Ochratoxin A in adult population of Lleida, Spain: Presence in blood plasma and consumption in different regions and seasons

Ochratoxin A (OTA) levels in blood plasma, as well as the consumption of possibly contaminated foodstuffs by adult inhabitants, were determined in three seasons in the plain and the mountain regions of the province of Lleida (Spain). Daily intake of the toxin was estimated in order to evaluate the exposure of the studied population. OTA was extracted from plasma through liquid-liquid extraction followed by immunoaffinity chromatography columns clean-up. Detection was done through HPLC-fluorescence, and limit of detection was 0.018 ng/mL. Consumption data of the participants were obtained by means of a food frequency questionnaire. Occurrence of OTA in plasma was 100%. Range was 0.06-10.92 ng/mL, and median was 0.50 ng/mL. Differences between genders, regions or seasons were not significant, whereas significant differences were found among age groups. Regarding food consumption, significant differences were found between genders, but not between age groups, regions, or seasons. OTA plasma levels were not correlated with food consumption. Distributions of the intake estimations based on plasma levels differed from those based on food consumption and contamination. Mean and median values of the daily intake estimations were below the latest provisional tolerable daily intake of 14 ng/kg bw/day, but some high percentiles were above it.     

Assessment of the exposure to ochratoxin A in the province of Lleida,Spain

Exposure to ochratoxin A (OTA) of 279 blood donors of nine localities of the province of Lleida (Spain) was assessed. OTA levels were detected in the blood plasma of the participants by HPLC-fluorescence detection with previous clean-up of the samples by immunoaffinity columns. Limit of detection was 0.075 ng/mL. Participants answered a questionnaire on consumption frequency of foods possibly contaminated with OTA. Foodstuffs were grouped: cereals and derived products, dried fruits and derived products, cacao and derived products, grape juice, wine, beer and coffee. The range of positive samples was 0.11–8.68 ng/mL and the median was 0.54 ng/mL. No differences were found between OTA plasma levels in men and women, neither in the different localities, but there were significant differences among three age groups. Highest consumed foods were cereals and derived products, followed by beer and wine. No correlation was found between food consumption and OTA plasma levels. OTA daily intake was estimated based on OTA plasma concentrations and on the food consumption data combined with food contamination data taken from the literature. Mean values were 1.69 and 1.96 ng/kg body weight/day, respectively. These values are below the latest proposed tolerable daily intake of 14 ng/kg body weight/day.     

Ochratoxin A in cereal-derived products in Turkey: Occurrence and exposure assessment

Eighty-three samples of cereal-derived products including 24 breakfast cereals, 24 cereal-based baby foods and 35 beers purchased from supermarkets and small shops of Adana in Turkey were analysed for the presence of ochratoxin A (OTA) using immunoaffinity column (IAC) clean-up and high performance liquid chromatography with fluorescence detection (HPLC–FD). The average OTA recoveries from spiked breakfast cereal and cereal-based baby food and beers samples were in the range of 79.33–83.86%, 72.93–80.34% and 76.47–83.11%, respectively. The relative standard deviations (RSD_r) of recoveries for breakfast cereals, cereal-based baby foods and beers were 1.1–3.39%, 2.56–8.37% and 5.73–13.61%, respectively.     

Effectiveness of mycotoxin sequestration activity of micronized wheat fibres on distribution of ochratoxin A in plasma,liver and kidney of piglets fed a naturally contaminated diet

A study was carried out to determine the ability of dietary micronized wheat fibres (MWF) to decrease the levels of ochratoxin A (OTA) in plasma, kidney and liver of piglets fed a naturally contaminated diet. A total of 96 piglets (weighting 11.4 ± 1.5 kg) were fed one of four different diets for 28 days. Diets included (1) control diet, (2) control diet with MWF (1%), (3) OTA naturally contaminated diet (117.45 ± 4.74 ng/g), (4) OTA naturally contaminated diet (118.13 ± 2.85 ng/g) with MWF (1%). No difference in feed efficiency (P > 0.05) could be observed between the different diets. The absolute weight of kidneys and liver were significantly higher in pigs fed the OTA-contaminated diet (diet 3) as compared to the control diet (diet 1) or to the control diet amended with MWF (diet 2) (P < 0.05). However the use of MWF (diet 4) significantly protected against these weight changes. A significant protective effect of MWF was also observed in terms of OTA concentration in plasma (45.6% decrease), kidney (40.8% decrease) and liver (26.5% decrease). These results suggest that the addition of MWF is effective in decreasing the bioavailability of OTA from contaminated diets in piglets.     

Variability and uncertainty assessment of patulin exposure for preschool children in Flanders.

The objective of the present study was to evaluate the patulin exposure of children consuming organic, handcrafted or conventional apple juice through a probabilistic approach and to evaluate the effectiveness of several risk management options aiming to reduce the risk for children due to patulin exposure. However, a large part of the data on patulin contamination of apple juice fell under the limit of detection (LOD). Different methods were tested to deal with these so-called left censored data and a uniform distribution with uncertain bounds was selected to handle this censorship. Variability and uncertainty assessment of patulin exposure showed that 0.9% [90% confidence interval (CI): 0.3-1.8%] of the children consuming only organic apple juice exceed the tolerable daily intake (TDI). For consumers of conventional and handcrafted apple juice this was respectively 0.1% [90% CI: 0-0.3%] and 0% [90% CI: 0-0.2%]. Reduction of the patulin contamination in apple juice to concentrations below 25 microg/kg reduced the percentage of the children exceeding the TDI to 0% [90%CI: 0-0.2%] for organic apple juice. Reduction of the apple juice consumption was less effective than a reduction of the patulin concentration in apple juice and is only useful when the patulin concentration of apple juice is below 25 microg/kg.     

Estimation of ochratoxin A in some Turkish populations: An analysis in urine as a simple,sensitive and reliable biomarker

In this study, ochratoxin A (OTA) occurrence and level in human urine samples, collected from four different regions of Turkey was analyzed by NaHCO₃ dilution, immunoaffinity column clean-up and high-performance liquid chromatography with fluorescent detection. For the repeatability of the method, RSD (%) values were found between 3.83 and 8.86, for the accuracy, the recovery values were found between 85.7% and 110.5% and limit of detection and limit of quantification of the method were measured as 0.006 and 0.018 ng mL⁻¹ respectively. For the analysis, first morning urine samples were collected and the results were adjusted with creatinine levels. From the total collected samples of 233 larger amounts of 83% was contaminated with OTA. Among the calculated to be OTA levels, positive sample rate, average OTA amount and the highest contamination was found in Ankara. (Positive sample rate; 90.1%, average OTA concentration; 14.34 ng g⁻¹ creatinine and highest OTA value; 75.60 ng g⁻¹ creatinine). In order to define the exposure profile of OTA in human a questionnaire was conducted among the voluntaries as well. But related to the gender, age, dietary habits, coffee consumption, smoking and alcohol habits of the volunteers, no correlation was found with the OTA exposure.     

Comparison of different exposure assessment methods to estimate the long-term dietary exposure to dioxins and ochratoxin A

Long-term exposures to dioxins (PCCD/F and dioxin-like PCBs) and ochratoxin A were calculated using food consumption data of the European concise database combined with concentration data of the Netherlands (NL) using a deterministic approach. To refine these assessments, exposures were also calculated using three long-term exposure models, observed individual means (OIM), Iowa State University Foods (ISUF), and betabinomial-normal (BBN) models, combined with individual food consumption data of NL. BBN and ISUF correct the variation in long-term exposure for the within-person variation, whereas OIM calculates the mean exposure over the days in the food consumption survey. Exposures obtained with the concise database were highest, and those obtained with OIM higher than with BBN and ISUF. Contribution of the major sources of exposure differed between the concise database and the three models. Given the constraints of the concise database, exposures obtained with this database should be interpreted as a first tier assessment. Preferably, refined assessments using models that correct the variation in long-term exposure for the within-person variation combined with individual food consumption data and national concentration data should be used to assess the long-term exposure. We recommend the use of BBN since it can model exposure distributions that depend on covariates.     

Simulated impact of a fish based shift in the population n--3 fatty acids intake on exposure to dioxins and dioxin-like compounds.

Due to the favourable health effects of LC n-3 PUFAs, marine products have been recognised as a food group of special importance in the human diet. However, seafood is susceptible to contamination by lipophilic organic pollutants. The objective of this study was to evaluate intake levels of PCDDs, PCDFs and dioxin-like PCBs, by a probabilistic Monte Carlo procedure, in relation to the recommendation on LC n-3 PUFAs given by Belgian Federal Health Council. Regarding the recommendation, two scenarios were developed differing in LC n-3 PUFAs intake: a 0.3 E% and a 0.46 E% scenario. Total exposure to dioxins and dioxin-like substances in the 0.3 E% LC n-3 PUFAs scenario ranges from 2.31 pg TEQ/kg bw/day at the 5th percentile, over 4.37 pg TEQ/kgbw/day at the 50th percentile to 8.41 pg TEQ/kgbw/day at the 95th percentile. In the 0.46 E% LC n-3 PUFAs scenario, 5, 50 and 95th percentile are exposed to 2.74, 5.52 and 9.98 pg TEQ/kgbw/day, respectively. Therefore, if the recommended LC n-3 PUFAs intake would be based on fish consumption as the only extra source, the majority of the study population would exceed the proposed health based guidance values for dioxins and dioxin-like substances.     

Ochratoxin A induces karyomegaly and cell cycle aberrations in renal tubular cells without relation to induction of oxidative stress responses in rats

Ochratoxin A (OTA) is a renal carcinogen that induces karyomegaly in target renal tubular cells of the outer stripe of the outer medulla (OSOM). This study was performed to clarify the relationship between oxidative stress and the karyomegaly-inducing potential involving cell cycle aberration of OTA in the OSOM. Rats were treated with OTA for 28 days in combination with enzymatically modified isoquercitrin (EMIQ) or α-lipoic acid (ALA) as antioxidants. OTA increased the mRNA levels of the antioxidant enzyme-related genes Gpx1, Gpx2, Gstm1 and Nfe2l2, but did not increase the levels of Gsta5, Keap1, Nqo1, Hmox1, Aldh1a1, Por, Prdx1 and Txn1. OTA also did not change the levels of thiobarbituric acid-reactive substances, glutathione disulfide/reduced glutathione, and the immunoreactive tubular cell distribution of nuclear factor erythroid 2-related factor 2 in the OSOM. Co-treatment with EMIQ or ALA did not cause any changes in these parameters. As previously reported, OTA increased cell proliferation activity, apoptosis and immunohistochemical cellular distributions of molecules suggestive of induction of DNA damage and cell cycle aberrations involving spindle checkpoint disruption and cell cycle arrest. However, co-treatment with EMIQ or ALA did not suppress these changes, and ALA co-treatment increased the cell proliferation activity induced by OTA. These results suggest that OTA facilitates cell cycling involving cell cycle aberrations and apoptosis as a basis of the mechanism behind the development of karyomegaly and subsequent carcinogenicity targeting the OSOM, without relation to induction of oxidative stress. On the other hand, ALA may promote the OTA-induced proliferation of carcinogenic target cells.     

The benchmark dose approach in food risk assessment: Is it applicable and worthwhile?

The benchmark dose (BMD) approach is being increasingly used in the area of food risk assessment because it offers several advantages compared to the conventional no-observed-adverse-effect-level approach. The aim of this work was to check the applicability of the BMD approach on toxicity data available from pesticides, mycotoxins and natural toxins.     

Effects of lovastatin on the pharmacokinetics of diltiazem and its main metabolite, desacetyldiltiazem, in rats: possible role of cytochrome P450 3A4 and P-glycoprotein inhibition by lovastatin

Objectives The purpose of this study was to examine the effects of lovastatin on cytochrome P450 (CYP) 3A4 and P‐glycoprotein (P‐gp) in vitro and then to determine the effects of lovastatin on the pharmacokinetics of diltiazem and its main metabolite, desacetyldiltiazem, in rats. Methods The pharmacokinetic parameters of diltiazem and desacetyldiltiazem were determined after orally administering diltiazem (12 mg/kg) to rats in the presence and absence of lovastatin (0.3 and 1.0 mg/kg). The effect of lovastatin on P‐gp as well as CYP3A4 activity was also evaluated. Key findings Lovastatin inhibited CYP3A4 enzyme activity with a 50% inhibition concentration of 6.06 µM. In addition, lovastatin significantly enhanced the cellular accumulation of rhodamine‐123 in MCF‐7/ADR cells overexpressing P‐gp. Compared with the control (given diltiazem alone), the presence of lovastatin significantly altered the pharmacokinetic parameters of diltiazem. The areas under the plasma concentration–time curve (AUC) and the peak concentration of diltiazem were significantly increased (P < 0.05, 1.0 mg/kg) in the presence of lovastatin. Consequently, the absolute bioavailability values of diltiazem in the presence of lovastatin (11.1% at 1.0 mg/kg) were significantly higher (P < 0.05) than that of the control group (7.6%). The metabolite–parent AUC ratio in the presence of lovastatin (1.0 mg/kg) was significantly (P < 0.05) decreased compared with the control group. Conclusions It might be considered that lovastatin resulted in reducing the first‐pass metabolism in the intestine and/or in the liver via inhibition of CYP3A4 and increasing the absorption of diltiazem in the intestine via inhibition of P‐gp by lovastatin.     

Arsenite and its metabolites, MMA and DMA, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA^III) and dimethylarsinous acid (DMA^III) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA^III induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA^III increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA^III induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.     

Effects of simvastatin on the pharmacokinetics of diltiazem and its main metabolite, desacetyldiltiazem, after oral and intravenous administration in rats: possible role of P-glycoprotein and CYP3A4 inhibition by simvastatin

The purpose of this study was to investigate the possible effects of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, simvastatin, on the pharmacokinetics of diltiazem and its main metabolite, desacetyldiltiazem, in rats. HMG-CoA reductase inhibitors and diltiazem are sometimes prescribed as a combination therapy for the prevention or treatment of cardiovascular diseases. The effect of simvastatin on P-glycoprotein (P-gp) and cytochrome P450 (CYP) 3A4 activity was evaluated. Simvastatin inhibited CYP3A4 enzyme activity in a concentration-dependent manner with a 50% inhibition concentration (IC(50)) of 3.0 μM. In addition, simvastatin significantly enhanced the cellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp. The pharmacokinetic parameters of diltiazem and desacetyldiltiazem were determined after oral and intravenous administration of diltiazem to rats in the presence and absence of simvastatin (0.3 and 1.0 mg/kg). The areas under the plasma concentration-time curve (AUC) and the peak concentration (C(max)) of diltiazem were significantly (p < 0.05, 1.0 mg/kg) increased by 45.2% and 35.2%, respectively, in the presence of simvastatin compared to control. Consequently, the absolute bioavailability (AB) values of diltiazem in the presence of simvastatin (1.0 mg/kg) were significantly (p < 0.05) higher (44.8%) than that of the control group. Moreover, the relative bioavailability (RB) of diltiazem was 1.21- to 1.45-fold greater than that in the control group. The metabolite-parent AUC ratio (MR) in the presence of simvastatin (1.0 mg/kg) significantly decreased compared to the control group. This result implied that simvastatin effectively inhibited the metabolism of diltiazem. The increase in diltiazem oral bioavailability might be attributable to enhanced absorption in the small intestine via the inhibition of P-gp and to reduced first-pass metabolism of diltiazem via the inhibition of the CYP3A subfamily in the small intestine and/or in the liver rather than renal elimination of diltiazem by simvastatin.     

Vanadate-induced activation of cytosolic phospholipase A2alpha in L929 cells: Roles of tyrosine kinase, protein kinase C, and extracellular signal-regulated kinase

Orthovanadate (Na3VO4), which acts as an inhibitor of protein tyrosine phosphatases, has a various pharmacological effects including the release of arachidonic acid (AA) from cells. We investigated roles of alpha-type cytosolic phospholipase A2 (cPLA2alpha), Src family kinases (Src) and protein kinase C (PKC) in the release of AA induced by Na3VO4 from a murine fibroblast cell line, L929. C12 cells, a variant of L929 that lacks expression of cPLA2alpha, were used along with a clone of C12 cells that are stably expressing cPLA2alpha (C12-cPLA2alpha cells). In the presence of a Ca2+ ionophore (10 microM A23187), 5 and 10mM Na3VO4 synergistically stimulated AA release from L929 and C12-cPLA2alpha cells, and to a much lesser extent from control C12 cells. The release of AA by Na3VO4/A23187 was inhibited by a selective cPLA2alpha inhibitor (3 microM pyrrophenone). The release of AA by Na3VO4/A23187 was significantly inhibited by a PKC inhibitor (10 microM GF109203X), in PKC-depleted cells, by a Src inhibitor (2 microM PP2) and by an inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2) kinase (10 microM U0126). The phosphorylation of ERK1/2 was stimulated by Na3VO4, and the response was significantly decreased by inhibitors of Src, PKC and ERK1/2 kinase. Our data show that Na3VO4 stimulates AA release largely via cPLA2alpha activation in Ca2+-dependent manner, and the cross-talk between Src and PKC and the ERK-dependent pathways are involved in Na3VO4-induced AA release from L929 cells.     

State of the art in benefit-risk analysis: Food and nutrition

Benefit-risk assessment in food and nutrition is relatively new. It weighs the beneficial and adverse effects that a food (component) may have, in order to facilitate more informed management decisions regarding public health issues. It is rooted in the recognition that good food and nutrition can improve health and that some risk may be acceptable if benefit is expected to outweigh it. This paper presents an overview of current concepts and practices in benefit-risk analysis for food and nutrition. It aims to facilitate scientists and policy makers in performing, interpreting and evaluating benefit-risk assessments.Historically, the assessments of risks and benefits have been separate processes. Risk assessment is mainly addressed by toxicology, as demanded by regulation. It traditionally assumes that a maximum safe dose can be determined from experimental studies (usually in animals) and that applying appropriate uncertainty factors then defines the ‘safe’ intake for human populations. There is a minor role for other research traditions in risk assessment, such as epidemiology, which quantifies associations between determinants and health effects in humans. These effects can be both adverse and beneficial. Benefit assessment is newly developing in regulatory terms, but has been the subject of research for a long time within nutrition and epidemiology. The exact scope is yet to be defined. Reductions in risk can be termed benefits, but also states rising above ‘the average health’ are explored as benefits. In nutrition, current interest is in ‘optimal’ intake; from a population perspective, but also from a more individualised perspective.In current approaches to combine benefit and risk assessment, benefit assessment mirrors the traditional risk assessment paradigm of hazard identification, hazard characterization, exposure assessment and risk characterization. Benefit-risk comparison can be qualitative and quantitative. In a quantitative comparison, benefits and risks are expressed in a common currency, for which the input may be deterministic or (increasingly more) probabilistic. A tiered approach is advocated, as this allows for transparency, an early stop in the analysis and interim interaction with the decision-maker. A general problem in the disciplines underlying benefit-risk assessment is that good dose-response data, i.e. at relevant intake levels and suitable for the target population, are scarce.It is concluded that, provided it is clearly explained, benefit-risk assessment is a valuable approach to systematically show current knowledge and its gaps and to transparently provide the best possible science-based answer to complicated questions with a large potential impact on public health.     

Hepatotoxic effect of ochratoxin A and citrinin,alone and in combination,and protective effect of vitamin E: In vitro study in HepG2 cell

Ochratoxin A (OTA) and citrinin (CTN) are the most commonly co-occurring mycotoxins in a wide variety of food and feed commodities. The major target organ of these toxins is kidney but liver could also be a target organ. The combined toxicity of these two toxins in kidney cells has been studied but not in liver cell. In this study HepG2 cells were exposed to OTA and CTN, alone and in combination, with a view to compare the molecular and cellular mechanisms underlying OTA, CTN and OTA + CTN hepatotoxicity. OTA and CTN alone as well as in combination affected the viability of HepG2 cells in a dose-dependent manner. OTA + CTN, at a dose of 20% of IC₅₀ of each, produced effect almost similar to that produced by either of the toxins at its IC₅₀ concentration, indicating that the two toxins in combination act synergistically. The cytotoxicity of OTA + CTN on hepatocytes is mediated by increased level of intracellular ROS followed/accompanied by DNA strand breaks and mitochondria-mediated intrinsic apoptosis. Co-treatment of vitamin E (Vit E) with OTA, CTN and OTA + CTN reduced the levels of ROS and the cytotoxicity. But the genotoxic effect of OTA and OTA + CTN was not completely alleviated by Vit E treatment whereas the DNA damage as caused by CTN when treated alone was obviated, indicating that OTA induces DNA damage directly whereas CTN induces ROS-mediated DNA damage and OTA + CTN combination induces DNA damage not exclusively relying on but influenced by ROS generation. Taken together, these findings indicate that OTA and CTN in combination affect hepatocytes at very low concentrations and, thereby, pose a potential threat to public and animal health.     

Similarities and differences in the coupling of human beta1- and beta2-adrenoceptors to Gs(alpha) splice variants

The human beta1-adrenoceptor (beta1AR) and beta2-adrenoceptor (beta2AR) couple to Gs-proteins to activate adenylyl cyclase (AC). There are differences in desensitization between the beta2AR and the originally cloned Gly389-beta1AR, but with respect to ternary complex formation, constitutive activity, and AC activation the picture is unclear. To learn more about the similarities and differences between the beta1AR and beta2AR, we analyzed coupling of the Gly389-beta1AR to the G(s(alpha)) splice variants Gs(alpha)L and Gs(alpha)S using beta1AR-Gs(alpha) fusion proteins expressed in Sf9 cells and compared the data with previously published data on beta2AR-Gs(alpha) fusion proteins (Seifert et al., J Biol Chem 1998;273:5109-16). Fusion ensures defined receptor/G-protein stoichiometry and efficient coupling. The agonist (-)-isoproterenol stabilized the ternary complex at beta1AR-Gs(alpha)S, beta1AR-Gs(alpha)L, beta2AR-Gs(alpha)S, and beta2AR-Gs(alpha)L with similar efficiency. beta1AR-Gs(alpha)L but not beta1AR-Gs(alpha)S showed the hallmarks of constitutive activity as assessed by increased potencies and efficacies of partial agonists and AC activation by the agonist-free receptor. Similar differences were observed previously for beta2AR-Gs(alpha)S and beta2AR-Gs(alpha)L. beta1AR-Gs(alpha)S and beta2AR-Gs(alpha)S were similarly efficient at activating AC, but beta1AR-Gs(alpha)L was approximately 4-fold more efficient at activating AC than beta2AR-Gs(alpha)L. Our data show that (i) the beta1AR and beta2AR are similarly efficient at stabilizing the ternary complex with Gs(alpha) splice variants, (ii) Gs(alpha)L confers constitutive activity to the beta1AR and beta2AR, and (iii) the beta1AR coupled to Gs(alpha)L is more efficient at activating AC than the beta2AR coupled to Gs(alpha)L. These data help us understand some of the discrepancies regarding similarities and differences between the beta1AR and beta2AR.