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.

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).

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.

An approach to the toxicity and toxicokinetics of aflatoxin B1 and ochratoxin A after simultaneous oral administration to fasted F344 rats.

Humans are exposed to the hepatotoxic aflatoxin B1 (AFB1) and nephrotoxic ochratoxin A (OTA) through diet. However, kinetic and toxicological data after their co-administration are scarce. In this study, a single oral dose of AFB1 (0.25 mg/kg bw)+OTA (0.5 mg/kgbw) was administered to fasted F344 rats. Blood, liver and kidney were harvested at different timepoints for mycotoxins quantification, relative weight calculation, clinical biochemistry and histopathology analysis. Toxicity parameters pointed to acute toxicity in liver due to AFB1. No remarkable toxicity was observed in kidneys or immunological organs. Maximum observed concentrations in plasma (Cmax) were at 10 min and 2 h for AFB1 and OTA, respectively. AFB1 plasma concentration could indicate a rapid absorption/ metabolism of the mycotoxin; and AFB1 liver and kidney concentrations were lower than LOQ and LOD, respectively. For OTA, Cmax was 4326.2 µg/L in plasma. In kidney and liver Cmax was reached at 8 h and concentrations were very similar between both organs at all timepoints. Due to the low levels of AFB1, the effect of OTA on AFB1 kinetics could not be assessed. However, AFB1 seems not to affect OTA kinetics, as its profile seems very similar to kinetic studies performed only with OTA in similar conditions.

A polyphenol-enriched cocoa extract reduces free radicals produced by mycotoxins.

Polyphenols are characterized by the presence of phenol units in the molecules. These compounds may show antioxidant ability by scavenging reactive oxygen species (ROS) of the free radical type. A polyphenol enriched cocoa extract (PECE) was obtained from cocoa seeds with 28% of procyanidins which were mainly epicatechin oligomers. PECE was very active as free radical scavenger against 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and tris(2,4,6-trichloro-3,5-dinitrophenyl)methyl (HNTTM) radicals; and the tris(2,3,5,6-tetrachloro-4-nitrophenyl)methyl (TNPTM) assay showed that the PECE might not be pro-oxidant. Thus it was considered a good candidate to be tested in in vitro models. It showed mild cytotoxic power on Hep G2 cells and induced ROS in a dose-dependent manner being weak oxidant only at high concentrations near the limit of solubility. The antioxidant properties were assayed in Hep G2 treated with the mycotoxins ochratoxin A (OTA) and/or aflatoxin B1 (AFB1). The PECE was not effective against AFB1 but it increased the cell viability and reduced significantly the amounts of ROS in cells treated with OTA or mixtures of AFB1+OTA. These results are coherent with the role of oxidative pathways in the mechanism of OTA and indicate that polyphenols extracted from cocoa may be good candidates as antioxidant agents.

Ochratoxin A reduces aflatoxin B1 induced DNA damage detected by the comet assay in Hep G2 cells.

Mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) can be present together in food commodities. These food contaminants are considered to be genotoxins, acting by different mechanisms. The aim of this work was to characterize combined genotoxic in vitro effects of both mycotoxins in Hep G2 cells. For this purpose, cytotoxicity was first determined in isolated and combined treatments in order to determine the dose range of genotoxicity studies. Co-exposure of cells to OTA+AFB1 for 24 h resulted in additive effects. Genotoxicity was determined in Hep G2 cells by the modified comet assay with restriction enzymes (endo III and FPG). Significant reactive oxygen species formation was detected in both single and combined treatments. AFB1 was genotoxic after 3 h with external metabolic activation (S9 mix) and after 24 h without metabolic activation. Co-exposure to OTA significantly decreased DNA damage induced by AFB1, not only in breaks and apurinic sites but also in FPG-sensitive sites. The apparent contradiction between additive cytotoxic effects and antagonic genotoxic effects may be explained if AFB1 and OTA compete for the same CYPs, yielding more ROS but less AFB1 adducts.

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.

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.

Influence of roasting and brew preparation on the ochratoxin A content in coffee infusion.

A study of the effect of coffee processing in the ochratoxin A (OTA) level has been carried out from the green beans to the drinking form. The analysis of OTA has been carried out by an in-house validated HPLC method with fluorescence detection. The limits of detection were 0.04 microg/kg for green and roasted coffee, and 0.01 microg/L for coffee brew. Thirty-six green coffee samples of different origin (Colombia, Costa Rica, Brazil, Vietnam, India and Uganda) were analysed. The highest concentrations of OTA were found in Vietnamese samples -- Robusta species treated by dry processing -- (range 0.64-8.05 microg/kg), that also showed the highest percentage of defective beans (7.6%). These contaminated samples were roasted in a process that controlled loss of weight and color, as in the industry. A mean reduction of 66.5% was obtained, but the reduction seems to be heterogeneous. Coffee brew was prepared by the three brewing processes more utilized in Europe: moka, auto-drip and espresso. A reduction of the OTA level has been attained, being greater when using a espresso coffee maker (49.8%) than when using auto-drip (14.5%) or moka brewing (32.1%). Therefore, the method of coffee brew preparation plays a key role in the final OTA human exposure.

Aplicación de la estrategia secuencial de la OCDE para evaluar la irritación y corrosión dérmica por un producto fitosanitario / Application of the OCDE sequential strategy for the evaluation of dermal irritation and corrosion

Con la intención de reemplazar los ensayos en animales y mejorar las propiedades predictivas, la Guía TG404 de la OCDE sobre irritación/corrosión dérmica aguda ha desarrollado una nueva estrategia secuencial que promueve el uso de métodos alternativos tales como los test en sistemas in vitro. Se ha aplicado el procedimiento de esta estrategia para evaluar la irritación y corrosión dérmica por un nuevo fertilizante con fines de registro, con el objetivo de comprobar sus ventajas en un estudio normalizado. Para evaluar la corrosión dérmica se ha utilizado el sistema in vitro validado EpiDerm™, epidermis reconstituída formada por queratinocitos humanos. Puesto que por el momento no existe un modelo in vitro validado para irritación dérmica, ésta fue evaluada en el sistema EpiDerm™ y mediante un método in vivo. En ambos ensayos in vitro se midió también la liberación de IL1α. El producto fue clasificado como no corrosivo y no irritante. Los resultados del estudio demuestran las ventajas de la estrategia secuencial propuesta por la OCDE en la evaluación de la corrosión e irritación por las siguientes razones: a) se adquiere un conocimiento previo sobre las propiedades físicoquímicas de los compuestos químicos con el fin de evitar la exposición innecesaria de los animales a sustancias corrosivas b) los métodos in vitro de corrosión dérmica pueden sustituir los animales de experimentación c) las modificaciones propuestas para el test in vivo reducen el sufrimiento de los animales y suponen un importante ahorro económico y de tiempo. No obstante, se requiere con urgencia disponer de métodos alternativos in vitro para evaluar la irritación cutánea

In order to replace the assays carried out on animals and improve the predictive properties, the Guideline TG404 of the OCDE on acute dermal corrosion/irritation has developed a new sequential strategy that promotes the use of alternative methods such as the tests in in vitro systems. The procedure of this strategy has been applied in order to evaluate the irritation and dermal corrosion produced by a new fertilizer with the objective of obtaining a patent and in order to determine the advantages of using said fertilizer in a normalized study. In order to evaluate the dermal corrosion, the validated in vitro system EpiDermTM, a reconstituted epidermis formed by human keratinocytes, has been used. Since, at this moment, no in vitro model which is validated for dermal irritation exists, this method was evaluated in the EpiDermTM system and by means of and in vivo method. In both in vivo assays, the release of IL1α was measured also. The product was classified as noncorrosive and nonirritant. The results of the study showed the advantages of the sequential strategy proposed by the OCDE in the evaluation of the corrosion and irritation due to the following reasons: a) prior knowledge is acquired regarding the physicochemical properties of the chemical compounds for the purpose of avoiding unnecessary exposure of corrosive substances to the animals; b) the in vitro methods of dermal corrosion can substitute the experimentation animals and c) the modifications proposed for the in vivo test reduce animal suffering and represent important time and economical savings. However, there is an urgent need for available alternative in vitro methods for the evaluation of cutaneous irritation

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.

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.

Validation of a high-performance liquid chromatography analytical method for ochratoxin A quantification in cocoa beans.

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in cocoa beans is described. OTA was extracted with methanol-3% sodium hydrogen carbonate solution and then purified with immunoaffinity columns before its analysis by HPLC. The validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within- and between-day variability) and recovery, robustness and uncertainty. Detection and quantification limits were 0.04 and 0.1 mug kg(-1), respectively. Recovery was 88.9% (relative standard deviation = 4.0%). This method was successfully applied to the measurement of 46 cocoa bean samples of different origins. A total of 63% of cocoa bean samples was contaminated with a level greater than the limit of detection. The means and medians obtained for cocoa bean were 1.71 and 1.12 mug kg(-1), respectively. Surveillance controls should be set up in both crops and factories involved in transformation processes to avoid this mycotoxin in final products.

¿Es la ocratoxina a una micotoxina mutagénica? / Is the ochratoxin A a mutagenic mycotoxine?

La ocratoxina A es una micotoxina producida por especies de los géneros Aspergillus y Penicillium que a través de los alimentos puede pasar al ser humano. Su órgano diana es el riñón pero también es hepatotóxica, inmunotóxica y teratogénica. Ha sido clasificada por la Agencia Internacional de Investigación contra el Cáncer (IARC) como posible carcinógeno humano (clase 2B) pero se desconoce si el mecanismo de acción transcurre a través de fenómenos genéticos o epigenéticos. En este artículo se revisan los datos de genotoxicidad y mutagenicidad de esta micotoxina. Aunque los primeros estudios en ensayos de reversión mutagénica con bacterias resultaron negativos, pronto se comprobó que administrada a animales de experimentación, la ocratoxina A inducía la formación de aductos especialmente en tejidos de riñón y vejiga urinaria de ratón. También se ha comprobado que esta micotoxina produce roturas monocatenarias en el ADN, da lugar a alteraciones cromosómicas e intercambios entre cromátidas hermanas e induce la síntesis de ADN fuera del período S, fenómeno indicativo de procesos de reparación. Se considera que la actividad genotóxica es dependiente de activación metabólica, en particular de varias isoformas P450, si bien los metabolitos genotóxicos no han sido aislados. Los últimos estudios realizados con ocratoxina A tritiada bajo diversas condiciones experimentales indican que el principal metabolito es el derivado monohidroxilado 4(R) - hidroxi-ocratoxina A y que ni la ocratoxina A ni este metabolito forman aductos con el ADN, por lo que su actividad genotóxica estaría más relacionada con procesos de citotoxicidad y peroxidación lipídica, los cuales podrían dar lugar a moléculas reactivas con los ácidos nucleicos (AU)

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Contribution to the study of ochratoxin A in Spanish wines.

With the aim of assessing ochratoxin A (OTA) contamination in wines from a Spanish northern region and the influence of harvest conditions, the following samples were analysed: 40 wines (28 red and 12 white) obtained from grapes cultivated in three different places of the northern Spanish region of Navarra, but in different years, 20 samples in 1997 and 20 in 1998. Wine samples were provided by a viticultural experimental station with very consistent and controlled cultural and enological practices. A high-performance liquid chromatography (HPLC) method with fluorescence detection and immunoaffinity columns was employed (LOD = 0.05 ng ml(-1); method recovery = 101%). Eighty five per cent of the samples from 1997 showed OTA levels >0.05 ng ml(-1) (range 0.056-0.316 ng ml(-1)) and 15% of the samples from 1998 showed OTA levels above the LOD (range 0.074-0.193 ng ml(-1)). Averages detected in 1997 positive samples were 0.185 ng OTA ml(-1) wine (SD = 0.023) in white wine samples (n = 6) and 0.160 ng ml(-1) (SD = 0.119) in the red wine samples (n = 11). These differences between contamination by OTA in the samples from the two different years were attributed to the different quality of the grapes, due to the bad climate in 1997. The possibility of the loss of the mycotoxin was excluded by the analysis of OTA in contaminated wine during 12 months. This study showed that OTA is stable in wine for at least 1 year.

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.