Blood plasma biomarkers of citrinin and ochratoxin A exposure in young adults in Bangladesh

Citrinin (CIT) and Ochratoxin A (OTA) are nephrotoxic mycotoxins which can co-occur in food commodities, resulting in internal exposure. Studies in many countries reported on the presence of OTA in human blood; however, such biomonitoring data for CIT is still scarce. This study was conducted to characterize both CIT and OTA biomarker levels in plasma of volunteers since food analysis data are insufficient to assess human exposure in Bangladesh. In total 104 blood samples were collected from university students in 2013 (sampling 1: n?=?64, midsummer) and 2014 (sampling 2: n?=?40, end winter) for analysis of CIT and OTA and their metabolites HO-CIT and OTα by LC-MS/MS and HPLC-FD techniques, respectively. CIT and HO-CIT were detected in 90% (max 2.70 ng/mL) and 85% (max 1.44 ng/mL) of all samples. Mean levels in sampling 2 (CIT 0.47 ng/mL; HO-CIT 0.40 ng/mL) were higher than in sampling 1 (0.25 ng/mL; 0.37 ng/mL) indicative of variable CIT exposure. OTA was present in all (max 6.63 ng/mL) and OTα in 98% (max 0.99 ng/mL) of the samples. In sampling 1, mean OTA (0.85 ng/mL) was higher than in sampling 2 (0.51 ng/mL); the reverse situation was found for OTα mean levels. The calculated dietary OTA intake among the students (mean 9.9; max 91.7 ng/kg bw/week) was lower than the tolerable weekly intake for this mycotoxin (120 ng/kg bw/week) set by EFSA. But frequent co-exposure to CIT should be considered, and the results of this study indicate the necessity to identify major sources of CIT and OTA intake in the Bangladeshi population.     

Exposure of neonates to ochratoxin A: first biomonitoring results in human milk (colostrum) from Chile

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OTα) were determined in milk and blood from nine lactating women who provided samples soon after delivery at a hospital in southern Chile. The analytical method applied liquid–liquid extraction with chloroform, and in the case of blood, an extra purification with solid phase extraction prior to HPLC analysis with fluorescence detection. OTA was detected in all human milk samples, with an average concentration of 106?±?45 ng/L (range 44–184 ng/L). Levels of OTα were 40?±?30 ng/L (LOQ 40 ng/L), but increased considerably upon enzymatic hydrolysis with ß-glucuronidase/sulfatase (up to 840?±?256 ng/L) in human milk. By contrast, there was no evidence for conjugates of OTA. The data on OTA in breast milk and levels reported in blood from women in Chile are indicative of an efficient lactational transfer of the mycotoxin. Infant exposure to OTA was estimated by considering their daily OTA intake with human milk at early stages of nursing. For the majority of milk samples, the calculated OTA intake of infants exceeded the tolerable daily intake (TDI) of 5 ng/kg body weight (bw)/day proposed by the Nordic Expert Group, and infant exposure approached the provisional tolerable doses of 14–16 ng/kg bw/day suggested by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) and by EFSA for adults. The present study documents and confirms the presence of OTA in human milk at levels where the TDI can be exceeded. These results point out the need to continue food and biological monitoring and to develop strategies, e.g. dietary recommendations to pregnant and lactating women, aimed to reduce OTA exposure in early periods of life.     

The mechanisms involved in ochratoxin A elimination by Yarrowia lipolytica Y‐2

Biological control of mycotoxin in cereals, fruits and vegetables have emerged as a promising method. In a previous study, Yarrowia lipolytica Y‐2 isolated by our research team showed biocontrol effect on the post‐harvest decay of grapes and ochratoxin A (OTA) elimination in polytoma medium. The aim of this study was to elucidate the possible mechanisms of OTA elimination by Y. lipolytica Y‐2. The results indicated that OTA elimination by Y. lipolytica Y‐2 was attributed to the degradation action of intracellular enzymes but not extracellular enzymes. A degradation product was identified as ochratoxin alpha (OTα) by liquid chromatography‐tandem mass spectrometry. The intracellular enzymes precipitated with 65% saturation of ammonium sulphate degrade OTA the most quickly and 97.2% OTA was degraded within 4 h. Analysis of this fraction showed that two proteins of carboxypeptidase were expressed in Y. lipolytica Y‐2 but not in Y. lipolytica Polh without the ability to degrade OTA. The results of the protein identification combined with product identification indicated that OTA was degraded to OTα by Y. lipolytica Y‐2 through the hydrolysis activity of carboxypeptidases. Additionally, many proteins of Y. lipolytica Y‐2 involved in stress response and reactive O₂ species elimination also played essential role in OTA degradation.     

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.     

Biomonitoring of concurrent exposure to ochratoxin A and citrinin in pregnant women in Bangladesh

Ochratoxin A (OTA) and citrinin (CIT) are both nephrotoxic and teratogenic in animals, and the occurrence of these mycotoxins in food may cause adverse health effects in humans. Data on the combined exposure to these food contaminants are still scarce, especially in pregnancy. Therefore, a biomonitoring study was conducted to determine the presence of urinary biomarkers of exposure to OTA and CIT in pregnant women in Bangladesh. In total, 54 spot urine samples were collected from residents of a rural and a suburban area of the Savar region in Dhaka district for analysis of OTA and CIT urinary biomarkers by previously validated HPLC-FD and LC-MS/MS methods. Most urines were positive for OTA and CIT biomarkers, with OTA being detected in 93 % (range 0.01–0.84 ng/mL) and CIT biomarkers in 87 % (range 0.02–6.93 ng/mL) of all samples. The mean levels of OTA were different between the rural (0.06?±?0.07 ng/mL) and suburban (0.15?±?0.19 ng/mL) study participants. CIT and its metabolite dihydrocitrinone (HO-CIT) were more than twofold higher in the rural (0.42?±?1.20 and 0.55?±?1.04 ng/mL, respectively) than the suburban (CIT 0.15?±?0.13 ng/mL; HO-CIT 0.23?±?0.18 ng/mL) participants. When a provisional daily intake for CIT was calculated, it exceeded the preliminary tolerable value set by European Food Safety Authority (0.2 μg/kg/day) in 9 % of the rural participants but in none of the urban participants. Urinary biomarker levels for OTA and CIT did not show significant association with intake of certain types of food consumed by the pregnant women, although total CIT biomarker levels were considerably higher among participants who consumed more rice in a day. Overall, this study indicates a frequent co-exposure to OTA and CIT among pregnant women in Bangladesh, at levels similar to those determined recently in the general population of this country.     

Effect of ochratoxin A and ochratoxin C on the monocyte and lymphocyte function

The effect of practically relevant mycotoxin concentrations on functions of immune cells was studied in in vitro experiments. Porcine mononuclear cells were exposed to a crudeAspergillus-ochraceus toxin containing OTA, a HPLC fraction identical with OTC derived from the crude toxin (RE2), as well as pure OTA and OTC in a concentration range from 0.46 to 3000 ng/ml. The influence of mycotoxin exposure on metabolic activity, mitogen induced proliferation, expression of the activation marker CD25 and the cell cycle of lymphocytes and on the formation of free oxygen radicals as well as the production of the cytokines IL-6 and TNF-α by monocytes was determined. Exposure to high concentrations of all mycotoxin preparations lead to non-specific suppression of the immune cell functions, which was related to cytotoxic effects. Low concentrations caused ambivalent reactions, especially on monocyte function. In general, the HPLC fraction RE2 had an up to 100-fold stronger effect than pure OTA. Ochratoxin-induced suppression of lymphocyte proliferation was not abrogated by phenylalanine or aspartame. The results indicate that immunomodulation can be caused by very low mycotoxin concentrations which are not related to clinical symptoms or loss of performance.     

Biomonitoring of ochratoxin A in grain workers

Handling agricultural commodities such as grain can result in an inhalation of mycotoxin-containing dusts. Ochratoxin A (OTA) is particularly well suited for biomonitoring studies due to its long half-life in blood, and served as a marker toxin to investigate whether or not exposure to dusts in occupational contexts may result in elevated OTA blood serum levels. OTA analysis was performed for blood samples (n=61) obtained from a cohort of male workers employed at granaries of several grain handling companies in Germany. OTA was analyzed in plasma extracts by HPLC with fluorimetric detection; calibration curves were run for each batch of samples collected between July 2005 and March 2006, and the level of detection was 0.05 ng/ml plasma. The OTA plasma levels of the 61 grain workers ranged between 0.07 ng/ml and 0.75 ng/ml. The mean (0.28±0.13 ng/ml) and median (0.26 ng/ml) OTA value for this cohort was similar to average values previously reported for the German population. Our results gave no indication that OTA in excess of those originating from typical dietary sources was ingested by these workers. Although measurable OTA concentrations have been found in dust samples collected at the corresponding workplaces (Mayeret al, this issue), the biomonitoring data do not provide evidence for a significant inhalatory burden of OTA in grain workers. Since deoxynivalenol and zearalenone were also detected in the dust samples in concentrations much higher than that of OTA, additional research should try to assess the potential relevance of an inhalation exposure to these mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Detoxification of ochratoxin A,a food contaminant: Prevention of growth of Aspergillus ochraceus and its production of ochratoxin A

The toxicity of ochratoxin A (OTA), a mycotoxin produced by fungi ofAspergillus orPenicillium genera is now well documented. Its nephrotoxicity, immunosuppression, teratogenicity, and carcinogenicity have been widely studied. Physical and biochemical methods have been studied to prevent these toxinogenicAspergillus andPenicillium from producing OTA, and/or to destroy the mycotoxin when already produced in a liquid or a solid medium. Repeated freezing at ? 20?C and thawing at + 26?C aleatory reduce OTA production in a liquid medium. Exposure to UV B for different periods of time is efficient in preventing OTA production in a liquid medium. Gamma-irradiation from 2 to 5 kGy gives good results in preventing the production of OTA or destroying it when already produced. Carboxypeptidase is very efficient at 5 units/50 ml in a liquid medium for cleaving the OTA already produced.     

Direct high-performance liquid chromatography determination of propofol and its metabolite quinol with their glucuronide conjugates and preliminary pharmacokinetics in plasma and urine of man

Propofol (P) is metabolized in humans by oxidation to 1,4-di-isopropylquinol (Q). P and Q are in turn conjugated with glucuronic acid to the respective glucuronides, propofol glucuronide (Pgluc), quinol-1-glucuronide (Q1G) and quinol-4-glucuronide (Q4G). Propofol and quinol with their glucuronide conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic hydrolysis. The glucuronide conjugates were isolated by preparative HPLC from human urine samples. The glucuronides of P and Q were present in plasma and urine, P and Q were present in plasma, but not in urine. Quinol in plasma was present in the oxidised form, the quinone. Calibration curves of the respective glucuronides were constructed by enzymic deconjugation of isolated samples containing different concentrations of the glucuronides. The limit of quantitation of P and quinone in plasma are respectively 0.119 and 0.138 μg/ml. The limit of quantitation of the glucuronides in plasma are respectively: Pgluc 0.370 μg/ml, Q1G 1.02 μg/ml and Q4G 0.278 μg/ml. The corresponding values in urine are: Pgluc 0.264 μg/ml, Q1G 0.731 μg/ml and Q4G 0.199 μg/ml. A pharmacokinetic profile of P with its metabolites is shown, and some preliminary pharmacokinetic parameters of P and Q glucuronides are given.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

Simultaneous Quantification of Multiple Urinary Naphthalene Metabolites by Liquid Chromatography Tandem Mass Spectrometry

Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.     

An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer

Ochratoxin A (OTA) is one of the most important mycotoxin contaminants of foods, particularly cereals and cereal products, with strict low regulatory levels (of ppb) in many countries worldwide. An electrochemical competitive aptamer-based biosensor for OTA is described. Paramagnetic microparticle beads (MBs) were functionalized with an aptamer specific to OTA, and were allowed to compete with a solution of the mycotoxin conjugated to the enzyme horseradish peroxidase (OTA-HRP) and free OTA. After separation and washing steps helped with magnetic separations, the modified MBs were localized on disposable screen-printed carbon electrodes (SPCEs) under a magnetic field, and the product of the enzymatic reaction with the substrate was detected with differential-pulse voltammetry. In addition to magnetic separation assays, other competitive schemes (direct/indirect aptasensors performed on the SPCEs surface or using gold nanoparticles functionalized with the aptamer) were preliminary tested, optimized and compared. The magnetic aptasensor showed a linear response to OTA in the range 0.78-8.74 ng mL(-1) and a limit of detection of 0.07±0.01 ng mL(-1), and was accurately applied to extracts of certified and spiked wheat samples with an RSD lower than about 8%.     

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.     

Ochratoxin A blood concentration in healthy subjects and bladder cancer cases from Pakistan

Astract  The mycotoxin ochratoxin A (OTA) is a public health issue in many countries. Data on OTA concentrations in foods and in blood are available for several European countries including the Balkan area, as well as for Canada and Japan. Yet, for developing countries such data are scarce. In this study we determined OTA blood levels as biomarker of exposure in bladder cancer patients and in healthy controls from Pakistan. OTA in blood was analyzed after extraction by HPLC with fluorescence detection (limit of detection: <0.03 ng/mL) in 96 patients and in 31 controls. Over 92% of all blood samples (87 patients, 30 controls) contained quantifiable amounts of OTA: The mean OTA concentrations were 0.33 ng/mL (SD 0.42; range: 0.03 to 3.41 ng/mL) in bladder cancer patients, and 0.31 ng/mL (SD 0.29; range: 0.04 to 1.25 ng/mL) in healthy controls. These OTA concentrations are comparable to those reported for the general population in the European Union. Presented at the 27^th Mykotoxin-Workshop, Dortmund Germany, done 13–15, 2005. The IfADo is accredited as WHO Cellaporating Center for Occupational Health.     

Oxidative damage and stress response from ochratoxin a exposure in rats

Ochratoxin A (OTA) is a mycotoxin found in some cereal and grain products.It is a potent renal carcinogen in male rats, although its mode of carcinogenic action is not known. Oxidative stress may play a role in OTA-induced toxicity and carcinogenicity.In this study, we measured several chemical and biological markers that are associated with oxidative stress response to determine if this process is involved in OTA-mediated toxicity in rats. Treatment of male rats with OTA (up to 2 mg/ 24 h exposure) did not increase the formation of biomarkers of oxidative damage such as the lipid peroxidation marker malondialdehyde in rat plasma, kidney, and liver, or the DNA damage marker 8-oxo-7,8-dihydro-2' deoxyguanosine in kidney DNA. However, OTA treatment (1 mg/kg) did result in a 22% decrease in alpha-tocopherol plasma levels and a 5-fold increase in the expression of the oxidative stress responsive protein haem oxygenase-1, specifically in the kidney. The selective alteration of these latter two markers indicates that OTA does evoke oxidative stress, which may contribute at least in part to OTA renal toxicity and carcinogenicity in rats during long-term exposure.     

PGFM (13,14-dihydro-15-keto-PGF2α) in pregnant and pseudo-pregnant Iberian lynx: A new noninvasive pregnancy marker for felid species

In mammals, uterine and placental prostaglandin F_2α is involved in the regulation of reproduction-related processes such as embryonic development, initiation of parturition, and resumption of ovarian activity. Prostaglandin F_2α (PGF_2α) is rapidly metabolized to its plasma metabolite PGFM (13,14-dihydro-15-keto-PGF_2α), which has also been detected in urine. Therefore, the current study aimed to develop and validate an efficient, quick, and inexpensive enzyme immunoassay (EIA) for PGFM estimation in urine of the Iberian lynx (Lynx pardinus) for pregnancy monitoring and for differentiation between pregnancy and pseudo-pregnancy. Urine samples collected from captive Iberian lynx (11 pregnant and 4 pseudo-pregnant cycles) were subjected directly to a PGFM EIA. The assay was validated for parallelism, precision, and stability of urinary PGFM. In addition, high-performance liquid chromatography (HPLC) immunograms and liquid chromatography-mass spectrometry (LCMS) were performed to identify PGFM within urine samples. Urinary PGFM levels before mating and after parturition were about 1.5 ng/mL. After Day 20 postmating, both pregnant and pseudo-pregnant females showed slight increase of hormone levels; in pseudo-pregnant females, this elevation did not exceed 7 ng/mL. A significant increase in pregnant females was observed after Day 45 postmating; urinary PGFM increased from 10 ng/mL at Day 45 toward a peak of 46.0 ± 19.3 ng/mL around parturition. First results show that PGFM is detectable in feces as well and follows similar courses as shown for urine. In conclusion, the presented and validated PGFM assay is an easy and reliable method for noninvasive pregnancy diagnosis in the Iberian lynx (and probably other felids) if applied approximately 20 d prior parturition in pure urine or fecal extracts. High PGFM levels in urine or fecal samples may allow a pregnancy diagnosis without knowledge of mating time, making the PGFM test applicable to free-ranging animals.     

A reproductive and developmental screening study of the fungal toxin ochratoxin A in Fischer rats

The presence of the mycotoxin ochratoxin A (OTA) in cereal grains is due to the growth of toxigenic Penicillium mold on stored crops. Human exposure to OTA is higher in infants, toddlers, and children than in adolescents and adults, based on exposure assessments of ng OTA consumed/kg body weight/day. Ochratoxin A is nephrotoxic and teratogenic in animals, but its effects on juveniles exposed during the reproduction and development period have not been studied. To address this, Fischer rats were exposed to 0, 0.16, 0.4, 1.0, or 2.5 mg OTA/kg diet throughout breeding, gestation, and lactation and its adverse effects were assessed in adult rats and their offspring on postnatal day (PND) 21. There were no effects on implantation but post-implantation fetotoxicity was observed in the 2.5 mg/kg dose group, corresponding to a calculated dose of 167.0 μg/kg bw/day in dams. Adverse effects on body and kidney weights and on clinical parameters indicative of renal toxicity were significant in adult rats exposed to 1.0 mg OTA/kg diet (55.2 and 73.3 μg/kg bw/day in adult males and females, respectively) and in PND21 rats at the 0.4 mg/kg dose (33.9 μg/kg bw/day in dams), suggesting that weanling rats were more sensitive to OTA than adults. Overall, nephrotoxicity was the primary effect of OTA in weanling rats exposed throughout gestation and lactation at sub-fetotoxic concentrations in diet.     

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.     

基于纳米颗粒的赭曲霉毒素A高灵敏酶联免疫检测方法的建立及应用

赭曲霉毒素(ochratoxins)主要是由青霉菌Penicillium和曲霉菌Aspergillus产生的有毒次级代谢产物,常见于发霉或发酵的农产品中,其中赭曲霉毒素A(ochratoxin A,OTA)毒性最强且最为普遍。OTA是粮食作物和饲料的重要污染物,在加工、储存或运输过程中均可产生,具有肾毒性和免疫毒性,可通过蓄积作用发挥毒性效应,对人类和动物健康造成严重威胁。本研究通过将OTA单克隆抗体包被于纳米磁珠(magnetic nanoparticles,MNPs)表面,获得具有免疫活性的磁珠抗体复合物(MNPs-Anti OTA),并制备生物素标记的偶联抗原OTA-BSA-Bio,后续采用链酶亲和素标记的纳米金颗粒(Strep-HRP-AuNPs)催化底物进行信号检测,最终建立了OTA高灵敏检测方法(MNPs-bs-AuNPs-ELISA)。在最优条件下,经计算该方法检测下限(IC10)为0.01ng/mL,检测区间(IC20-IC80)为0.02-0.73ng/mL,半数抑制率(IC50)为0.13ng/mL。与OTA类似物OTB、OTC交叉反应性为4.3%和8.1%,对其他常见真菌毒素AFB1、ZEN、FB1、DON、CIT和PAT均无交叉反应。玉米、面粉和大豆样本中的加标回收率可达85.6%-115.7%,对天然样本中OTA含量的检测结果表明,该方法与LC-MS/MS相关性良好。本研究建立的MNPs-bs-AuNPs-ELISA可满足谷物及饲料样本中OTA的快速、高灵敏度定量检测,成本较低,具有很好的应用前景。     

The effects of ochratoxin A on lipid peroxidation and antioxidant enzymes: a protective role of melatonin

Various studies indicate that the mycotoxin ochratoxin A (OTA) is a carcinogenic, genotoxic, teratogenic, immunotoxic, and nephrotoxic agent. In the present study, the activities of some enzymes in the serum and liver of rats with ochratoxicosis and the effects of melatonin on these enzymes were investigated. Rats were divided into three equal groups, each consisting of eight rats; control, OTA (289 microg/kg per day) and OTA + melatonin groups for this study. In the OTA treated group, the level of lipid peroxidation (LPO) and the activities of glutathione peroxidase (GSH-Px) were increased in the liver and serum in comparison with the control group. The activities of catalase (CAT) and superoxide dismutase (SOD) were significantly changed in the serum when compared with controls. Our results showed structural tissue damage in the liver in OTA-treated rats. Melatonin decreased the OTA-induced damage to support the antioxidant defense system and/or with free radical scavenger action.