Julie Vanden Bussche

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.—Norouzitallab, P., Baruah, K., Vandegehuchte, M., Van Stappen, G., Catania, F., Vanden Bussche, J., Vanhaecke, L., Sorgeloos, P., Bossier, P. Environmental heat stress induces epigenetic transgenerational inheritance of robustness in parthenogenetic Artemia model. FASEB J. 28, 3552–3563 (2014). www.fasebj.org

Ultra-high performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant

Phytohormones are key signalling biomolecules and are of particular interest because of their regulating role in numerous physiological and developmental plant processes. Since the plant response to a given stimulus results amongst others from the complex interaction between phytohormones, there is a mounting interest for multiple phytohormone analysis. Therefore, with the primary aim of profiling the hormonal status of the tomato plant, a generic extraction protocol and an U-HPLC-Orbitrap-MS analysis were developed and validated for both tomato fruit and leaf tissue. To this end, eight phytohormones were considered, i.e. gibberellic acid, indol-3-acetic acid, abscisic acid, jasmonic acid, salicylic acid, zeatin, N6-benzyladenine and epibrassinolide, representing the major hormonal classes. The sample pre-treatment involved liquid extraction with a buffer of methanol, ultrapure water and formic acid (75:20:5, v/v/v), after which the extract was purified by means of an Amicon® Ultra centrifugal unit. Subsequently, analytes were chromatographically separated on a sub-2 μm particles Nucleodur Gravity C18 column and detected by an Exactive™ high-resolution mass spectrometer. Validation of the analytical method demonstrated that linearity (≥0.99), precision (CV≤15%) and mean corrected recovery (between 80% and 110%) performed well for the majority of the eight targeted phytohormones. In addition, the generic nature of the extraction protocol and the full scan approach of the Orbitrap mass spectrometer allowed metabolomic profiling of the hormonal status of the tomato plant.

Ultra-high performance liquid chromatography-tandem mass spectrometry in high-throughput confirmation and quantification of 34 anabolic steroids in bovine muscle.

An ultra-high performance liquid chromatography tandem mass spectrometry multi-residue method for the determination of 34 anabolic steroids (10 estrogens including stilbenes, 14 androgens and 10 gestagens) in meat of bovine origin is reported. The extraction and clean-up procedure involved homogenization with methanol, defatting with hexane, liquid/liquid extraction with diethylether and finally SPE clean-up with coupled Si and NH(2) cartridges. The analytes were separated on a 1.9 μm Hypersil Gold column (100×2.1 mm) and quantified on a triple quadrupole mass spectrometer (TSQ Vantage) operating simultaneously in both positive and negative atmospheric pressure chemical ionisation (APCI) modes. This analytical procedure was subsequently validated according to EU criteria (CD 2002/657/EC), resulting in decision limits and detection capabilities ranging between 0.04 and 0.88 μg kg(-1) and 0.12 and 1.9 μg kg(-1), respectively. The method obtained for all, natural and synthetic steroids, adequate precisions and intra-laboratory reproducibilities (relative standard deviation below 20%), and the linearity ranged between 0.991 and 0.999. The performance characteristics fulfill the recommended concentrations fixed by the Community Reference Laboratories. The developed analysis is sensitive, and robust and therefore useful for confirmation and quantification of anabolic steroids for research purposes and residue control programs.

Nitrite curing of chicken, pork, and beef inhibits oxidation but does not affect N‑nitroso compound (NOC)-specific DNA adduct formation during in vitro digestion

Uncured and nitrite-cured chicken, pork, and beef were used as low, medium, and high sources of heme-Fe, respectively, and exposed to an in vitro digestion model simulating the mouth, stomach, duodenum, and colon. With increasing content of iron compounds, up to 25-fold higher concentrations of the toxic lipid oxidation products malondialdehyde, 4-hydroxy-2-nonenal, and other volatile aldehydes were formed during digestion, together with increased protein carbonyl compounds as measurement of protein oxidation. Nitrite curing of all meats lowered lipid and protein oxidation to the level of oxidation in uncured chicken. Strongly depending on the individual fecal inoculum, colonic digestion of beef resulted in significantly higher concentrations of the NOC-specific DNA adduct O(6)-carboxymethyl-guanine compared to chicken and pork, whereas nitrite curing had no significant effect. This study confirms previously reported evidence that heme-Fe is involved in the epidemiological association between red meat consumption and colorectal cancer, but questions the role of nitrite curing in this association.

High resolution Orbitrap mass spectrometry in comparison with tandem mass spectrometry for confirmation of anabolic steroids in meat

A prominent trend which has been observed in recent years in the analysis of veterinary drugs and growth-promoting agents is the shift from target-oriented procedures, mainly based on liquid chromatography coupled to triple-quadrupole mass spectrometry (LC-QqQ-MS), towards accurate mass full scan MS (such as time of flight (ToF) and Fourier Transform (FT) Orbitrap MS). In this study the applicability of high resolution single-stage-Orbitrap-MS for confirmatory analysis of growth-promoting agents in meat was compared to that of a QqQ-MS. Validation according to CD 2002/657/EC demonstrated that steroid analysis based on Orbitrap MS, operating at a resolution of 50,000 FWHM, is indeed capable to compete with QqQ-MS in terms of selectivity/specificity, while providing excellent linearity (for most compounds >0.99) but somewhat inferior sensitivity. Indeed, CCαs reached from 0.04-0.88μgkg(-1) for the 34 anabolic steroids upon MS/MS detection, while upon Orbitrap MS detection a range of 0.07-2.50μgkg(-1) was observed. Using QqQ-MS adequate precision was obtained since relative standard deviations, associated with the repeatability and intra-laboratory reproducibility, were below 20%. In the case of Orbitrap MS, for some compounds (i.e. some estrogens) this threshold was exceeded and thus poor precision was observed, which is possibly caused by the lack in sensitivity. Overall, it may be concluded that Orbitrap-MS offers an adequate performance in terms of linearity and precision but lacks in sensitivity for some of the compounds.

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.

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.

Ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry detection of naturally occurring thiouracil in urine of untreated livestock, domesticated animals and humans

Thiouracil belongs to the xenobiotic thyreostats, which are growth-promoting agents illegally used in animal production. Recently it has been reported that thiouracil is suspected to have a natural origin. The European Union of Reference Laboratory guidance paper of 2007 acknowledged this by stating that thiouracil concentrations below 10 µg l−1 might have a natural origin derived from the consumption of Brassicaceae. The present research aimed at endorsing this possible natural occurrence. Urine samples of animals (livestock and domesticated) with known and unknown clinical backgrounds were analysed for thiouracil with a newly developed ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometric analysis method without derivatisation. In addition, a small-scale 9-day human experiment with Brassicaceae vegetables was performed to investigate if this natural prevalence could be extrapolated to the human population. The untreated animals had thiouracil concentrations below 10 µg l−1 acknowledging the alleged natural occurrence of thiouracil. As for the humans, in 66.7% of the urine samples thiouracil was found above the CCα of 2.2 µg l−1. However, the correlation with the Brassicaceae diet proved to be non-significant (p = 0.095). Nevertheless, these results clearly demonstrate the natural occurrence of thiouracil in urine of animals and humans. The exact origin of this natural thiouracil trace still needs to be identified.

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.