DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR BASED ON SCREEN-PRINTED ELECTRODES FOR OCHRATOXIN A IN PORK MEAT SAMPLES

Ochratoxin A (OTA) is a nephrotoxic, immunosuppressive and teratogenic mycotoxin produced by As- pergillus and Penicillium spp. fungi during food storage. OTA can be detected in cereal products, coffee, wine, beer, cheese and in poultry and pork meat. Many detection techniques, such as liquid chromatography coupled with immunoaffinity column or solid phase extraction cleanup, have been used for OTA determina- tion in different samples (1). In recent years electrochemical techniques have been used for the rapid and accurate detection of OTA (2). The aim of the present study was to develop a new analytical method for OTA quantitative detection in pork meat based on electrochemical sensing, using graphite-based screen-printed electrodes and differential pulse voltammetry (DPV) as detection technique. Experiments were performed with an electrochemical transducer Palmsens, monitored with a personal computer using PSTrace software (Palm Instrument BV, Houten, The Netherlands) for data acquisition and subsequent analysis. The electrochemical assays were performed with miniaturized disposable graphite based screen-printed electrodes (EcoBioServices & Researches s.r.l., Florence, Italy). The effect of pH (range 2-7) and of ionic strength (KCl concentration range 10-200 mM) of the supporting electrolyte solution (acetate buffer) on the DPV peak current and potentials was investigated to optimize the DPV method. The effect of the DPV parameters on OTA oxidation peak was studied. Potential pulse amplitude (Epulse) was evaluated in the range of 10-100 mV. Step height was evaluated in the range of 2-10 mV. The influence of the scan rate was examined in the range of 0.005-0.1V/s. Standard addition method was applied for quantitative analysis. The method was applied for OTA determination in spiked pork meat samples. Results were compared with those provided by a reference HPLC method. The OTA peak current increased with increasing acetate buffer pH (from 2.0 to 7), thus pH of 7.0 for the supporting electrolyte solution was chosen. Concentrations of 75 mM KCl in the supporting electrolyte was selected. The optimization of DPV parameters indicated that best results for voltammograms were obtained from 0 to 1.1 V by using 5 mV potential step, 50 mV potential pulse, 0.01 V/sec scan rate and 0.07 sec time pulse; each scan was performed after an equilibrium time of 30 sec. Calibration graphs of peak height against concentration for OTA by DPV were plotted over the range 25-1000 μg/l in the supporting electrolyte with a LOQ of 25 μg/l. The findings obtained with voltammetric-based sensing were in good agreement with results obtained by HPLC analysis but matrix effects have been detected at lower OTA concentrations indicating the need of more selective extraction procedure. The proposed method is more rapid and inexpensive in comparison with the classical methods for OTA analysis, and can be considered a promising alternative for the evaluation of OTA in meat. 1) Turner et al, Anal Chim Acta, 2009, 632 ,168-180. 2) Prieto-Simón et al, TrAC, 2007, 26, 689-702

Gill histopathology in zebrafish model following exposure to aquacultural disinfectants

The effect of acute exposure of four disinfectants commonly used in aquacultural practice (formalin, potassium permanganate, benzalkonium chloride and malachite green) was studied on the histological structure of adult zebrafish (Danio rerio) gills. Groups of 8 individuals were exposed to a dose of each disinfectant corresponding to the therapeutic dose (TD) and five folds of the therapeutic dose (5xTD). Gills of all exposed zebrafish showed a higher occurrence of histopathological changes. These alterations included a slightly focal proliferation of interlamellar cells with obliteration of interlamellar spaces, mild infiammatory reaction with leucocyte infiltration and lifting of the epithelial layer from gill lamellae. Fish exposed to potassium permanganate showed more severe histopathological changes consisting of necrotic change of lamellar cells, distorsion and apical necrosis of secondary lamellae

DETERMINATION OF OCHRATOXIN A IN FARMED FISH BY ENZYMATIC DIGESTION (ED) COUPLED TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH A FLUORESCENCE DETECTOR (HPLC-FLD)

Several studies have demonstrated that fish feeds contain significant concentrations of chemical contaminants, many of which can bioaccumulate and bioconcentrate in fish tissues (1).  The serious concern regarding the use of fish meal and fish oil in the the aquaculture industry has led to extensive search of alternative raw materials for aquafeeds. The most obvious alternatives are oils and proteins of plants origin. The use of these alternative feed ingredients can introduce contaminants that were previously not associated with fish farming such as mycotoxins (2). Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by various Aspergillus and Penicillium species with nephrotoxic, carcinogenic, immunotoxic and teratogenic potential (3). OTA has been found in several food commodities, including cereals and can also be present in food of animal origin as a result of carryover from contaminated feed (3). The aim of the present study was to determine OTA concentrations in muscle, kidney and liver of 10 seabream and 10 seabass of farmed origin collected on the market. Analysis will be performed by using an enzymatic digestion (ED) method coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). Fish tissues were digested for 1 hour at 37°C with a 1% pancreatin solution in a phosphate buffer and then cleaned up with ethylacetate. After being evaporated to dryness and re-dissolved, the sample was processed using HPLC-FLD. The method was validated for: specificity, recovery, trueness, selectivity, linearity, limit of detection (LOD) and limit of quntification (LOQ), repeatability and reproducibility. Recoveries of analytical method were higher than 85 % for all the matrices. Intra and inter-day repeatability expressed as relative standard deviation were less than 9%. The LOD and LOQ for liver and muscles samples were 0.001 and 0.002 μg/kg, respectively. The LOD and LOQ for kidney samples were 0.01 and 0.02 μg/kg, respectively. The highest concentrations of OTA were found in kidney of the 20 fishes analyzed (rang

OCHRATOXIN A RESIDUES IN HUNTED WILD BOAR (SUS SCROFA) FROM TUSCANY

Ochratoxin A (OTA) is a secondary toxic metabolite synthesized by Aspergillus or Penicillium species, which can contaminate various crops. The International Agency for Research on Cancer classified OTA as a group 2B possible human carcinogen. OTA is nephrotoxic, mutagenic, teratogenic and immunosuppressive. OTA can also be present in meat of animals where its presence comes as a result of animal feeding with contaminated grain and feed mixtures. The Italian Ministry of Health Circular No 10, dated 9 June 1999, establishes, as a guideline, a maximum value of 1 μg/kg OTA for swine meat and meat products. The significant increase in the wild boar population has resulted in an increased prevalence of wild boar meat, offal and ready-made products in the food industry. The aim of the present study was to determine OTA concentrations in muscle, kidney and liver of wild boar hunted in Tuscany region. A total of twenty wild boars (male n=11 female n=9) were collected in the Province of Pisa from November 2014 to April 2015, animals have been slaughtered and the carcass weight were determined (from a min. of 14.9 kg and a max. of 72.0 kg). Samples of kidney, liver and muscles from each wild boar were collected and analyzed with an enzymatic digestion clean-up and high-pressure liquid chromatography with fluorescence detection method (1). The highest levels of OTA were found in the kidneys of the twenty wild boar analyzed (0.07- 2.01 μg/kg, mean 0.58±0.63 μg/kg). The levels found in the liver ranged between 0.08- 1.93 μg/kg, (mean 0.53±0.60). The lowest concentrations were found in muscle (0.04- 0.77 μg/kg, mean 0.24±0.24). In eight samples of the tissue samples examined in this study (4 kidney and corresponding 4 liver), the levels of OTA were higher than the guideline level (1 μg/kg) established by the Italian Ministry of Health. The present results are in agreement with a previous study conducted in Calabria in wild boars (2). Swine are particularly sensitive to OTA, kidneys showed the highest accumulation of the latter 101 Società Italiana delle Scienze Veterinarie toxin, followed by liver and muscle tissue, finally the lowest accumulation is represented in adipose tissue. The present results showed the same type of accumulation in wild boar. Traditionally in Tuscany, as in other regions, wild boar meats are used to produce niche products, especially coppa and salami. In agreement with the research of Monaci et al. (3), dried wild boar meat may contribute to overall OTA intake by carry-over effects into processed meats. Monitoring the quality of meat destined for transformation is a priority in order to decrease the possibility of toxin carry-over to humans. The present study confirms that contamination of meat products by OTA represents a potential emerging source of OTA for distinct segments of the Italian population, who are significant consumers of locally-produced wild boar specialties. 1. Luci G., Vanni M., Ferruzzi G., Mani D., Intorre L., Meucci V. 2016. MethodsX 3: 171-177. 2. Bozzo G., Ceci E., Bonerba E., Di Pinto A., Tantillo G., De Giglio E. 2012. Toxins (Basel) 4: 1440-1450. 3. Monaci L., Tantillo G., Palmisano F. 2004. Analytical and Bioanalytical Chemistry 378: 1777- 1782

Determination of ochratoxin A in pig tissues using enzymatic digestion coupled with high-performance liquid chromatography with a fluorescence detector

We present a new method for the rapid analysis of ochratoxin A (OTA) in pig tissues (muscle, liver and kidney) using enzymatic digestion (ED) coupled to high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). OTA was digested with a 1% pancreatin solution in a phosphate buffer and then cleaned with ethylacetate. After being evaporated to dryness and re-dissolved, the sample was determined using HPLC-FLD. The method was validated taking into account the currently permitted limit of 1 μg/kg OTA in pork meat and derived products in Italy. The recovery was higher than 90%. Intra- and inter-day repeatability expressed as RSD were less than 7%. The LOD and LOQ were 0.001 and 0.002 μg/kg, respectively. Our method is more efficient, easier, and cheaper than conventional clean-up procedures (liquid–liquid extraction)

Serum levels of Ochratoxin A in dogs with chronic kidney disease (CKD): a retrospective study

Ochratoxin A (OTA) is a mycotoxin produced by secondary metabolism of several fungi belonging to the genera Aspergillus and Penicillium. OTA is potentially nephrotoxic, neurotoxic, immunotoxic and carcinogenic in several animal species and in humans. This toxin has been detected in several human food and animal feed. The aim of this study was to determine OTA in blood samples of healthy and affected by chronic kidney disease (CKD) dogs. CKD group showed higher incidence of OTA-positivity than healthy dogs (96% vs. 56%) and a significantly higher median value of OTA plasma concentration (0.008 ng/ml vs. 0.144 ng/ml). No significant correlation was observed between OTA levels and creatinine values in CKD dogs. This is first study regarding OTA detection in plasma samples of healthy and CKD dogs; the presence of this toxin is higher in nephropatic patients but is not yet clear, if it is correlated with progression of the disease

Ecotoxicological properties of ketoprofen and the S(+)‐enantiomer (dexketoprofen): Bioassays in freshwater model species and biomarkers in fish PLHC‐1 cell line

The increased use of non-steroidal anti-inflammatory drugs (NSAIDs) has resulted in their ubiquitous presence in the environment. The toxicological properties of these two widely prescribed NSAIDs, namely - racemic ketoprofen (rac-KP) and its enantiomer S(+)-ketoprofen (dexketoprofen, DKP) were evaluated. Firstly, by acute and chronic toxicity tests using three representative model organisms (Vibrio fischeri, Pseudokirchneriella subcapitata and Ceriodaphnia dubia). Secondly, by evaluating the responses of biotransformation systems and multidrug resistance associated proteins (MRP1/MRP2) using the PLHC-1 fish hepatic cell-line. Toxicity data from both acute and chronic DKP exposure indicated higher sensitivity through inhibition of bioluminescence and algal growth and through increased mortality/immobilization compared to rac-KP exposure. The growth inhibition test showed that rac-KP and DKP exhibited different values for EC50 (240.2 µg/L and 65.6 µg/L, respectively). Furthermore, rac-KP and DKP did not exert cytotoxic effects in PLHC-1 cells, and produced compound-, time- and concentration-specific differential effects on CYP1A and GST levels. For CYP1A, the effects of rac-KP and DKP differed at transcriptional and catalytic level. Exposure to rac-KP and DKP modulated MRP1 and MRP2 mRNA levels and these effects were also dependent on compound, exposure time and concentration of the individual drug. The present study revealed for the first time, the interactions between these NSAIDs and key detoxification systems, and different sensitivity to the racemic mixture compared to its enantiomer. This article is protected by copyright. All rights reserved

Novel organ-specific effects of Ketoprofen and its enantiomer, dexketoprofen on toxicological response transcripts and their functional products in salmon.

Racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are widely used non-steroidal anti-inflammatory drugs (NSAIDs), and commonly detected in the aquatic environment. The present study has evaluated the toxicological effects of RS-KP and S(+)-KP on biotransformation and oxidative stress responses in gills and liver of Atlantic salmon. Fish were exposed for 10 days using different concentrations of RS-KP (1, 10 and 100 μg/L) and S(+)-KP (0.5, 5 and 50 μg/L). Biotransformation and oxidative stress responses were analysed at both transcript and functional levels. In the gills, significant inhibitory effect at transcriptional and enzymatic levels were observed for biotransformation and oxidative stress responses. On the contrary, biotransformation responses were significantly increased at transcriptional and translational levels in the liver, while the associated enzymatic activities did not parallel this trend and were inhibited and further demonstrated by principal component analysis (PCA). Our findings showed that both compounds produced comparable toxicological effects, by producing organ-specific effect differences. RS-KP and S(+)-KP did not bioaccumulate in fish muscle, either due to rapid metabolism or excretion as a result of their hydrophobic properties. Interestingly, the inhibitory effects observed in the gills suggest that these drugs may not undergo first pass metabolism, that might result to downstream differences in toxicological outcomes

Novel organ-specific effects of Ketoprofen and its enantiomer, dexketoprofen on toxicological response transcripts and their functional products in salmon

Racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are widely used non-steroidal anti-inflammatory drugs (NSAIDs), and commonly detected in the aquatic environment. The present study has evaluated the toxicological effects of RS-KP and S(+)-KP on biotransformation and oxidative stress responses in gills and liver of Atlantic salmon. Fish were exposed for 10 days using different concentrations of RS-KP (1, 10 and 100 μg/L) and S(+)-KP (0.5, 5 and 50 μg/L). Biotransformation and oxidative stress responses were analysed at both transcript and functional levels. In the gills, significant inhibitory effect at transcriptional and enzymatic levels were observed for biotransformation and oxidative stress responses. On the contrary, biotransformation responses were significantly increased at transcriptional and translational levels in the liver, while the associated enzymatic activities did not parallel this trend and were inhibited and further demonstrated by principal component analysis (PCA). Our findings showed that both compounds produced comparable toxicological effects, by producing organ-specific effect differences. RS-KP and S(+)-KP did not bioaccumulate in fish muscle, either due to rapid metabolism or excretion as a result of their hydrophobic properties. Interestingly, the inhibitory effects observed in the gills suggest that these drugs may not undergo first pass metabolism, that might result to downstream differences in toxicological outcomes

Aflatoxins M1 and M2 in the milk of donkeys fed with naturally contaminated diet

For its nutritional composition, donkey milk is an excellent alternative to breast milk for infants suffering from cow’s milk allergies. Even in donkeys, a passage of aflatoxin from contaminated feed to milk could occur, as reported by many authors in other dairy species, but there are no studies on this topic. This work was aimed at studying the excretion of aflatoxin M1 (AFM1) and M2 (AFM2) in milk after feeding trials with contaminated feed. Six donkeys, at the end of lactation, received a diet with naturally contaminated corn containing 202 and 11 μg.kg−1 of aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2), respectively. Individual milk samples were analyzed for AFM1 and AFM2 for 15 days after the contaminated feed administration. Amounts of AFM1 and AFM2 were detected in the milk. The steady state condition was reached after 6 days. No AFM1 or AFM2 were detected in milk after 28 h from the last contaminated feed administration. The carryover from AFB1 to AFM1 and from AFB2 to AFM2 was found to be 0.02 and 0.31%, respectively. The results obtained in this study are thus a further step toward understanding the possible carryover of aflatoxin in donkey milk