Spontaneous occurrence of ochratoxin A residues in porcine kidney and serum samples in Poland

During the period 1 April 1983 to 31 July 1984, 214,700 swine were processed in a slaughterhouse in Poznań, Poland. Of these pigs, 122 (0.057%) exhibited macroscopical kidney changes typical for mycotoxic porcine nephropathy. Ochratoxin A was found in kidneys from 52 of these pigs. Porcine serum samples not biased for nephropathy were collected at random in the same slaughterhouse. Of 388 samples, 148 exhibited ochratoxin A residues from 1 to 520 ng/ml. Significant increases in nephropathy and ochratoxin A frequencies were observed during the spring of 1984.     

Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys and blood of Polish swine.

Kidneys showing renal changes characteristic for mycotoxic porcine nephropathy were collected during the period 1 April 1982 to 31 March 1983 from 225,000 swine processed in a large slaughterhouse in the district of Poznań, Poland. Of 113 kidneys suspected of mycotoxic porcine nephropathy, 27 exhibited ochratoxin A levels from traces to 23 ng/g. In 17 kidneys the level of the toxin was lower than 2 ng/g. Increased frequency of ochratoxin A presence and its level in kidneys were observed during the spring. Of 195 porcine blood samples collected at random, 36 exhibited toxin levels from 3 to 270 ng/ml.     

Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys and blood of Polish swine

Kidneys showing renal changes characteristic for mycotoxic porcine nephropathy were collected during the period 1 April 1982 to 31 March 1983 from 225,000 swine processed in a large slaughterhouse in the district of Poznań, Poland. Of 113 kidneys suspected of mycotoxic porcine nephropathy, 27 exhibited ochratoxin A levels from traces to 23 ng/g. In 17 kidneys the level of the toxin was lower than 2 ng/g. Increased frequency of ochratoxin A presence and its level in kidneys were observed during the spring. Of 195 porcine blood samples collected at random, 36 exhibited toxin levels from 3 to 270 ng/ml.     

Monitoring of residues of ochratoxin A in blood and kidney of chicken using HPLC-FLD

Detoxification of ochratoxin A can be achieved by chemical or enzymatic hydrolyzation, the products of such reactions are ochratoxin α and phenylalanine. Ochratoxin α like ochratoxin A, is a fluorescing molecule, therefore sensitive analysis is possible at very low concentration levels. Methods have been established that make it possible to look for residues of ochratoxin A and its main metabolite ochratoxin α in blood and tissues at very low concentration levels. Plasma is extracted by the use of small amounts of chloroform; the extract is cleaned with water and afterwards evaporated to dryness]. The residue is re-dissolved and analysed by HPLC-FLD. Using this method a limit of detection of 0.5μg/l for both ochratoxin A and ochratoxin α can be reached.     

Natural occurrence of ochratoxin A and citrinin in food stuffs in Egypt

Natural occurrence of ochratoxin A (OA) and citrinin in cereals (274 samples) and animal tissues (250 samples) have been investigated during a period of more than 2 years. OA was found in cereals and animal tissues while citrinin was found in cereals only. The highest level of OA (up to 80.0 μg/kg) was found in yellow corn, 52.8% of contaminated samples while respectively 55.9% and 39.4% of barley and rice samples were contaminated with citrinin, with the highest level up to 100.0 and 27.92 μg/kg for barley and rice respectively. The frequent contamination of animal kidney with OA (28% positive out of 150 tested) average concentration 12.33 μg/kg. 2% of liver and 4% of muscles tissue were observed.     

A survey of porcine kidneys and chicken liver for ochratoxin A in Spain

Contamination studies by ochratoxin A on pork kidney and chicken liver has been carried out in Catalonia (Spain). 73% of the pork kidney samples analyzed did not contain an amount of ochratoxin A over our detection limit (0.5 ng/g) whereas only 7% had contamination higher than 1 ng/g. None of the chicken samples analyzed were contaminated by this toxin above the detection limit. All contamination levels found are below the maximum levels accepted by several countries for this kind of material. A confirmative test is necessary before discarding false positive samples.     

A French monitoring programme for determining ochratoxin A occurrence in pig kidneys

Ochratoxin A is a carcinogen and nephrotoxin which can enter the food chain resulting in human exposure. As pig herds are exposed to ochratoxin A through their feed, their kidneys, livers and pork meat are considered as a possible route of exposure for humans. France, an important producer of pork and pork products, set up a national monitoring programme which included the training of six routine public laboratories in the analysis of ochratoxin A using an immunoaffinity step followed by a HPLC-fluorimetric detection. The programme randomly sampled 300 healthy and 100 nephropathic pig kidneys in 1997 and 710 healthy pig kidneys in 1998. Less than 10% of samples were significantly contaminated by ochratoxin A : in the 1997 survey, 1% of samples contained 0.40-1.40 microg kg(-1) of ochratoxin A and in the 1998 survey 7.6 % exhibited ochratoxin A levels in the range 0.5-5 microg kg(-1). In the case of nephropathic kidneys, only traces of ochratoxin A (0.16 to 0.48 microg kg(-1)) were detected in six samples out of 100. Even if not a major route of exposure for humans, pigs are clearly exposed to this mycotoxin and monitoring of pork products and of feed for swine is necessary.     

Evidence of ochratoxin A conjugates in urine samples from infants and adults

Ochratoxin A (OTA), a mycotoxin with nephrotoxic and carcinogenic properties, is an important contaminant of food and feed. Analysis of OTA in human biological fluids (blood, urine, or breast milk) has documented frequent exposure to this mycotoxin, yet at quite variable levels in different population groups across the world. Urine is the preferred matrix in biomonitoring since sample collection is non-invasive and better accepted by study participants. As only a small fraction of the ingested OTA is excreted in urine, determination of urinary OTA requires sensitive analytical techniques, and phase-II-metabolites should be also considered as biomarkers of exposure. Yet, data published so far on the presence of OTA-glucuronide/sulfate in human urine have been contradictory. In this study, urines (n = 38) from two groups of breastfed infants (German and Turkish) and from German adults were now analysed for the presence of OTA glucuronides or sulfates by an indirect method, i.e. by comparing the levels of OTA (aglycone) in urines without and after enzymatic hydrolysis with ß-glucuronidase/arylsulfatase. Additionally, ochratoxin A-8-β-glucuronide and open lactone ochratoxin A-8-β-glucuronide were synthesized to serve as reference materials for metabolite analysis. Attempts for definitive confirmation of glucuronides of OTA via direct identification in LC–MS/MS analysis were hampered by the lower ionizability of the conjugates compared to the parent compound. Considerable increases in OTA levels were found after enzymatic hydrolysis in several (not all) urine samples and provide clear evidence for the excretion of OTA-conjugates. The latter observation is of importance, since OTA phase-II-metabolites may escape detection when direct methods are applied for urinary biomarker analysis. In conclusion, enzymatic hydrolysis of urine samples is highly advisable in order to avoid an underestimation of the OTA-exposure.     

Conversion of ochratoxin C into ochratoxin A in vivo.

The conversion of ochratoxin C to ochratoxin A was studied in rats after oral and intravenous administration. The concentration of ochratoxin A in the blood as a function of time was the same after oral administration of equivalent amounts of either ochratoxin C or ochratoxin A. The maximum ochratoxin A concentrations were measured 60 min after administration. Given intravenously, ochratoxin C was also converted to ochratoxin A. Maximum concentrations were reached after 90 min. It is concluded that ochratoxin C is readily converted to ochratoxin A after both oral and intravenous administration. There is reason to believe that a comparable toxicity of the two toxins is based upon this conversion and that only interference with the biotransformation mechanisms may cause a difference in their toxicity.     

The occurrence of ochratoxin A in dust collected from a problem household

Accumulated dust samples were collected from the heating ducts in a household where signs resembling ochratoxin poisoning in animals occurred. Several Penicillium spp. and Aspergillus ochraceous had been identified previously from air samples taken from this house. A composite sample from six collected samples was examined by HPLC, and it was determined that 58 ppb of ochratoxin A was present in this sample. A second set of six samples was collected and determinations were made by HPLC of the ochratoxin content in each sample. All samples, including one sample of dirt from a crawl space, yielded at least a trace of ochratoxin A; however, one sample of dust collected from the heating ducts yielded over 1500 ppb of ochratoxin A, and another sample of dust from a different heating duct yielded 306 ppb of ochratoxin A. Ochratoxin A was confirmed in all samples by LC-MS, and ochratoxin was evident in the samples by TLC analysis. This is believed to be the first report of finding ochratoxin inhouse dust. This revised version was published online in June 2006 with corrections to the Cover Date.     

A survey on the occurrence of ochratoxin A in feeds for swine and laying hens

Results of a 2-year (2009–2010) survey on the occurrence of ochratoxin A (OTA) in swine feed and in feed for laying hens in Portugal are reported. A total of 664 samples (478 swine feed, 186 feed for laying hens) were analyzed by a HPLC method using fluorescence detection with 2 μg kg^?1 as detection limit. In swine feed, 31 samples (6.49%) were positive for OTA. In feed for laying hens, 12 samples (6.45%) were OTA-positive. The average levels of contamination were low, with median values of positive samples at 3–4 μg kg^?1 in both years and both commodities, although a few samples contained exceptionally high levels (maximum 130 μg kg^?1). Only the maximum level sample (swine feed) contained OTA at a concentration exceeding the European Commission guidance value. The remaining OTA concentrations found in feed samples were much lower than the guidance values.     

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.     

Novel highly-performing immunosensor-based strategy for ochratoxin A detection in wine samples

The increasing concern about ochratoxin A (OTA) contamination of different food and feedstuffs demands high-performing detection techniques for quality assessment. Two indirect competitive enzyme-linked immunosorbent assay (ELISA) strategies were investigated for the development of OTA electrochemical immunosensors based on different OTA immobilisation procedures. Immunosensors based on avidin/biotin-OTA showed enhanced performance characteristics compared to those based on the adsorption of bovine serum albumin (BSA)-OTA conjugate. Performance of polyclonal (PAb) and monoclonal (MAb) antibodies against OTA was compared, showing at least one-order of magnitude lower IC(50) values when working with MAb. Alkaline phosphatase (ALP)- and horseradish peroxidase (HRP)-labelled secondary antibodies were evaluated. Both conjugates led to similar results when working with OTA standard solutions in buffer. However, whereas electroactive interferences present in spiked wine samples did not affect HRP-labelled immunosensors (4% slope deviation), they were likely oxidised at 0.225 V versus Ag/AgCl, the working potential for ALP-labelled immunosensors (25% slope deviation). Considering 80% of antibody binding as the limit of detection, values of 0.7 and 0.3 ng/mL for HRP- and ALP-labelled immunosensors respectively, validate these immunosensors as useful screening tools to assess OTA levels in wine.     

Uptake and genotoxic effects of ochratoxin A in cultured porcine urinary bladder epithelial cells

Uptake of radiolabelled ochratoxin A (OTA) into porcine urinary bladder epithelial cells (PUBEC) was measured at neutral (pH 7.5) or acidic (pH 5.0) conditions. Genotoxicity of OTA was evaluated with the Comet assay and cytotoxicity with the neutral red uptake assay. At acidic pH-conditions, the bladder cells were able to take up more OTA than at neutral conditions. Cytotoxic effects were not increased at pH 5.0 compared to pH 7.5, but higher OTA uptake correlated with stronger genotoxic effects in the Comet assay at pH 5.0 compared to pH 7.5. These results demonstrate that uptake of OTA has to be regarded as an important factor for the toxicity of OTA as adverse effects depend on the amount of OTA taken up by the cells. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

The beta-glucosidases of porcine kidney.

Some of the properties of a partially purified particle bound and soluble beta-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble beta-glucosidase (1) hydrolyzed 4-methylumbelliferyl-beta-D-glucoside (4-MU-beta-D-glucoside) 17 alpha-estradiol 3beta-glucoside. 17 alpha-estradiol 17beta-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5-7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone beta-glucoside being the most effective. In contrast, a particulate beta-glucodidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-beta-glucosidase and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-beta-D-glucoside or glucosylceramide as the glucosyl donor, and [14C]ceramide as acceptor.     

Development of stable isotope dilution assays for ochratoxin A in blood samples

Two new stable isotope dilution assays were developed for the quantification of ochratoxin A in human blood samples for exposure studies. The methods based on two different sample extraction and cleanup procedures including liquid–liquid extraction with following immunoaffinity chromatography (IA) as well as a dispersive solid-phase extraction (DSPE) method. For detection, LC–MS/MS was applied. For the first time, exact quantitation of the reference compound ochratoxin A was performed by quantitative NMR spectroscopy (qNMR). Additionally, a comparison of different blood-drawing procedures revealed no differences for heparin plasma and serum whereas citrate plasma gave significantly lower results for the mycotoxin. Limits of detection (LOD: 0.02 ng/g (IA) vs 0.03 ng/g (DSPE)), limits of quantification (LOQ: 0.07 ng/g (IA) vs 0.08 ng/g (DSPE)), relative recovery (?94%), precision, and linearity indicated excellent performance of the developed methods.     

Analysis of serum and seminal plasma after feeding ochratoxin A with breeding boars.

The aim of the experiment was to investigate whether or not ochratoxin A (OA) can be detected in seminal plasma after feeding the toxin in five and 10 times of the human tolerable daily intake with breeding boars and how toxin profiles of serum and seminal plasma correspond to each other. In addition to that, the effect of the toxin challenge on motility and longevity of boar semen was also evaluated. OA from samples was analyzed by microplate ELISA. Percentage of progressive motility of spermatozoa was determined initially and after 24, 48, 96, 120 and 144 h of storage. OA appeared in serum and seminal plasma shortly after toxin application had started. Significant reduction of initial motility and impaired longevity was observed after toxin withdrawal. These findings suggest that OA might have the potential to affect sperm production and semen quality of boars, but further research is required to elucidate whether OA exerts direct effect on germinal epithelium or disturbs sperm cell maturation only.     

Ochratoxin A in porcine blood and in consumed feed samples

The aim of the study was to estimate occurrence of ochratoxin A (OA) in feeds and the metabolite residues in porcine blood serum in Poland. Samples were collected in the period from February to May, 1999, in the southern Wielkopolska region. Altogether 40 and 45 samples of feed and porcine blood serum, respectively, were analyzed for OA. Percentage of samples contaminated with OA, both in case of feeds and blood, collected in the winter season was considerably higher than that for the spring season. The percentages for feeds were as follows: 47.6 and 26.3 %, while for porcine serum: 66.7 and 50.0 %, respectively winter and spring. In 25 % of cases ochratoxin A was present in both types of investigated material (feed, blood), whereas in 27.5 % of samples this metabolite was detected in blood only, or in 7.5 % only in the feed. The presence of OA was found neither in the feed nor in the serum in 40 % of all cases. In subgroups (feed, blood) the concentration in the whole collective of positive samples were in the range 0.3–13.5 ng/g and 0.3–69.5 ng/ml, respectively, while median values were 2.3 ng/g and 6.0 ng/ml. Only one feed and three porcine serum samples, were found to be contaminated at concentration levels higher than 10 ng/g or 10 ng/ml.