epc -0.90 V). The most potent cytotoxins to cells in culture were the 6,7-dichloro and 6,7-difluoro derivatives 6i and 6l, which were 30-fold more potent than Tirapazamine. The 6(7)-fluoro and 6(7)-chloro compounds, 6e and 6h, showed the greatest hypoxia selectivity. Four of the compounds, 63, 6f, 6h and 6i, killed the inner cells of multicellular tumor spheroids in vitro. In vivo Balb/c mice tolerated a dose of these four compounds twice the size of that of Tirapazamine. This study demonstrates that quinoxaline 1,4-di-N-oxides could provide useful hypoxia-selective therapeutic agents.

Nonclonal chromosomal aberrations induced by anti-tumoral regimens in childhood cancer: relationship with cancer-related genes and fragile sites.

Cytogenetic studies were performed on 80 pediatric cancer patients to observe the chromosomal damage, both quantitative and qualitative, induced by chemotherapy. Peripheral blood lymphocytes (PBL) (n = 127) were obtained at diagnosis, during treatment, at remission, and at relapse, and chromosome analysis performed utilizing G-banding standard procedures. The results show a significant increase in the number of altered karyotypes (P = 0.03) in the samples during treatment, returning to values that were similar to those at diagnosis at 2-year remission. Most of the chromosomal aberrations (CA) detected during the chemotherapy regimens were nonclonal, unbalanced (75%), and involved chromosomes 1, 3, 5, 6, 11, 12, 16, and 17 most frequently. There was also a marked increase of CA in samples at relapse with very similar features (type and distribution) to those detected during treatment. There was a good correlation between the chromosomal breakpoints in our series and fragile sites (58%), oncogene (75%), and tumor suppressor gene (33%) loci described in the literature. The results obtained suggest that cytostatic drugs induce a transient increase in chromosome fragility occurring at several cancer-associated breakpoints.

Determination of ochratoxin A in wine using liquid-phase microextraction combined with liquid chromatography with fluorescence detection.

A liquid-liquid microextraction technique (LPME) has been applied to the extraction of ochratoxin A (OTA) from wine prior to its quantification by HPLC-fluorescence detection. OTA was extracted from wine, through 1-octanol immobilized in the pores of a porous hollow fiber, and introduced into 1-octanol inside the fiber. Recovery was 77%. The method was adequate for quantification of OTA in wine at levels within the range 0.25-10 ng/ml with a LOD of 0.2 ng/ml, and can be a simple and inexpensive alternative to the use of inmunoaffinity columns in order to quantify OTA levels in wine.

Quantitative structure-mutagenic activity relationships of triazino indole derivatives.

The mutagenicity of 3-(4'-benzylidenamino)-5H-1,2,3-triazin[5,4-b]-indol-4-one derivatives, new compounds with considerable platelet antiaggregating activity, was assayed with the Ames test using the Salmonella typhimurium strains TA97, TA98, TA100 and TA102. The adaptive least-squares method (ALS method) was used to carry out a quantitative structure-activity relationship (QSAR) analysis. Three equations, based on 10 congeners, were found for strains TA97, TA98 and TA100. The results suggest that lipophilicity of the substituent decreases the mutagenicity of the series.

New quinoxalinecarbonitrile 1,4-di-N-oxide derivatives as hypoxic-cytotoxic agents.

We report the synthesis and biological in vitro activities of 16 new 2-quinoxalinecarbonitrile 1,4-di-N-oxides. These compounds present new basic lateral chains (piperazines and anilines) in the 3 position as well as different substituents in the 6 and/or 7 positions of the quinoxaline ring. Among piperazine derivatives, 4b (a 7-chloro-3-(4-methylpiperazin-1-yl) derivative) was the most potent (P = 0.5 x10(-6) M). In general, aniline derivatives were more potent and selective than the former, compound 12b (with a 4-(methylphenyl)amino moiety in the 3 position and a chlorine atom in the 7 position) being the best one (P = 3 x 10(-6) 16).

1,2,5-Oxadiazole N-oxide derivatives as potential anti-cancer agents: synthesis and biological evaluation. Part IV.

Several new 1,2,5-oxadiazole N-oxide derivatives and some deoxygenated analogues were synthesized to be tested as potential selective hypoxic cell cytotoxins. Compounds prepared were designed in order to gain insight into the mechanism of action of this kind of cytotoxin. Compounds were tested in oxia and hypoxia and they proved to be non-selective. 3-Cyano-N(2)-oxide-4-phenyl-1,2,5-oxadiazole showed the best cytotoxic activity in oxia. The cytotoxicity observed for these derivatives could be explained in terms of the electronic characteristics of the 1,2,5-oxadiazole substituents. Electrochemical and ESR studies were performed on the more cytotoxic derivative.

Induction of micronuclei in V79 cells after combined treatments with heterocyclic aromatic amines.

Heterocyclic aromatic amines (HAs) appear in foods rich in proteins when subjected to different cooking processes. These amines have been demonstrated to be mutagenic in bacteria; in eucaryotic cells, controversial results have been referred. The objective of this study is to evaluate the clastogenic and/or aneugenic capacity of three HAs--2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), and 2-amino-3-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)--in isolated as well as in combined treatments. The micronucleus test in vitro was used on V79 cells in the presence and absence of metabolic activation. The duration of the treatment was 2 h, and cytochalasin B was added for 21 h to stop cytokinesis; then, micronuclei (MN) were counted in binucleated cells. In the presence of metabolic activation, the three amines showed a significant increase in the number of MN with respect to the negative control. The PhIP amine presented the highest values and it also resulted slightly active in the absence of metabolic activation, although these differences have not been considered to be significant. The combined treatments of these amines have shown that the effects attributed to them when administered together are those that are expected for a possible additive effect; the effect attributed to each HA separately is not potentiated nor inhibited.

Immunotoxic effects of Ochratoxin A in Wistar rats after oral administration.

Ochratoxin A (OTA) is a mycotoxin produced by species of the genus Aspergillus and Penicillium. Human exposure has been demonstrated worldwide and its origin seems to be the intake of contaminated foods. The kidneys are the target organ of this mycotoxin. Immunotoxic and genotoxic effects of OTA were investigated in Wistar male rats (aged 12 weeks), treated by gavage with 50, 150 or 450 microg OTA/kg body weight for 28 days, in the context of a general toxicity study, which was designed following the recommendations of OECD guideline 407. At the end of the study, the mean plasma concentration of the mycotoxin was determined, several immune function assays were performed and bone marrow smears were obtained and stained in order to analyse micronuclei in polychromatic erytrocytes. Mean plasma concentration was found to be 187, 600 and 807 microg/L, respectively. At the highest dose, a decrease in body weight gain was observed. Histopathological investigations revealed tubulonephrosis and acute tubular necrosis in the kidneys of the animals treated with OTA. The frequency and severity of the lesions increased with the dose. The response of splenocytes to sheep red blood cells was decreased in a dose-dependent manner; however, nonstatistically significant differences were obtained. The natural killer cell activity was strongly affected by OTA treatment. Cytotoxic T lymphocyte activity was lower in the animals exposed to 50 microg OTA/kg b.w. but was not modified in the groups exposed to 150 and 450 microg OTA/ kg b.w. The bacteriolytic capability of macrophages was significantly reduced in groups exposed to 50 and 450 microg OTA/ kg b.w. The number of micronuclei in bone marrow polychromatic erytrocytes did not vary significantly with respect to the control at any dose, but a false negative result can not be ruled out because the exposure doses were much lower than those recommended in OECD guideline 474.

Synthesis and antituberculosis activity of some new 2-quinoxalinecarbonitriles.

Tuberculosis, an ancient disease undergoing recent control by public hygiene and drug therapy, has experienced a recrudescence throughout the world. New and effective therapies are rapidly needed to combat infections caused by these strains. Some new 2-quinoxalinecarbonitriles have been synthesized and tested as antituberculosis agents and interesting results have been obtained from the first screening.