Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle: A parallel-like randomized design.

In the European Union, the use of thyreostats for animal fattening purposes has been banned and monitoring plans have been established to detect potential abuse. However, this is not always straightforward as thyreostats such as thiouracil may also have a semi-endogenous origin. Therefore, this study aimed at defining urinary metabolites, which may aid in defining the origin of detected thiouracil. Hereto, a parallel-like randomized in vivo study was conducted in which calves (n = 8) and cows (n = 8) were subjected to either a control treatment, rapeseed-enriched diet to induce semi-endogenous formation, or thiouracil treatment. Urine samples (n = 330) were assessed through metabolic fingerprinting, employing liquid-chromatography and Q-ExactiveTM Orbitrap mass spectrometry. Urinary fingerprints comprised up to 40,000 features whereby multivariate discriminant analysis was able to point out significant metabolome differences between treatments (Q2(Y) ≥ 0.873). Using the validated models, a total of twelve metabolites (including thiouracil) were assigned marker potential. Combining these markers into age-dependent biomarker panels rendered a tool by which sample classification could be improved in comparison with thiouracil-based thresholds, and this during on-going thiouracil treatment (specificities ≥ 95.2% and sensitivities ≥ 85.7%), post-treatment (sensitivities ≥ 80% for ≥ 24 h after last administration), and simulated low-dose thiouracil treatment (exogenous thiouracil below 30 ng µL-1). Moreover, the metabolic relevance of revealed markers was supported by the suggested identities, for which a structural link with thiouracil could be determined in most cases. The proposed biomarker panels may contribute to a more justified decision-making in monitoring thiouracil abuse.

Pharmacokinetic and urinary profiling reveals the prednisolone/cortisol ratio as a valid biomarker for prednisolone administration.

BACKGROUND: In Europe, synthetic corticosteroids are not allowed in animal breeding for growth-promoting purposes. Nevertheless, a high prevalence of non-compliant urine samples was recently reported for prednisolone, however, without any indication of unauthorized use. Within this context, 20ß-dihydroprednisolone and the prednisolone/cortisol ratio have been suggested as potential tools to discriminate between exogenous and endogenous urinary prednisolone. In this study, the validity of these strategies was verified by investigating the plasma pharmacokinetic and urinary excretion profiles of relevant glucocorticoids in bovines, subjected to exogenous prednisolone treatment or tetracosactide hexaacetate administration to induce endogenous prednisolone formation. Bovine urine and plasma samples were analysed by liquid chromatography and mass spectrometry. RESULTS: Based on the plasma pharmacokinetics and urinary profiles, 20ß-dihydroprednisolone was confirmed as the main prednisolone-derived metabolite, being detected in the biological fluids of all 12 bovines (plasma AUC0-inf of 121 h µg L-1 and urinary concentration > 0.695 µg L-1). However, this metabolite enclosed no potential as discriminative marker as no significant concentration differences were observed upon exogenous prednisolone treatment or tetracosactide hexaacetate administration under all experimental conditions. As a second marker tool, the prednisolone/cortisol ratios were assessed along the various treatments, taking into account that endogenous prednisolone formation involves the hypothalamic-pituitary-adrenal axis and is associated with an increased cortisol secretion. Significantly lower ratios were observed in case of endogenous prednisolone formation (i.e. ratios ranging from 0.00379 to 0.129) compared to the exogenous prednisolone treatment (i.e. ratios ranging from 0.0603 to 36.9). On the basis of these findings, a discriminative threshold of 0.260 was proposed, which allowed classification of urine samples according to prednisolone origin with a sensitivity of 94.2% and specificity of 99.0%. CONCLUSION: The prednisolone/cortisol ratio was affirmed as an expedient strategy to discriminate between endogenous and exogenous prednisolone in urine. Although the suggested threshold value was associated with high specificity and sensitivity, a large-scale study with varying experimental conditions is designated to optimize this value.

Ursodeoxycholic Acid and Its Taurine- or Glycine-Conjugated Species Reduce Colitogenic Dysbiosis and Equally Suppress Experimental Colitis in Mice.

The promising results seen in studies of secondary bile acids in experimental colitis suggest that they may represent an attractive and safe class of drugs for the treatment of inflammatory bowel diseases (IBD). However, the exact mechanism by which bile acid therapy confers protection from colitogenesis is currently unknown. Since the gut microbiota plays a crucial role in the pathogenesis of IBD, and exogenous bile acid administration may affect the community structure of the microbiota, we examined the impact of the secondary bile acid ursodeoxycholic acid (UDCA) and its taurine or glycine conjugates on the fecal microbial community structure during experimental colitis. Daily oral administration of UDCA, tauroursodeoxycholic acid (TUDCA), or glycoursodeoxycholic acid (GUDCA) equally lowered the severity of dextran sodium sulfate-induced colitis in mice, as evidenced by reduced body weight loss, colonic shortening, and expression of inflammatory cytokines. Illumina sequencing demonstrated that bile acid therapy during colitis did not restore fecal bacterial richness and diversity. However, bile acid therapy normalized the colitis-associated increased ratio of Firmicutes to Bacteroidetes Interestingly, administration of bile acids prevented the loss of Clostridium cluster XIVa and increased the abundance of Akkermansia muciniphila, bacterial species known to be particularly decreased in IBD patients. We conclude that UDCA, which is an FDA-approved drug for cholestatic liver disorders, could be an attractive treatment option to reduce dysbiosis and ameliorate inflammation in human IBD.IMPORTANCE Secondary bile acids are emerging as attractive candidates for the treatment of inflammatory bowel disease. Although bile acids may affect the intestinal microbial community structure, which significantly contributes to the course of these inflammatory disorders, the impact of bile acid therapy on the fecal microbiota during colitis has not yet been considered. Here, we studied the alterations in the fecal microbial abundance in colitic mice following the administration of secondary bile acids. Our results show that secondary bile acids reduce the severity of colitis and ameliorate colitis-associated fecal dysbiosis at the phylum level. This study indicates that secondary bile acids might act as a safe and effective drug for inflammatory bowel disease.

Effect of oxidation and catalytic reduction of trace organic contaminants on their activated carbon adsorption.

The combination of ozonation and activated carbon (AC) adsorption is an established technology for removal of trace organic contaminants (TrOCs). In contrast to oxidation, reduction of TrOCs has recently gained attention as well, however less attention has gone to the combination of reduction with AC adsorption. In addition, no literature has compared the removal behavior of reduction vs. ozonation by-products by AC. In this study, the effect of pre-ozonation vs pre-catalytic reduction on the AC adsorption efficiency of five TrOCs and their by-products was compared. All compounds were susceptible to oxidation and reduction, however the catalytic reductive treatment proved to be a slower reaction than ozonation. New oxidation products were identified for dinoseb and new reduction products were identified for carbamazepine, bromoxynil and dinoseb. In terms of compatibility with AC adsorption, the influence of the oxidative and reductive pretreatments proved to be compound dependent. Oxidation products of bromoxynil and diatrizoic acid adsorbed better than their parent TrOCs, but oxidation products of atrazine, carbamazepine and dinoseb showed a decreased adsorption. The reductive pre-treatment showed an enhanced AC adsorption for dinoseb and a major enhancement for diatrizoic acid. For atrazine and bromoxynil, no clear influence on adsorption was noted, while for carbamazepine, the reductive pretreatment resulted in a decreased AC affinity. It may thus be concluded that when targeting mixtures of TrOCs, a trade-off will undoubtedly have to be made towards overall reactivity and removal of the different constituents, since no single treatment proves to be superior to the other.

Validated High Resolution Mass Spectrometry-Based Approach for Metabolomic Fingerprinting of the Human Gut Phenotype.

Fecal samples are an obvious choice for metabolomic approaches, since they can be obtained noninvasively and allow one to study the interactions between the gut microbiota and the host. The use of ultrahigh performance liquid chromatography hyphenated to Orbitrap high-resolution mass spectrometry (UHPLC-Orbitrap HRMS) in this field is unique. Hence, this study relied on Orbitrap HRMS to develop and validate a metabolic fingerprinting workflow for human feces and in vitro digestive fluids. After chemometric sample extraction optimization, an aqueous dilution appeared necessary to comply to the dynamic range of the MS. The method was proven "fit-for-purpose" through a validation procedure that monitored endogenous metabolites in quality control samples, which displayed in both matrices an excellent linearity (R(2) > 0.990), recoveries ranging from 93% to 105%, and precision with coefficients of variation (CVs) < 15%. Finally, feces from 10 healthy individuals and 13 patients diagnosed with inflammatory bowel disease were subjected to metabolomic fingerprinting. 9553 ions were detected, as well as differentiating profiles between Crohn's disease and ulcerative colitis by means of (orthogonal) partial least-square analysis ((O)PLS)-DA (discriminate analysis) models. Additionally, samples from the dynamic gastrointestinal tract simulator (SHIME (Simulator of the Human Intestinal Microbial Ecosystem) platform) were analyzed resulting in 6446 and 5010 ions for the proximal and distal colonic samples, respectively. Supplementing SHIME feed with antibiotics resulted in a significant shift (P < 0.05) of 27.7% of the metabolites from the proximal data set and 34.3% for the distal one. As a result, the presented fingerprinting approach provided predictive modeling of the gastrointestinal metabolome in vivo and in vitro, offering a window to reveal disease related biomarkers and potential insight into the mechanisms behind pathologies.

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts.

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O(6)-methylguanine (O(6)-MeG), O(6)-carboxymethylguanine (O(6)-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.

The impact of stress on the prevalence of prednisolone in bovine urine: A metabolic fingerprinting approach.

Recent studies support the hypothesis that the glucocorticoid prednisolone can be formed from cortisol under influence of stress. To evaluate this hypothesis, urine samples of supposedly non-stressed bovines (at the farm) and bovines subjected to two different forms of stress, i.e. upon slaughter (natural stress) or following administration of a synthetic analog of the adrenocorticotropic hormone (pharmacologically-induced stress) were analysed, and their urinary cortisol and prednisolone levels evaluated. At the farm, none of the examined samples exhibited urinary prednisolone levels higher than the CCα (0.09 µg L(-1)). Upon slaughter or following synthetically induced stress, significantly positive correlations between cortisol and prednisolone could be demonstrated, 0.52 and 0.69, respectively. Of all prednisolone-positive urine samples (n=84), only one showed a prednisolone levels (i.e. 6.45 µg L(-1)) above the threshold level of 5 µg L(-1) suggested by the European Reference Laboratories. Subsequently, an untargeted analysis was performed (metabolic fingerprinting) to characterize the urinary metabolite patterns related to the three different cattle groups. In this context, multivariate statistics assigned a total of 169 differentiating metabolites as playing a key role in the urinary pattern in response to stress. Three of these ions were defined as steroids using an in-house created database. As a result, the metabolic fingerprinting approach proved to be a powerful tool to classify unknown bovine urine samples that tested positive for prednisolone, while providing information about the stress status of the animal.

Increased oxidative and nitrosative reactions during digestion could contribute to the association between well-done red meat consumption and colorectal cancer.

Uncured and nitrite-cured pork were subjected, raw, cooked (65 °C, 15 min) or overcooked (90 °C, 30 min), to an in vitro digestion model, which includes mouth, stomach, duodenum, and colon phases. Heating of uncured meat resulted in a pronounced increase in lipid and protein oxidation products throughout digestion. Nitrite-curing had an antioxidant effect during digestion, but this effect disappeared when the meat was overcooked, resulting in up to ninefold higher 4-hydroxy-2-nonenal concentrations compared with digested nitrite-cured raw and cooked pork. Colonic digesta contained significantly higher concentrations of the NOC-specific DNA adduct O(6)-carboxy-methylguanine when pork underwent a more intense heating procedure, independent of nitrite-curing, depending strongly on the fecal inoculum used. Since processed meats are usually nitrite-cured, the present study suggests that overcooking processed meat is likely to result in the formation of genotoxic compounds during digestion and should, therefore, be avoided.

Steroids in the equine oviduct: synthesis, local concentrations and receptor expression.

Steroids play an important role in mammalian reproduction and early pregnancy. Although systemic changes in steroid concentrations have been well documented, it is not clear how these correlate with local steroid concentrations in the genital tract. We hypothesised that, in the horse, the preimplantation embryo may be subjected to high local steroid concentrations for several days. Therefore, we measured progesterone, 17-hydroxyprogesterone, 17?-oestradiol, testosterone and 17?-testosterone concentrations in equine oviductal tissue by ultra-HPLC coupled with tandem mass spectrometry, and progesterone, 17?-oestradiol, oestrone and testosterone concentrations in oviduct fluid by radioimmunoassay, with reference to cycle stage and side of ovulation. Progesterone concentrations were high in oviductal tissue and fluid ipsilateral to the ovulation side during dioestrus, whereas other steroid hormone concentrations were not influenced by the side of ovulation. These results suggest that the high ipsilateral progesterone concentration is caused by: (1) contributions from the follicular fluid in the oviduct and diffusion of follicular fluid steroids after ovulation; (2) local transfer of steroids via blood or lymph; (3) local synthesis of progesterone in the oviduct, as evidenced by the expression of steroidogenic enzymes; and (4) a paracrine contribution from follicular cells. These data provide a basis for the study of the importance of endocrine and paracrine signalling during early embryonic development in the horse.

Global cytosine methylation in Daphnia magna depends on genotype, environment, and their interaction.

The authors characterized global cytosine methylation levels in 2 different genotypes of the ecotoxicological model organism Daphnia magna after exposure to a wide array of biotic and abiotic environmental stressors. The present study aimed to improve the authors' understanding of the role of cytosine methylation in the organism's response to environmental conditions. The authors observed a significant genotype effect, an environment effect, and a genotype × environment effect. In particular, global cytosine methylation levels were significantly altered after exposure to Triops predation cues, Microcystis, and sodium chloride compared with control conditions. Significant differences between the 2 genotypes were observed when animals were exposed to Triops predation cues, Microcystis, Cryptomonas, and sodium chloride. Despite the low global methylation rate under control conditions (0.49-0.52%), global cytosine methylation levels upon exposure to Triops demonstrated a 5-fold difference between the genotypes (0.21% vs 1.02%). No effects were found in response to arsenic, cadmium, fish, lead, pH of 5.5, pH of 8, temperature, hypoxia, and white fat cell disease. The authors' results point to the potential role of epigenetic effects under changing environmental conditions such as predation (i.e., Triops), diet (i.e., Cryptomonas and Microcystis), and salinity. The results of the present study indicate that, despite global cytosine methylation levels being low, epigenetic effects may be important in environmental studies on Daphnia.

Toward a new European threshold to discriminate illegally administered from naturally occurring thiouracil in livestock.

Thiouracil is a thyrostat inhibiting the thyroid function, resulting in fraudulent weight gain if applied in the fattening of livestock. The latter abuse is strictly forbidden and monitored in the European Union. Recently, endogenous sources of thiouracil were identified after frequently monitoring low-level thiouracil positive urine samples and a "recommend concentration" (RC) of 10 µg/L was suggested by the EURL to facilitate decision-making. However, the systematic occurrence of urine samples exceeding the RC led to demands for international surveys defining an epidemiologic threshold. Therefore, six European member states (France, Poland, The Netherlands, United Kingdom, Norway, and Belgium) have shared their official thiouracil data (2010-2012) collected from bovines, porcines, and small livestock with 95 and 99% percentiles of 8.1 and 18.2 µg/L for bovines (n = 3894); 7.4 and 13.5 µg/L for porcines (n = 654); and 7.4 µg/L (95% only) for small livestock (n = 85), respectively. Bovine percentiles decreased with the animal age (nonadults had significantly higher levels for bovines), and higher levels were observed in male bovines compared to female bovines.

A validated UHPLC-MS/MS method to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses.

Doping control is a main priority for regulatory bodies of both the horse racing industry and the equestrian sports. Urine and blood samples are screened for the presence of hundreds of forbidden substances including anabolic-androgenic steroids (AASs). Based on the suspected endogenous origin of some AASs, with ß-boldenone as the most illicit candidate, this study aimed to improve the knowledge of the naturally present AAS in horse urine. To this extent, a novel ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated according to the Association of Official Racing Chemists (AORC) and European Commission (EC) guidelines, proving the power of this new method. Low limits of detection (0.2 ng/mL), good reproducibility (percentage of standard deviation (%RSD) < 10%), high recovery (94.6 to 117.1%), selectivity and specificity, and a linear response (confirmed with R(2) > 0.99 and lack-of-fit analysis) were obtained for all included AASs. With this method, urine samples of 105 guaranteed untreated horses (47 geldings, 53 mares, and 5 stallions serving as a control) were screened for ß-boldenone and five related natural steroids: androstadienedione (ADD), androstenedione (AED), alpha-testosterone (αT), beta-testosterone (ßT), and progesterone (P). Progesterone, ß-testosterone, and α-testosterone were detected in more than half of the horses at low concentrations (<2 ng/mL). Occasionally, not only testosterone and progesterone but also low concentrations of AED, ADD, and boldenone (Bol) were found (0.5-5 ng/mL). Graphical Abstract A sensitive, new and fully validated UHPLC-MS/MS method has been developed that is able to quantify low levels of anabolic-androgenic steroids naturally present in urine of untreated horses (mares and geldings).

Validation of a quantitative method using liquid chromatography coupled to multiple mass spectrometry for thiouracil in feedstuffs used in animal husbandry.

The use of thyreostatic drugs, like thiouracil (TU), in animal production has been banned for over three decades by the European Union, due to potential teratogenic and carcinogenic effects of its residues upon human consumption. Besides, thyreostats induce water retention in livestock, causing fallacious weight gain and deterioration of meat quality. Development of more competent analytical methods gave rise to sporadic TU detection in urine of untreated animals, questioning the actual synthetic origin TU. Research showed that TU can be formed upon digestion of Brassicaceae feeds in vivo and in vitro, which called for a means of differentiation between endogenous formation of TU and illicit administration. Therefore, in the present study, a routinely applicable liquid chromatography (LC) ion trap multiple mass spectrometry (MS(2)) method for TU analysis in animal feeds was optimised and validated, according to CD 2002/657/EC. A fractional factorial Plackett-Burman design was used to optimise the extraction procedure for TU from Brassicaceae and non-Brassicaceae feeds. This resulted in the discrimination of five influential factors (amount of feed, myrosinase, pH 7 buffer, 3-iodobenzyl bromide and elution solvent), for which the most optimal conditions were perfected. The limit of quantification for TU amounted 0.5 ng g(-1). The individual recoveries for TU ranged between 90.9 and 99.7%. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed, i.e. ≤6.0 and ≤5.2%, respectively, for TU. Excellent linearity was proven based on determination coefficient (R(2) ≥ 0.99) and lack-of-fit test (F test, α = 0.05). Subsequently, a selection of feeds sampled during European national monitoring campaigns were evaluated with the present method showing concentrations ranging from 0.32 to 20.60 ng g(-1), demonstrating the relevance of the method in the analysis of TU from animal feeds.

Properties governing the transport of trace organic contaminants through ion-exchange membranes.

Ion exchange membranes could provide a solution to the selective separation of organic and inorganic components in industrial wastewater. The phenomena governing the transport of organics through the IEM however, are not yet fully understood. Therefore, the transport of trace organic contaminants (TOrCs) as a model for a wide variety of organic compounds was studied under different conditions. It was found that in the absence of salt and external potential, the chemical equilibrium is the main driver for TOrC-transport, resulting in the transport of mainly charged TOrCs. When salt is present, the transport of TOrCs is hampered in favor of the NaCl transport, which shows a preferential interaction with the membranes due to its small size, high mobility and concentration. It is hypothesized that electrostatic interactions and electron donor/acceptor interactions are the main drivers for TOrC transport and that transport is mainly diffusion driven. This was confirmed in the experiments with different current densities, where the external potential seemed to have only a minor influence on the transport of TOrCs. It is only when the salt becomes nearly completely depleted that the TOrCs are transported as charge carriers. This shows that it is very difficult to get preferential transport of organic compounds due to the diffusive nature of their transport.

Thiouracil-Forming Bacteria Identified and Characterized upon Porcine In Vitro Digestion of Brassicaceae Feed.

In recent years, the frequent detection of the banned thyreostat thiouracil (TU) in livestock urine has been related to endogenous TU formation following digestion of glucosinolate-rich Brassicaceae crops. Recently, it was demonstrated that, upon in vitro digestion of Brassicaceae, fecal bacteria induce TU detection in livestock (porcine livestock > bovines). Therefore, the present study was intended to isolate and identify bacteria involved in this intestinal TU formation upon Brassicaceae digestion and to gain more insight into the underlying mechanism in porcine livestock. Twenty porcine fecal inocula (gilts and multiparous sows) were assessed through static in vitro colonic-digestion simulations with rapeseed. After derivatization and extraction of the fecal suspensions, TU was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS(2)). On average, lower TU concentrations were observed in fecal colonic simulations in gilts (8.35 ng g(-1) rapeseed ± 3.42 [mean ± standard deviation]) than in multiparous sows (52.63 ng g(-1) ± 16.17), which correlates with maturation of the gut microbial population with age. Further exploration of the mechanism showed cell-dependent activity of the microbial conversion and sustained TU-forming activity after subjection of the fecal inoculum to moderate heat over a time span of up to 30 min. Finally, nine TU-producing bacterial species were successfully isolated and identified by a combination of biochemical and molecular techniques as Escherichia coli (n = 5), Lactobacillus reuteri (n = 2), Enterococcus faecium (n = 1), and Salmonella enterica subsp. arizonae (n = 1). This report demonstrates that endogenous formation of TU is Brassicaceae induced and occurs under colonic conditions most likely through myrosinase-like enzyme activity expressed by different common intestinal bacterial species.

O6-carboxymethylguanine DNA adduct formation and lipid peroxidation upon in vitro gastrointestinal digestion of haem-rich meat.

SCOPE: Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO). METHODS AND RESULTS: In this study, the NOC-derived DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O(6)-CMG formation (p < 0.05). MDA concentrations proved to be positively correlated (p < 0.0004) with haem content of digested meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose-response association with O(6)-CMG (p = 0.004) and MDA (p = 0.008) formation. CONCLUSION: The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption.

Fat content and nitrite-curing influence the formation of oxidation products and NOC-specific DNA adducts during in vitro digestion of meat.

The effects of fat content and nitrite-curing of pork were investigated on the formation of cytotoxic and genotoxic lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal, volatile simple aldehydes), protein oxidation products (protein carbonyl compounds) and NOC-specific DNA adducts (O6-carboxy-methylguanine) during in vitro digestion. The formation of these products during digestion is suggested to be responsible for the association between red meat and processed meat consumption and colorectal cancer risk. Digestion of uncured pork to which fat was added (total fat content 5 or 20%), resulted in significantly higher lipid and protein oxidation in the mimicked duodenal and colonic fluids, compared to digestion of pork without added fat (1% fat). A higher fat content also significantly favored the formation of O6-carboxy-methylguanine in the colon. Nitrite-curing of meat resulted in significantly lower lipid and protein oxidation before and after digestion, while an inconsistent effect on the formation of O6-carboxy-methylguanine was observed. The presented results demonstrate that haem-Fe is not solely responsible for oxidation and nitrosation reactions throughout an in vitro digestion approach but its effect is promoted by a higher fat content in meat.

Validation of a confirmatory method for lipophilic marine toxins in shellfish using UHPLC-HR-Orbitrap MS.

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion ((13)C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software.

Validated ultra high performance liquid chromatography-tandem mass spectrometry method for quantitative analysis of total and free thyroid hormones in bovine serum.

Thyroid hormones are essential hormones for regulating growth and development. Methods to accurately monitor low-levels (ppb-ppt) of these hormones in serum are needed to assess overall health, both from a clinical perspective as from environmental contaminant or drug exposures. In general, the separation of the free thyroid hormone fraction from animal sera is performed through labour intensive equilibrium dialysis, while detection of total and free thyroid hormone fractions in animals is done with commercially available radioimmunoassays (RIAs). This study reports newly developed analysis methods for both the total and free fractions of triiodothyronine (T3), reverse-triiodothyronine (rT3) and thyroxin (T4) from bovine serum, with a much higher specificity and selectivity than commercially available RIAs. The bovine serum extraction procedures of total and free T3, rT3, T4 were optimised with fractional factorial designs and consisted of, respectively, deproteinisation followed by liquid-liquid extraction, 30 kDA ultracentrifugation and solid phase extraction. Both free and total thyroid hormone UHPLC-HESI-MS/MS based analysis methods were successfully validated. The limits of quantification for T4, rT3 and T3 amounted respectively 0.04 ng mL(-1), 0.05 ng mL(-1), 0.03 ng mL(-1) for the total fraction, and 6.6 pg mL(-1), 2.6 pg mL(-1) and 2.7 pg mL(-1) for the free fraction. Individual recoveries of total and free thyroid hormone fractions ranged between 95.6 and 106.3% and 92.1 and 106.5%. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed, i.e. respectively ≤8.0% and ≤7.3% for the total and free fractions. Excellent linearity (R(2)≥0.99) and lack-of-fit was proven for both fractions. In conclusion, these methods show excellent in-house performance and possibilities for elaboration to application in other animal sera (e.g. feline, canine, equine).

Environmental heat stress induces epigenetic transgenerational inheritance of robustness in parthenogenetic Artemia model.

The notion that phenotypic traits emerging from environmental experiences are heritable remains under debate. However, the recent report of nonmendelian transgenerational epigenetic inheritance, i.e., the inheritance of traits not determined by the DNA sequence, might make such a phenomenon plausible. In our study, by carrying out common garden experiments, we could provide clear evidences that, on exposure to nonlethal heat shocks, a parental population of parthenogenetic (all female) Artemia (originating from one single female) experiences an increase in levels of Hsp70 production, tolerance toward lethal heat stress, and resistance against pathogenic Vibrio campbellii. Interestingly, these acquired phenotypic traits were transmitted to three successive generations, none of which were exposed to the parental stressor. This transgenerational inheritance of the acquired traits was associated with altered levels of global DNA methylation and acetylated histones H3 and H4 in the heat-shocked group compared to the control group, where both the parental and successive generations were reared at standard temperature. These results indicated that epigenetic mechanisms, such as global DNA methylation and histones H3 and H4 acetylation, have particular dynamics that are crucial in the heritability of the acquired adaptive phenotypic traits across generations.