A. López de Cerain

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.

Occurrence of ochratoxin A in cocoa beans: Effect of shelling

The aim of this study was to investigate the influence of the shelling process on the presence of ochratoxin A (OTA) in cocoa samples. Twenty-two cocoa samples were analysed for the determination of OTA before (cocoa bean) and after undergoing manual shelling process (cocoa nib). In order to determine OTA contamination in cocoa samples, a validated high-performance liquid chromatography (HPLC) method with fluorescence detection was used for the quantitative analysis of ochratoxin A (OTA). In both types of samples, OTA was extracted with methanol–3% sodium hydrogen carbonate solution and then purified using immunoaffinity columns prior to HPLC analysis. Due to the fact that different recovery values were obtained for OTA from both types of samples, a revalidation of the method in the case of cocoa nibs was needed. Revalidation was based on the following criteria: Selectivity, limits of detection and quantification (0.03 and 0.1 µg kg−1, respectively), precision (within-day and between-day variability) and recovery 84.2% (RSD = 7.1%), and uncertainty (30%). Fourteen of the twenty-two cocoa bean samples (64%) suffered a loss of OTA of more than 95% due to shelling, six samples suffered a loss of OTA in the range 65–95%, and only one sample presented a reduction of less than 50%. The principal conclusion derived from this study is that OTA contamination in cocoa beans is concentrated in the shell; therefore, improvements of the industrial shelling process could prevent OTA occurrence in cocoa final products.

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 µg/kg for green and roasted coffee, and 0.01 µg/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 µg/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.

Exposure to Ochratoxin a in Europe: Comparison with a Region of Northern Spain

AbstractHuman exposure to Ochratoxin A (OTA) has been detected in Europe and various other countries by analysing human blood. With the aim of determining if such exposure also occurs in Spain, blood samples were collected from healthy donors and from patients undergoing haemodialysis from a region of northern Spain. OTA is routinely analysed by HPLC with fluorescence detection, after validation of the analytical method. The percentage of positive samples was 53.3% in healthy people (n=75), and 77.8% in patients (n=72) (Detection limit = 0.52 ng/ml of plasma). The mean concentration was 0.71 ng/ml for healthy people, and 1.97 ng/ml for the patients. The difference between the two values was statistically significant (p<0.001). After a multivariate adjustment by the multiple linear regression method, and taking into account potentially interfering effects of age, sex and month of extraction, significantly lower levels where found during the months of June and October. No variation depending on age or sex w...

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.

Carbonyl reductase and NADPH cytochrome P450 reductase activities in human tumoral versus normal tissues.

The use of bioreductive agents in enzyme-directed bioreductive therapy has been proposed to take advantage not only of hypoxia in tumours, but also of the presence of reductases that metabolise such compounds. In this study, we studied the activities of NADPH cytochrome P450 reductase (P450R) and carbonyl reductase (CR) in 17 human lung tumours and 18 human breast tumours, together with the corresponding normal tissues. For lung cancer but not for breast cancer there was a significant difference in the CR activity between normal and tumour tissue. CR activity was increased with respect to the normal tissue between 2-fold and 40-fold indicating heterogeneity in tumour samples. No relationship was found between CR activity and the histological type, tumoral grade or TNM stage of the tumours. Although some variation in P450R activity in tumoral versus normal tissues was found in the majority of the samples studied, no significant differences could be demonstrated.

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 μg 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 μg 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.

A different kinetic profile of ochratoxin A in mature male rats.

Ochratoxin A (OTA) is a mycotoxin that causes renal tumors in rodents, particularly in male rats. The present work explored the impact of gender and age on OTA toxicokinetics in F344 rats after a single oral dose (0.5mg/kg b.w.). OTA plasma concentrations were analysed with a validated HPLC-FLD method and a population approach (NONMEM VI) was used to perform the kinetic analysis and the one year exposure simulation (0.21 mg/kg daily). Maximum observed OTA concentration (CMAX(obs)) was at 2h in all groups except in mature females (6h). Mature females reached higher CMAX(obs) than males of the same age. Apparent volume of distribution, but not apparent total plasma clearance, increased significantly with body weight (P<0.01) resulting in the following values for the terminal plasma half life (h) in males: 219 (young), 264 (matures) and females: 191 (young), 205 (matures). In addition mature males showed a significant lower relative bioavailability. The simulation showed similar plasma concentrations in males and females after two-months. Thus, toxicokinetic does not seem to explain sex-differences in toxicity in long-term studies. However, the age and weight should be taken into account in short-term toxicological studies if sex-differences are studied.

Spray-drying of poly(anhydride) nanoparticles for drug/antigen delivery

Gantrez AN nanoparticles can be obtained by desolvation of the poly(anhydride) with an aqueous medium, followed by a purification step and freeze-drying. In order to simplify this method, a spray-drying procedure was evaluated here. For this purpose, suspensions of nanoparticles were mixed with carbohydrates before their drying. Lactose, in a saccharide/polymer ratio of 2, was found to be the most suitable excipient regarding the physicochemical characteristics and stability of the resulting nanoparticles. These conditions can be successfully applied to the preparation of conventional, combined cyclodextrin/polyanhydride or pegylated nanoparticles. The capabilities of the new procedure were also studied by the characterization of nanoparticles loaded with either an antigenic extract (HS from Brucella ovis) or atovaquone. In all cases, the new formulations displayed similar physico-chemical characteristics to those of nanoparticles obtained by lyophilization. In summary, the spray-drying procedure can be an adequate alternative to lyophilization in the preparative process of poly(anhydride) nanoparticles.

A high-performance liquid-chromatographic method for the determination of ochratoxin a in human plasma

SummaryA high-performance liquid-chromatographic method is described for the quantitative determination of the mycotoxin ochratoxin A (OTA) in human plasma. The assay involves extraction with chloroform and sodium bicarbonate then HPLC with fluorescence detection. The method was validated in terms of selectivity, recovery, linearity, precision (within-day and between-day variability), accuracy, detection and quantification limits, and the stability of OTA in plasma and treated samples. The limit of detection was 0.4 ng mL−1 of OTA in methanol, corresponding to 0.52 ng ml−1 OTA in plasma. This assay was successfully applied for the determination of OTA levels in human plasma.