J. Bello

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.

DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours.

The level of expression of enzymes that can activate or detoxify bioreductive agents within tumours has emerged as an important feature in the development of these anti-tumour compounds. The levels of two such reductase enzymes have been determined in 19 human non-small-cell lung tumours and 20 human breast tumours, together with the corresponding normal tissue. DT-diaphorase (DTD) enzyme levels (both expression and activity) were determined in these samples. Cytochrome b5 reductase (Cytb5R) activity was also assessed. With the exception of six patients, the levels of DTD activity were below 45 nmol min(-1) mg(-1) in the normal tissues assayed. DTD tumour activity was extremely variable, distinguishing two different groups of patients, one with DTD activity above 79 nmol min(-1) mg(-1) and the other with levels that were in the same range as found for the normal tissues. In 53% of the lung tumour samples, DTD activity was increased with respect to the normal tissue by a factor of 2.4-90.3 (range 79-965 nmol min[-1] mg[-1]). In 70% of the breast tumour samples, DTD activity was over 80 nmol min(-1) mg(-1) (range 83-267 nmol min[-1] mg[-1]). DTD expression measured by Western blot correlated well with the enzyme activity measured in both tumour and normal tissues. The levels of the other reductase enzyme, Cytb5R, were not as variable as those for DTD, being in the same range in both tumour and normal tissue or slightly higher in the normal tissues. The heterogeneous nature of DTD activity and expression reinforces the need to measure enzyme levels in individual patients before therapy with DTD-activated bioreductive drugs.

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...

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.

Determination of ochratoxin A in pig liver-derived pa?te´s by high-performance liquid chromatography

A solid phase extraction procedure followed by HPLC analysis with fluorescence detection has been developed for ochratoxin A (OTA) in pâtés derived from pig liver. After a previous extraction of OTA with 60% acetonitrile, all the samples were purified through C8 columns. The percentage recovery was 85.7% and the lower limits calculated for accurate detection and quantitation were 0.56ng/g and 0.84ng/g respectively. The HPLC procedure showed a good linearity in the interval of equivalent OTA concentrations of 0.84 to 3ng/g. Values <10% were obtained for precision in HPLC determinations performed (n=3) and (n=9). Stability of calibration standards and samples during the analytical procedure was also demonstrated. This method was successfully applied to 38 pig-derived pâtés and three samples were found to be positive with OTA levels above the detection limit. The highest concentration (1.77ng/g) has been found in a home-made pâté.

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.

Induction of micronuclei in V79 cells after combined treatments with heterocyclic aromatic amines.

Heterocyclic aromatic amines (HAs) appear in foods rich in proteins when subjected to different cooking processes. These amines have been demonstrated to be mutagenic in bacteria; in eucaryotic cells, controversial results have been referred. The objective of this study is to evaluate the clastogenic and/or aneugenic capacity of three HAs--2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx), and 2-amino-3-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)--in isolated as well as in combined treatments. The micronucleus test in vitro was used on V79 cells in the presence and absence of metabolic activation. The duration of the treatment was 2 h, and cytochalasin B was added for 21 h to stop cytokinesis; then, micronuclei (MN) were counted in binucleated cells. In the presence of metabolic activation, the three amines showed a significant increase in the number of MN with respect to the negative control. The PhIP amine presented the highest values and it also resulted slightly active in the absence of metabolic activation, although these differences have not been considered to be significant. The combined treatments of these amines have shown that the effects attributed to them when administered together are those that are expected for a possible additive effect; the effect attributed to each HA separately is not potentiated nor inhibited.