Metabolomic Profiling of In Vivo Plasma Responses to DioxinAssociated Dietary Contaminant Exposure in Rats: Implications for Identification of Sources of Animal and Human Exposure

Dioxin contamination of the food chain typically occurs when cocktails of combustion residues or polychlorinated biphenyl (PCB) containing oils become incorporated into animal feed. These highly toxic compounds are bioaccumulative with small amounts posing a major health risk. The ability to identify animal exposure to these compounds prior to their entry into the food chain may be an invaluable tool to safeguard public health. Dioxin-like compounds act by a common mode of action and this suggests that markers or patterns of response may facilitate identification of exposed animals. However, secondary co-contaminating compounds present in typical dioxin sources may affect responses to compounds. This study has investigated for the first time the potential of a metabolomics platform to distinguish between animals exposed to different sources of dioxin contamination through their diet. Sprague-Dawley rats were given feed containing dioxin-like toxins from hospital incinerator soot, a common PCB oil standard and pure 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (normalized at 0.1 µg/kg TEQ) and acquired plasma was subsequently biochemically profiled using ultra high performance liquid chromatography (UPLC) quadropole time-of-flight-mass spectrometry (QTof-MS). An OPLS-DA model was generated from acquired metabolite fingerprints and validated which allowed classification of plasma from individual animals into the four dietary exposure study groups with a level of accuracy of 97-100%. A set of 24 ions of importance to the prediction model, and which had levels significantly altered between feeding groups, were positively identified as deriving from eight identifiable metabolites including lysophosphatidylcholine (16:0) and tyrosine. This study demonstrates the enormous potential of metabolomic-based profiling to provide a powerful and reliable tool for the detection of dioxin exposure in food-producing animals.

Arsenate causes differential acute toxicity to two P-deprived genotypes of rice seedlings (Oryza sativa L.)

Significant genotypic difference in response to arsenate toxicity in rice (Oryza sativa) was investigated in root elongation, arsenate uptake kinetics, physiological and biochemical response and arsenic (As) speciation. Uptake kinetics data showed that P-deprived genotype 94D-54 had a little higher As uptake than P-deprived 94D-64, but the difference was not large enough to cause acute toxicity in P-deprived 94D-54. There was no difference in tissue P concentrations between the two genotypes under P deficient conditions. In addition, arsenic speciation in plant tissues (using high performance liquid chromatography-inductively coupled plasma mass spectrometry) was not different between P pretreatments and between genotypes. P-deprived genotype 94D-54 suffered much higher stress induced by arsenate toxicity than P-deprived genotype 94D-64, in terms of lipid peroxidation, tissue H2O2 concentrations and exosmosis of K, P and As. However, P-deprived 94D-54 also had higher overproduction of enzymatic antioxidants (with higher GPX, SOD, CAT) and NPT (non-protein thiols) than P-deprived 94D-64. It appeared that, the higher sensitivity of P-deprived 94D-54 to arsenate toxicity might cause the overproduction of NPT, thus leading to the depletion of GSH and to the accumulation of H2O2. The differential sensitivity of the two genotypes has major implications for breeding rice for As affected paddy soil.

Spatial distribution of arsenic and temporal variation of its concentration in rice

P>In order to gain insights into the transport and distribution of arsenic (As) in intact rice (Oryza sativa) plants and its unloading into the rice grain, we investigated the spatial distribution of As and the temporal variation of As concentration in whole rice plants at different growth stages. To the best of our knowledge, this is the first time that such a study has been performed.

Inductively coupled plasma mass spectroscopy (ICP-MS) and high-performance liquid chromatography (HPLC)-ICP-MS were used to analyze total As concentration and speciation. Moreover, synchrotron-based X-ray fluorescence (SXRF) was used to investigate in situ As distribution in the leaf, internode, node and grain.

Total As concentrations of vegetative tissues increased during the 2 wk after flowering. The concentration of dimethylarsinic acid (DMA) in the caryopsis decreased progressively with its development, whereas inorganic As concentration remained stable. The ratios of As content between neighboring leaves or between neighboring internodes were c. 0.6. SXRF revealed As accumulation in the center of the caryopsis during its early development and then in the ovular vascular trace.

These results indicate that there are different controls on the unloading of inorganic As and DMA; the latter accumulated mainly in the caryopsis before flowering, whereas inorganic As was mainly transported into the caryopsis during grain filling. Moreover, nodes appeared to serve as a check-point in As distribution in rice shoots.

Accumulation, Subcellular Distribution and Toxicity of Copper in Earthworm (Eisenia fetida) in the Presence of Ciprofloxacin

Land application of wastes from concentrated animal feeding operations results in accumulation of copper (Cu) and antimicrobials in terrestrial systems. Interaction between Cu and antimicrobials may change Cu speciation in soil solution, and affect Cu bioavailability and toxicity. In this study, earthworms were exposed to quartz sand percolated with different concentrations of Cu and ciprofloxacin (CIP). Copper uptake by earthworms, its subcellular partition, and toxicity were studied. An increase in the applied CIP decreased the free Cu ion concentration in external solution and mortalities of earthworm, while Cu contents in earthworms increased. Copper and CIP in earthworms were fractionated into five fractions: a granular fraction (D), a fraction consisting of tissue fragments, cell membranes, and intact cells (E), a microsomal fraction (F), a denatured proteins fraction (G), and a heat-stable proteins fraction (H). Most of the CIP in earthworms was in fraction H. Copper was redistributed from the metal-sensitive fraction E to fractions D, F, G, and H with increasing CIP concentration. These results challenge the free ion activity model and suggested that Cu may be partly taken up as Cu-CIP complexes in earthworms, changing the bioavailability, subcellular distribution, and toxicity of Cu to earthworms.

Green tea supplementation increases glutathione and plasma antioxidant capacity in adults with the metabolic syndrome

Green tea, a popular polyphenol-containing beverage, has been shown to alleviate clinical features of the metabolic syndrome. However, its effects in endogenous antioxidant biomarkers are not clearly understood. Thus, we tested the hypothesis that green tea supplementation will upregulate antioxidant parameters (enzymatic and nonenzymatic) in adults with the metabolic syndrome. Thirty-five obese participants with the metabolic syndrome were randomly assigned to receive one of the following for 8 weeks: green tea (4 cups per day), control (4 cups water per day), or green tea extract (2 capsules and 4 cups water per day). Blood samples and dietary information were collected at baseline (0 week) and 8 weeks of the study. Circulating carotenoids (a-carotene, ß-carotene, lycopene) and tocopherols (a-tocopherol, ?-tocopherol) and trace elements were measured using high-performance liquid chromatography and inductively coupled plasma mass spectroscopy, respectively. Serum antioxidant enzymes (glutathione peroxidase, glutathione, catalase) and plasma antioxidant capacity were measured spectrophotometrically. Green tea beverage and green tea extract significantly increased plasma antioxidant capacity (1.5 to 2.3 µmol/L and 1.2 to 2.5 µmol/L, respectively; P <.05) and whole blood glutathione (1783 to 2395 µg/g hemoglobin and 1905 to 2751 µg/g hemoglobin, respectively; P <.05) vs controls at 8 weeks. No effects were noted in serum levels of carotenoids and tocopherols and glutathione peroxidase and catalase activities. Green tea extract significantly reduced plasma iron vs baseline (128 to 92 µg/dL, P <.02), whereas copper, zinc, and selenium were not affected. These results support the hypothesis that green tea may provide antioxidant protection in the metabolic syndrome.

Maternal Vitamin D Status in Type 1 Diabetic Pregnancy: Impact on Neonatal Vitamin D Status and Association with Maternal Glycaemic Control

Objective: The first aim of this study was to assess 25-hydroxy vitamin D (25OHD) concentrations in women with type 1 diabetes (T1DM) during pregnancy, post-delivery and also foetal (cord blood) 25OHD concentrations and to examine relationships between these. The second aim of the study was to investigate potential interactions between maternal body mass index (BMI) and foetal vitamin D status. A further study aim was to examine potential relationships between maternal 25OHD and glycosylated haemoglobin (HbA_1c) throughout pregnancy.

Research Design and Methods: This was an observational study of 52 pregnant controls without diabetes and 65 pregnant women with T1DM in a university teaching hospital. Maternal serum 25OHD was measured serially throughout the pregnancy and post-delivery. Cord blood 25OHD was measured at delivery. 25OHD was measured by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Results: Vitamin D deficiency (25OHD <25 nmol/L) was apparent in both the T1DM subjects and controls at all 3 pregnancy trimesters. Vitamin D levels in all cord blood were <50 nmol/L. Maternal 25OHD correlated positively with cord 25OHD at all 3 trimesters in the T1DM group (p= 0.02; p<0.001; p<0.001). 25OHD levels within cord blood were significantly lower for women with diabetes classified as obese vs. normal weight at booking [normal weight BMI <25 kg/m² vs. obese BMI >30 kg/m² (nmol/L±SD); 19.93±11.15 vs. 13.73±4.74, p= 0.026]. In the T1DM group, HbA_1c at booking was significantly negatively correlated with maternal 25OHD at all 3 trimesters (p= 0.004; p = 0.001; p= 0.05).

Conclusion: In T1DM pregnancy, low vitamin D levels persist throughout gestation and post-delivery. Cord blood vitamin D levels correlate with those of the mother, and are significantly lower in obese women than in their normal weight counterparts. Maternal vitamin D levels exhibit a significant negative relationship with HbA_1c levels, supporting a potential role for this vitamin in maintaining glycaemic control. 

Validation study to compare effects of processing protocols on measured Nε_(carboxymethyl)lysine and Nε_(carboxyethyl)lysine in blood.

Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured Ne_(carboxy-methyl)lysine (CML), the best characterised AGE, and its homolog, Ne_(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra_performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.

Development and single laboratory validation of an optical biosensor assay for tetrodotoxin detection as a tool to combat emerging risks in European seafood

Tetrodotoxin (TTX) is a potent neurotoxin emerging in European waters due to increasing ocean temperatures. Its detection in seafood is currently performed as a consequence of using the Association of Analytical Communities (AOAC) mouse bioassay (MBA) for paralytic shellfish poisoning (PSP) toxins, but TTX is not monitored routinely in Europe. Due to ethical and performance-related issues associated with this bioassay, the European Commission has recently published directives extending procedures that may be used for official PSP control. An AOAC-accredited high-performance liquid chromatography (HPLC) method has now been accepted by the European Union as a first action screening method for PSP toxins to replace the MBA. However, this AOAC HPLC method is not capable of detecting TTX, so this potent toxin would be undetected; thereby, a separate method of analysis is required. Surface plasmon resonance (SPR) optical biosensor technology has been proven as a potential alternative screening method to detect PSP toxins in seafood. The addition of a similar SPR inhibition assay for TTX would complement the PSP assay in removing the MBA. The present report describes the development and single laboratory validation in accordance with AOAC and IUPAC guidelines of an SPR method to be used as a rapid screening tool to detect TTX in the sea snail Charonia lampas lampas, a species which has been implicated in 2008 in the first case of human TTX poisoning in Europe. As no current regulatory limits are set for TTX in Europe, single laboratory validation was undertaken using those for PSP toxins at 800 µg/kg. The decision limit (CCa) was 100 µg/kg, with the detection capability (CCß) found to be =200 µg/kg. Repeatability and reproducibility were assessed at 200, 400, and 800 µg/kg and showed relative standard deviations of 8.3, 3.8, and 5.4 % and 7.8, 8.3, and 3.7 % for both parameters at each level, respectively. At these three respective levels, the recovery of the assay was 112, 98, and 99 %.

SPR immunosensor for the detection of okadaic acid in mussels using magnetic particles as antibody carriers

Protein G-coated magnetic particles (MPs) were used as immobilisation supports for an antibody against okadaic acid (MAb(OA)) and carriers into a surface plasmon resonance (SPR) device for the development of a direct competitive immunosensor for okadaic acid (OA). SPR analysis of MAb(OA)-MP conjugates demonstrated that conjugations were successful with complete immobilisation of all the antibody biomolecules onto the MPs. Moreover, MAb(OA)-MP conjugates provided up to 11-fold higher SPR signals, compared to free MAb(OA). The use of conjugates in the direct competition assay provided a 3-fold lower LOD mu g/L (2.6 mu g of OA/L, equivalent to 12 mu g of OA/kg mussel meat). The presence of mussel matrix did not interfere in the OA quantification as seen in the calibration curves. Mussel samples, obtained from Ebro Delta's bays (NW Mediterranean) during a diarrheic shellfish poisoning (DSP) event and in the presence of Dinophysis sacculus, an OA producer, in the shellfish production area, were analysed with the MP-based SPR immunosensor. The OA contents correlated with those obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (y = 0.984x -5.273, R-2 = 0.789, p <0.001) and by mouse bioassay (MBA).

Natural Toxicants: Tetrodotoxin

Tetrodotoxin (tetrodotoxin) is a potent neurotoxin, which shuts down electrical signaling in nerves by blocking the voltage-gated sodium channel proteins in nerve cell membranes. It was originally discovered in puffer fish but is found in a range of animal species and thought to be produced by bacteria. The toxin can be lethal to humans being 10 000 times more potent than cyanide. Human fatalities have been attributed to the ingestion of this toxin through consumption of puffer fish, a delicacy in Japan and other regions, and other marine species. The effects of tetrodotoxin poisoning onset quickly and include shortness of breath, numbness, tingling, light-headedness, paralysis, and irregular heartbeat. Treatment usually consists of respiratory assistance as no antidote has been developed. The accepted method of analysis for tetrodotoxin is the mouse bioassay, although recently more ethical assays have been developed including high performance liquid chromatography, biosensor and enzyme-linked immunosorbant assay.

Monitoring a toxic bloom of Alexandrium minutum using novel microarray and multiplex surface plasmon resonance biosensor technology

Blooms of Alexandrium occur annually during the summer months in the North Channel of Cork Harbour on the south coast of Ireland. This study monitored an extensive bloom of the toxin producing Alexandrium minutum during the summer of 2011 with the use of the MIDTAL (Microarrays for the Detection of Toxic Algae) microarray and a prototype multiplex surface plasmon resonance (multi SPR) biosensor. Microarray signal intensities and toxin results from three testing platforms of the prototype multi SPR biosensor, commercial (CER) enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) were compared against light microscopy counts. The main aim was to demonstrate the use of these methodologies to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying the harmful phytoplankton community and their toxins in natural water samples. Both the microarray signals and multi SPR biosensor results followed a significant trend with light microscopy results and both techniques indicated detection limits of <4000 cells of A. minutum in natural seawater samples.

Evolving to the optoelectronic mouse for phycotoxin analysis in shellfish

Despite ethical and technical concerns, the in vivo method, or more commonly referred to mouse bioassay (MBA), is employed globally as a reference method for phycotoxin analysis in shellfish. This is particularly the case for paralytic shellfish poisoning (PSP) and emerging toxin monitoring. A high-performance liquid chromatography method (HPLC-FLD) has been developed for PSP toxin analysis, but due to difficulties and limitations in the method, this procedure has not been fully implemented as a replacement. Detection of the diarrhetic shellfish poisoning (DSP) toxins has moved towards LC-mass spectrometry (MS) analysis, whereas the analysis of the amnesic shellfish poisoning (ASP) toxin domoic acid is performed by HPLC. Although alternative methods of detection to the MBA have been described, each procedure is specific for a particular toxin and its analogues, with each group of toxins requiring separate analysis utilising different extraction procedures and analytical equipment. In addition, consideration towards the detection of unregulated and emerging toxins on the replacement of the MBA must be given. The ideal scenario for the monitoring of phycotoxins in shellfish and seafood would be to evolve to multiple toxin detection on a single bioanalytical sensing platform, i.e. 'an artificial mouse'. Immunologically based techniques and in particular surface plasmon resonance technology have been shown as a highly promising bioanalytical tool offering rapid, real-time detection requiring minimal quantities of toxin standards. A Biacore Q and a prototype multiplex SPR biosensor have been evaluated for their ability to be fit for purpose for the simultaneous detection of key regulated phycotoxin groups and the emerging toxin palytoxin. Deemed more applicable due to the separate flow channels, the prototype performance for domoic acid, okadaic acid, saxitoxin, and palytoxin calibration curves in shellfish achieved detection limits (IC20) of 4,000, 36, 144 and 46 μg/kg of mussel, respectively. A one-step extraction procedure demonstrated recoveries greater than 80 % for all toxins. For validation of the method at the 95 % confidence limit, the decision limits (CCα) determined from an extracted matrix curve were calculated to be 450, 36 and 24 μg/kg, and the detection capability (CCβ) as a screening method is ≤10 mg/kg, ≤160 μg/kg and ≤400 μg/kg for domoic acid, okadaic acid and saxitoxin, respectively.

Development of a planar waveguide microarray for the monitoring and early detection of five harmful algal toxins in water and cultures

A novel multiplex microarray has been developed for the detection of five groups of harmful algal and cyanobacterial toxins found in marine, brackish, and freshwater environments including domoic acid (DA), okadaic acid (OA, and analogues), saxitoxin (STX, and analogues), cylindrospermopsin (CYN) and microcystins (MC, and analogues). The sensitivity and specificity were determined and feasibility to be used as a screening tool investigated. Results for algal/cyanobacterial cultures (n = 12) and seawater samples (n = 33) were compared to conventional analytical methods, such as high performance liquid chromatography (HPLC) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Detection limits for the 15 min assay were 0.37, 0.44, 0.05, 0.08, and 0.40 ng/mL for DA, OA, STX, CYN, and MC, respectively. The correlation of data obtained from the microarray compared to conventional analysis for the 12 cultures was r(2) = 0.83. Analysis of seawater samples showed that 82, 82, 70, 82, and 12% of samples were positive (>IC20) compared to 67, 55, 36, 0, and 0% for DA, OA, STX, CYN, and MC, respectively, for conventional analytical methods. The discrepancies in results can be attributed to the enhanced sensitivity and cross-reactivity profiles of the antibodies in the MBio microarray. The feasibility of the microarray as a rapid, easy to use, and highly sensitive screening tool has been illustrated for the five-plex detection of biotoxins. The research demonstrates an early warning screening assay to support national monitoring agencies by providing a faster and more accurate means of identifying and quantifying harmful toxins in water samples.

A randomised controlled trial of increasing fruit and vegetable intake and how this influences the carotenoid concentration and activities of PON-1 and LCAT in HDL from subjects with type 2 diabetes

Background

High density lipoproteins (HDL) have many cardioprotective roles; however, in subjects with type 2 diabetes (T2D) these cardioprotective properties are diminished. Conversely, increased fruit and vegetable (F&V) intake may reduce cardiovascular disease risk, although direct trial evidence of a mechanism by which this occurs in subjects with T2D is lacking. Therefore, the aim of this study was to examine if increased F&V consumption influenced the carotenoid content and enzymes associated with the antioxidant properties of HDL in subjects with T2D.

Eighty obese subjects with T2D were randomised to a 1- or ≥6-portion/day F&V diet for 8-weeks. Fasting serum was collected pre- and post-intervention. HDL was subfractionated into HDL₂ and HDL₃ by rapid ultracentrifugation. Carotenoids were measured in serum, HDL₂ and HDL₃ by high performance liquid chromatography. The activity of paraoxonase-1 (PON-1) was measured in serum, HDL₂ and HDL₃ by a spectrophotometric assay, while the activity of lecithin cholesterol acyltransferase (LCAT) was measured in serum, HDL₂ and HDL₃ by a fluorometric assay.

In the ≥6- vs. 1-portion post-intervention comparisons, carotenoids increased in serum, HDL₂ and particularly HDL₃, (α-carotene, p = 0.008; β-cryptoxanthin, p = 0.042; lutein, p = 0.012; lycopene, p = 0.016), as did the activities of PON-1 and LCAT in HDL₃ (p = 0.006 and 0.044, respectively).

To our knowledge, this is the first study in subjects with T2D to demonstrate that increased F&V intake augmented the carotenoid content and influenced enzymes associated with the antioxidant properties of HDL. We suggest that these changes would enhance the cardioprotective properties of this lipoprotein.

Validation study to compare effects of processing protocols on measured N (ε)-(carboxymethyl)lysine and N (ε)-(carboxyethyl)lysine in blood

Epidemiological studies show that elevated plasma levels of advanced glycation end products (AGEs) are associated with diabetes, kidney disease, and heart disease. Thus AGEs have been used as disease progression markers. However, the effects of variations in biological sample processing procedures on the level of AGEs in plasma/serum samples have not been investigated. The objective of this investigation was to assess the effect of variations in blood sample collection on measured N (ε)-(carboxymethyl)lysine (CML), the best characterised AGE, and its homolog, N (ε)-(carboxyethyl)lysine (CEL). The investigation examined the effect on CML and CEL of different blood collection tubes, inclusion of a stabilising cocktail, effect of freeze thaw cycles, different storage times and temperatures, and effects of delaying centrifugation on a pooled sample from healthy volunteers. CML and CEL were measured in extracted samples by ultra-performance liquid chromatography-tandem mass spectrometry. Median CML and CEL ranged from 0.132 to 0.140 mM/M lys and from 0.053 to 0.060 mM/M lys, respectively. No significant difference was shown CML or CEL in plasma/serum samples. Therefore samples collected as part of epidemiological studies that do not undergo specific sample treatment at collection are suitable for measuring CML and CEL.