Ochratoxin A (OTA) contamination in dried figs was investigated using high performance liquid chromatography (HPLC) with fluorescence detection after extraction with methanol and orthophosphoric acid and clean up by an immunoaffinity column. The limit of detection for OTA was 0.12mugkg(-1). One hundred and fifteen samples were taken during the drying stage from 7 different districts in the Aegean Region in 2003 and 2004. Fifty-five (47.2%) of the 115 samples were found to contain detectable levels of ochratoxin A, ranging from 0.12 to 15.31mugkg(-1). However, the OTA level for a majority of the samples was low, with only 4 samples containing OTA exceeding 1mugkg(-1). The calculated overall median for the OTA level was below the limit of detection and the overall mean was estimated as 0.52mugkg(-1). Frequency of ochratoxin A contamination in dried figs harvested in 2003 and 2004 are 47 and 50%, respectively. Highest contamination ratio was determined in dried figs from Erbeyli (60%), followed by Selcuk (56%), and Ortaklar (50%).

The molecular pathogenesis of hepatocellular carcinoma, a tumour characterized by a vast clinical heterogeneity, remains unexplored outside Europe and Eastern Asia. We analysed by direct sequencing or loss of heterozygosity assay, the common targets of genomic alterations in 42 hepatocellular carcinomas collected in western North-Africa. Overall, genomic instability was uncommon, allelic losses affecting mostly chromosomes 1p, 4q, 8p and 17p (24-28% of cases). CTNNB1 and TP53 were infrequently mutated (9 and 17% of cases, respectively). Surprisingly, TP53 mutation R249S, diagnostic of aflatoxin B1 exposure, usually frequent in Africa, was exceptional (one case), indicating that in western North-Africa, hepatocellular carcinoma genetics differs markedly from that of the remainder of the continent.

Ochratoxin A (OTA) is a nephrotoxin and carcinogen that is associated with Balkan endemic nephropathy and urinary tract tumors. OTA crosses the placenta and causes adducts in the liver and kidney DNA of newborns. Because the testis and kidney develop from the same embryonic tissue, we reasoned that OTA also may cause adducts transplacentally in the testis. We tested the hypothesis that acute exposure to OTA, via food and via exposure in utero, causes adducts in testicular DNA and that these lesions are identical to those that can be produced in the kidney and testis by the consumption of OTA. Adult mice received a single dose of OTA (from 0-1,056 microg/kg) by gavage. Pregnant mice received a single i.p. injection of OTA (2.5 mg/kg) at gestation day 17. DNA adducts were determined by (32)P-postlabeling. Gavage-fed animals sacrificed after 48 hours accumulated OTA in kidney and testis and showed DNA adducts in kidney and testis. Some OTA metabolites isolated from the tissues were similar in both organs (kidney and testis). The litters of mice exposed prenatally to OTA showed no signs of overt toxicity. However, newborn and 1-month old males had DNA adducts in kidney and testis that were chromatographically similar to DNA adducts observed in the kidney and testis of gavage-fed adults. One adduct was identified previously as C8-dG-OTA adduct by LC MS/MS. No adducts were observed in males from dams not exposed to OTA. Our findings that in utero exposure to OTA causes adducts in the testicular DNA of male offspring support a possible role for OTA in testicular cancer.

This review addresses the unresolved aetiology of several nephropathies and associated upper tract tumours diagnosed all over the world, but especially in the Balkan regions. Studies conducted over the last 35 years point to mycotoxins, mainly ochratoxin A (OTA) as the main culprit. Recent theories however have implicated aristolochic acids (AA). The aim of this review is to put forward arguments in favour of the mycotoxin theory and to show the incoherence of the AA theory. It discusses the differences between the epidemiology of Balkan endemic nephropathy (BEN) and aristolochic acid nephropathy (AAN); OTA and AA carcinogenicity; clinical and pathological effects induced by OTA and AA; sources of OTA contamination (food, air, drinking water); OTA- and AA-DNA adduct formation; the role of genetic polymorphisms; and the risk for young children.

The potent renal carcinogenicity of ochratoxin A (OTA) in rats, principally in the male, raises questions about mechanism. Chromatographic evidence of DNA adducts after (32)P-postlabeling analysis contrasts with experimental attempts to demonstrate the absence of OTA in such adducts. Proffered schemes for alternative epigenetic mechanisms in OTA carcinogenicity remain unsatisfying, while structural data substantiating DNA-OTA adducts has also been lacking. We report refined (32)P-postlabeling methodology revealing one principal adduct isolated in small amounts from the kidneys of all five Fischer and five Dark Agouti rats to which OTA had been given on four consecutive days. We also describe structural data for the principal adduct from OTA/DNA interaction in vitro and its subsequent preparative isolation by the postlabeling methodology (as C-C8 OTA 3'dGMP), essentially creating an ochratoxin B-guanine adduct. Reasoning for the unsuitability of experimental protocols in published evidence claiming nongenotoxicity of OTA is given. In vivo exposure of renal DNA to cycles of adduction with OTA, necessarily protracted for carcinogenesis to occur, can reasonably explain an occasional focal neoplasm from which metastasizing carcinoma could develop.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium that is widely found as a contaminant of food products. The toxin is a renal carcinogen in male rats, the cause of mycotoxicoses in pigs and has been associated with chronic human kidney diseases. Bioactivation has been implicated in OTA-mediated toxicity, although inconsistent results have been reported, due, in part, to the difficulty in detecting OTA metabolites in vivo. Liquid chromatography (LC) coupled with fluorescence detection (FLD) is the most widely used analytical detection method for OTA. Under acidic conditions the toxin generates blue fluorescence (465nm) that is due to an excited state intramolecular proton transfer (ESIPT) process that generates an emissive keto tautomer. Disruption of this ESIPT process quenches fluorescence intensity and causes a blue shift in emission maxima. The aim of the present study was to determine the impact of the C5-chlorine atom, the lactone moiety and the amide bond on OTA fluorescence and derive optical parameters for OTA metabolites that have been detected in vitro. Our results highlight the limitations of LC/FLD for OTA metabolites that do not undergo ESIPT. For emissive derivatives, our absorption and emission data improves the sensitivity of LC/FLD (3-4-fold increase in the limit of detection (LOD)) for OTA analogues bearing a C5-OH group, such as the hydroquinone (OTHQ) metabolite and the glutathione conjugate of OTA (OTA-GSH). This increased sensitivity may facilitate the detection of OTA metabolites bearing a C5-OH group in biological fluids and enhance our understanding of OTA-mediated toxicity.

Wheat samples were taken at different stages of germination characterized by their falling number (which is a relevant indicator of germination) from 400 to 60 s. Each batch was treated by the Oxygreen process, a treatment by ozone, in a closed sequential batch reactor. Leucotriene B4 (LTB 4) was induced by germination, but ozone treatment did not increase this effect. Extract obtained from these wheat batches was applied on human epithelial bronchial cells. Wheat extract from nongerminated wheat did not induce any DNA adduct. More the wheat germination gets underway, more DNA adducts are observed. In contrast, germination did not affect the cell viability. Ozonization of wheat exemplified genotoxic effects only if the wheat was germinated. The implication of hydroxamic acids is discussed. In conclusion, ozonization of wheat, of high milling quality, does not pose any problem.

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To find out whether ochratoxin A (OTA), citrinin (CIT), aristolochic acids (AA) are etiologic agents of Balkan endemic nephropathy (BEN) or Chinese herbal nephrotoxicity, and associated urinary tract tumor (UTT), we have compared (i) in human kidney cell culture, the DNA adduct formation and persistence of OTA/CIT and AA adducts (ii) analyzed DNA adduct in several tumors from human kidney suspected to be exposed to either OTA and CIT, or AAs (iii) analyzed OTA, CIT, and AA in food. In kidney cell cultures, formation of specific OTA-DNA adduct and AA-DNA adduct were detected in the same range (around 10 adducts/10(9) nucleotides) and were time- and dose-dependent. After 2 days all disappeared. DNA adduct related to OTA and CIT are found in human kidney tissues from Balkans, France, and Belgium whereas no DNA adducts related to AA could be found in any tumors of BEN patients from Croatia, Bulgaria, or Serbia. No DNA adduct was found in kidney biopsy or necropsy of the French women suspected to be exposed to AA. OTA and CIT are more frequently found in rural area. AA was never detected. All these plead for implication of mycotoxins, especially OTA, in BEN and UTT.

Ochratoxin A (OTA) is a ubiquitous mycotoxin produced by fungi of improperly stored food products. OTA is nephrotoxic and is suspected of being the main etiological agent responsible for human Balkan endemic nephropathy (BEN) and associated urinary tract tumours. Striking similarities between OTA-induced porcine nephropathy in pigs and BEN in humans are observed. International Agency for Research on Cancer (IARC) has classified OTA as a possible human carcinogen (group 2B). Currently, the mode of carcinogenic action by OTA is unknown. OTA is genotoxic following oxidative metabolism. This activity is thought to play a central role in OTA-mediated carcinogenesis and may be divided into direct (covalent DNA adduction) and indirect (oxidative DNA damage) mechanisms of action. Evidence for a direct mode of genotoxicity has been derived from the sensitive (32)P-postlabelling assay. OTA facilitates guanine-specific DNA adducts in vitro and in rat and pig kidney orally dosed, one adduct comigrates with a synthetic carbon (C)-bonded C8-dG OTA adduct standard. In this paper, our current understanding of OTA toxicity and carcinogenicity are reviewed. The available evidence suggests that OTA is a genotoxic carcinogen by induction of oxidative DNA lesions coupled with direct DNA adducts via quinone formation. This mechanism of action should be used to establish acceptable intake levels of OTA from human food sources.

Mould incidence and aflatoxin B1 (AFB1) and ochratoxin A (OTA) contamination as well as proximate composition and minerals content of maize kernels from Swat Valley, North West Frontier Province of Pakistan was studied during the year, 2007. Results indicated that the mean moisture content of the kernels was within the recommended safe storage levels of 15%. Across the whole valley, Aspergillus, Fusarium, Penicillium and Rhizopus were the most predominant fungal genera identified and amongst the mycotoxigenic species, Aspergillus flavus had the highest incidence. AFB1 content ranged from none to 30.92mug/kg with the average values of 14.94 and 16.22mug/kg for Upper and Lower Swat regions, respectively. Similar trend was observed for OTA with the contamination level ranged from <0.001 to 7.32mug/kg. A significant numbers of samples contained AFB1 and OTA levels above the safe limits as recommended by the USFDA and EU but on the average the results were within the safe limit. These results indicate that maize consumers in Swat Valley may be exposed to the danger of aflatoxins and ochratoxins poisoning. Thus, there is a need for policy makers to establish and enforce maize quality standards and regulations related to moulds and mycotoxins across the area.

AIMS: The aim of this study was to identify fungal populations in unroasted cocoa beans stored in Spain in order to evaluate the ochratoxin A (OTA)-production ability of certain Aspergillus isolates. METHODS AND RESULTS: Twenty batches of cocoa beans from different origins and with different OTA content were selected for this study. Three Aspergillus carbonarius and 13 Aspergillus niger aggregate strains isolated from these cocoa bean samples were selected to evaluate their OTA synthesis ability, being the only A. carbonarius isolates which are OTA producers [<limit of detection (LOD)= 3520 microg kg(-1) culture medium; LOD = 6 microg kg(-1) culture medium]. CONCLUSIONS: No correspondence was found between the OTA levels in cocoa beans and the presence of OTA-producing fungi. Nonetheless, some samples contained A. carbonarius with a high OTA-producing ability and, consequently, specific fungal controls should be set up during storage to avoid this toxin. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxigenic fungi in cocoa beans are not well understood. This study attempted to identify these fungi and evaluate their OTA-producing ability.

Five hundred and ten strains of filamentous fungi were isolated from Lebanese grapes during 2005 at veraison and harvesting periods. Four hundred eighty-seven isolates belonged to the Aspergillus spp.(95.5%) and 23 belonged to the Penicillium spp.(4.5%). Black aspergilli constituted 56.9%(52.2% Aspergillus niger aggregates, 2.9% Aspergillus japonicus and 1.8% Aspergillus carbonarius) while the isolation rate of Aspergillus flavus the none habitual member of grape mycobiota was 43.1% of the total Aspergillus spp. isolated. All isolates were tested for the ability to produce the Ochratoxin A (OTA) and the Aflatoxin B1 (AFB1). A. carbonarius showed that it is the only species able to produce the OTA with a production ability of 100% and a maximum concentration reaching 8.38mug/g CYA. As for the aflatoxigenic ability, 43.4% of A. flavus isolates produced this mycotoxin with a maximum production reaching 22.6mug/g CYA while none of the other isolates showed a production capacity of this mycotoxin. Forty-seven samples of must produced from the collected grapes were also analyzed. None of these samples was contaminated by OTA at a detectable limit while 40% of these same samples were found to contain AFB1 with concentrations ranging from 0.01 to 0.46mugl(-1).

A survey on the occurrence on grape of fungi species in 2001 and their capability to produce ochratoxin A (OTA) and naphtho-gamma-pyrones (NGPs) was conducted in different vineyards from several French viticulture regions. The total numbers of fungal isolates, from setting to harvest, were 732. The Aspergillus genus was essentially represented by section Nigri (98.53%) and it was predominant (74.72%) when compared to Penicillium (25.27%). Approximately one third (30.46%) of the fungal isolates were OTA producers, and 94.17% belong to black aspergilli; Aspergillus carbonarius was the main OTA producer. Moreover, 8.33% of isolates (belong to A. carbonarius and A. niger) were NGP producers. However, none of the Penicillium spp. or other Aspergillus spp. isolates can produces NGP derivatives under the conditions used. No other study on NGPs production by fungi isolated from grapes has been reported. In the second part, a novel NGP, named aurasperone G ( 1), was isolated from the fermentation broth of the culture extracts of Aspergillus niger C-433, strain producer of OTA, along with the known compound aurasperone F ( 2). The chemical structure of the new polyketide was proposed based on complete (1)H and partial (13)C, COSY, HMQC, 1D NOE NMR spectra as well as UV and MS spectra. This new NGP was not reported before in nature or prepared synthetically.

Olive oil, the most important dietary fat source of the Mediterranean diet, can be contaminated by mycotoxins. An efficient analytical method for the simultaneous determination of aflatoxin B1 (AFB1) and ochratoxin A (OTA) in olive oil is reported. Thirty commercial samples of virgin olive oil, purchased in olive-press plants and supermarkets in southern Italy and North Africa, were analysed to verify the analytical procedure and monitor mycotoxin contamination. A simple, rapid and economic method was set up and tested for both the extractive step and the clean-up procedures for simultaneous AFB1 and OTA determination in olive oil. Data obtained showed that OTA was detected with high frequency (80%) in samples from both geographical areas (up to 17.0 ng g-1), while AFB1 was found from three of four samples from North Africa (up to 2.4 ng g-1). In addition,'not labelled' oil samples proved to be more contaminated by OTA then 'labelled' samples (mean values of 2.47 and 0.66 ng g-1, respectively). These findings indicate that olive oil can be significantly contaminated by mycotoxins and confirm that a scrupulous application of European Regulation 1019/2002 (European Commission 2002), which prohibits the sale of non-labelled olive oil, is strongly recommended. Conventional qualitative parameters such as peroxide number, spectrophotometric evaluation and acid values were not correlated with mycotoxin occurrence.

This paper reports the results of an extensive survey on the occurrence of filamentous fungi isolated from wine-grapes in Lebanon and to test their ability to produce ochratoxin A (OTA) and aflatoxin B1 (AFB1) on CYA culture medium, in order to assess their potential for producing these mycotoxins on grapes. From the 470 grapes samples taken during season 2004, 550 fungi strains were isolated with 490 belonging to Aspergillus spp. and 60 belonging to Penicillium spp. All these isolated fungi starins were tested for their ability to produce OTA and AFB1. Aspergillus carbonarius shows that it is the only species able to produce OTA with a production percentage reaching 100% and a maximum concentration of 52.8 mug/g of Czapek yeast extract agar (CYA). In its turn, Aspergillus flavus was considered as the only AFB1-producing species with production percentage of 45.3% and a maximum concentration reaching 40 mug/g CYA. A total of 47 handmade musts produced from the collected grapes were also analyzed in order to correlate the presence of OTA in must and the occurrence of filamentous fungi on grapes; 57.4% were contaminated with OTA at low level with concentrations ranging between 0.011 and 0.221 mug OTA L(-1). The analysis of these must samples was not performed with regard to AFB1. Seventy samples of finish red wine were also assayed for OTA content. The results showed that 42 of the tested samples (60%) were found to be positive for OTA with low levels (0.012-0.126 mug OTA L(-1)).

Aflatoxin B(1)(AFB(1)) is a carcinogenic metabolite produced by certain Aspergillus species. Ochratoxin A (OTA) is classified as "possible carcinogen" and it is a metabolite of Aspergillus ochraceus and Penicillium verrucosum. Fungi contaminate natural and processed olives which support AFB(1) and OTA production. The aim of this study was to compare and investigate AFB(1) and OTA production in three different varieties of damaged olives. For each variety two different treatments were applied:(1) olives with natural microflora and (2) olives inoculated with A. parasiticus after natural microflora elimination. AFB(1) and OTA have been extracted simultaneously from olives, purified with immunoaffinity columns and quantitated by HPLC using fluorescence detector. The recoveries and detection limits of AFB(1) and OTA were 94% and 0.15ng AFB(1)g(-1) and 102.7%, 0.41ng OTAg(-1) respectively. Results showed that, meanwhile OTA was not found in any olive sample, AFB(1) production within the three varieties of olives with natural microflora was significantly (P0.05) different regarding their substrate and time of incubation (18 days). AFB(1) production in two different varieties of black olives after inoculation by A. parasiticus was not significantly higher compared with control samples. On the contrary, AFB(1) production in green olives was stimulated after the 12th day. Additionally, investigation on the occurrence of AFB(1) and OTA in 30 samples of olives and olive pasta from Athens market showed OTA's presence in two samples of olives contaminated at the levels of 1.18 and 1.86ng OTAg(-1). Aflatoxin B(1) was found at levels 0.15-1.13ng AFB(1)g(-1) in all samples tested.

During the 2003 and 2004 olive oil production campaigns in Morocco, 136 samples from spoiled olive and olive cake were analyzed and 285 strains were isolated in pure culture. Strains included 167 mesophilic strains belonging to ten genera: Penicillium, Aspergillus, Geotrichum, Mucor, Rhizopus, Trichoderma, Alternaria, Acremonium, Humicola, Ulocladium as well as 118 thermophilic strains isolated in 2003 and 2004, mainly belonging to six species: Aspergillus fumigatus, Paecilomyces variotii, Mucor pusillus, Thermomyces lanuginosus, Humicola grisea, and Thermoascus aurantiacus. Penicillium and Aspergillus, respectively, 32.3 and 26.9% of total isolates represented the majority of mesophilic fungi isolated. When considering total strains (including thermotolerant strains) Aspergillus were the predominant strains isolated; follow-up studies on mycotoxins therefore focused primarily on aflatoxins (AFs) and ochratoxin A (OTA) from the latter strains. All isolated Aspergillus flavus strains (9) and Aspergillus niger strains (36) were studied in order to evaluate their capacity to produce AFs and OTA, respectively, when grown on starch-based culture media. Seven of the nine tested A. flavus strains isolated from olive and olive cake produced AF B1 at concentrations between 48 and 95 mug/kg of dry rice weight. As for the A. niger strains, 27 of the 36 strains produced OTA.

A total of 93 Portuguese grape samples destined for wine production were examined for the presence of ochratoxin A (OTA) and the OTA producing fungi Aspergillus carbonarius and A. niger aggregate. Samples came from 11 vineyards from four winemaking regions in the North and South of the Portuguese mainland, during the harvest seasons of 2001, 2002 and 2003. Grapes were examined at 3 maturation stages, from setting to the harvesting period, to evaluate when contamination with OTA producing fungi and OTA synthesis occur. The detection of fungi in grape samples was made by plating methods with and without surface disinfection. OTA was formed by 14% of the 650 isolates tested. Most of the OTA producing strains (96%) were isolated at harvest time. At this stage, the percentage of grape samples with OTA producing strains detected without surface disinfection was 56%. With surface disinfection, A. carbonarius was isolated from 10% of the samples. OTA was detected in grapes at the 3 maturation stages. The average OTA concentrations in 60 samples at pea berry (28 samples), early veraison (22 samples) and ripe berry (20 samples) were 263, 149 and 35 ng/kg, respectively. Experiments with an A. carbonarius strain demonstrated that OTA production differs significantly with the composition of the berries at different maturation stages (P<0.001), with a mean value of OTA production at pea berry, early veraison and ripe berry of 3402, 1530 and 22 mug/kg, respectively. The production of OTA by A. carbonarius was correlated positively and negatively with the total acidity of grapes (r(s)=0.855, P<0.001) and reducing sugars content (r(s)=-0.835, P<0.001), respectively. Our data demonstrate that OTA synthesis in grapes occurs since early maturation stages.

A 3-year survey was conducted to assay the number of Aspergillus Section Nigri isolates and in vitro ochratoxin A (OTA) production capacity in 10 vineyards in Israel. The survey included field sampling of two wine cultivars,'Sauvignon Blanc' and 'Cabernet Sauvignon' as well as the table grape cultivar 'Superior'. A total of 2114 isolates were analyzed and of those 161 isolates were shown to produce OTA. The major finding was that Aspergillus carbonarius (336 tested strains) is the most consistent producer of OTA, with approximately 35% of the isolates identified as positive in vitro. In comparison, 3.1% of other isolates from the Aspergillus niger aggregate (of 1432 strains) produced OTA in vitro. In contrast, none of the 346 tested strains with a uniseriate head morphology produced OTA. The incidence of infected berries was very low before veraison, while at harvest, this frequency was twice as high. In general, the composition of black Aspergilli did not differ during berry development. Generally, more OTA-producing isolates were isolated from the surface of table grapes cv.'Superior' compared to 'Sauvignon Blanc'. None of the samples collected at harvest contained traces of OTA in the juice. This study shows that grapes in Israel are contaminated with ochratoxigenic species which represent a risk of OTA contamination.