Quantitative analysis of binary and ternary organo-mineral solid dispersions by Raman spectroscopy for robotic planetary exploration missions on Mars.

The Mars 2020 and ExoMars 2022, rover-based missions are specifically dedicated to the search for evidence of life and will both utilise Raman spectrometers on the surface of Mars. Raman spectroscopy is indeed a valuable analytical technique for planetary exploration that enables in situ characterisation of rocks and soils collected directly from the surface or retrieved as cores and subsequently crushed when extracted from the subsurface with a drill. On Mars, the miniaturised spectrometers will interrogate ancient geological deposits, in order to try and identify hydrated or aqueously altered minerals and organic matter to assess the habitability of Mars. While the identification of relevant hydrous minerals and organic components is the primary analytical objective of the missions, quantifying their abundances would be of particular significance for interpreting past geological conditions (e.g. formation or alteration processes) and for ascertaining the putative presence of biosignatures. Therefore, we have developed quantitative models that enable the quantification of both mineral proportions from crushed mixtures of geological components and spiked mixtures containing organic analytes dispersed in mineral matrices. Based on data normalisation with appropriate standards (internal and external), we demonstrate that robust quantitative models can be (1) applied for solid dispersions of various complexities relevant to planetary exploration; and (2) applied to different Raman set-ups, including an instrument representative of the ExoMars Raman Laser Spectrometer. With important Raman-active minerals (calcite, gypsum, baryte, quartz), we demonstrate that using a correction factor Fϕ2/ϕ1, based on the ratio of apparent Raman scattering coefficients, the relative proportion of minerals in binary mixtures can be accurately determined. Regarding the organics, evaluated in clay-rich sediments (Fe-smectite) and crushed rocks of coarse-grained fraction (>100µm), we establish calibration curves in the concentration range 2-20 wt% for non-resonant compounds (L-cysteine, phthalic acid, adenine) and even lower (<1 wt%) for pre-resonant anthracene. Despite large levels of heterogeneity, the Raman analyses of these solid dispersions verify that quantitative Raman analyses can be performed in the context of robotic exploration studies.

Volatiles profile of the different parts of Algerian Bupleurum plantagineum Desf. by headspace solid-phase microextraction and hydrodistillation.

The volatiles from a rare and endemic plant Bupleurum plantagineum Desf collected from Béjaia (Algerian National Park) were analyzed using HS-SPME-GC/MS. The main factors affecting this extraction technique, including fibre coating, ionic forces (salt adding), temperature and time of extraction were performed in order to reach a maximum of sensitivity. The best results were obtained by PDMS fiber using the powdered leaves at specific temperatures for 25 min, without salt adding. On the other hand, the volatile composition of leaves, stems and roots were determined with a clear prevalence of sesquiterpene hydrocarbons of 45.66, 31.99 and 70.28% respectively. A qualitative comparison with essential oil from hydrodistillation was also performed showing differences essentially in minor components.

On the Habitability of Desert Varnish: A Combined Study by Micro-Raman Spectroscopy, X-ray Diffraction, and Methylated Pyrolysis-Gas Chromatography-Mass Spectrometry.

In 2020, the ESA ExoMars and NASA Mars 2020 missions will be launched to Mars to search for evidence of past and present life. In preparation for these missions, terrestrial analog samples of rock formations on Mars are studied in detail in order to optimize the scientific information that the analytical instrumentation will return. Desert varnishes are thin mineral coatings found on rocks in arid and semi-arid environments on Earth that are recognized as analog samples. During the formation of desert varnishes (which takes many hundreds of years), organic matter is incorporated, and microorganisms may also play an active role in the formation process. During this study, four complementary analytical techniques proposed for Mars missions (X-ray diffraction [XRD], Raman spectroscopy, elemental analysis, and pyrolysis-gas chromatography-mass spectrometry [Py-GC-MS]) were used to interrogate samples of desert varnish and describe their capacity to sustain life under extreme scenarios. For the first time, both the geochemistry and the organic compounds associated with desert varnish are described with the use of identical sets of samples. XRD and Raman spectroscopy measurements were used to nondestructively interrogate the mineralogy of the samples. In addition, the use of Raman spectroscopy instruments enabled the detection of ß-carotene, a highly Raman-active biomarker. The content and the nature of the organic material in the samples were further investigated with elemental analysis and methylated Py-GC-MS, and a bacterial origin was determined to be likely. In the context of planetary exploration, we describe the habitable nature of desert varnish based on the biogeochemical composition of the samples. Possible interference of the geological substrate on the detectability of pyrolysis products is also suggested. Key Words: Desert varnish-Habitability-Raman spectroscopy-Py-GC-MS-XRD-ExoMars-Planetary science. Astrobiology 17, 1123-1137.

Metabolic inhibitors accentuate the anti-tumoral effect of HDAC5 inhibition.

The US FDA approval of broad-spectrum histone deacetylase (HDAC) inhibitors has firmly laid the cancer community to explore HDAC inhibition as a therapeutic approach for cancer treatment. Hitting one HDAC member could yield clinical benefit but this required a complete understanding of the functions of the different HDAC members. Here we explored the consequences of specific HDAC5 inhibition in cancer cells. We demonstrated that HDAC5 inhibition induces an iron-dependent reactive oxygen species (ROS) production, ultimately leading to apoptotic cell death as well as mechanisms of mitochondria quality control (mitophagy and mitobiogenesis). Interestingly, adaptation of HDAC5-depleted cells to oxidative stress passes through reprogramming of metabolic pathways towards glucose and glutamine. Therefore, interference with both glucose and glutamine supply in HDAC5-inhibited cancer cells significantly increases apoptotic cell death and reduces tumour growth in vivo; providing insight into a valuable clinical strategy combining the selective inhibition of HDAC5 with various inhibitors of metabolism as a new therapy to kill cancer cells.

Validation of a gas chromatography-triple quadrupole mass spectrometry method for confirmatory analysis of dioxins and dioxin-like polychlorobiphenyls in feed following new EU Regulation 709/2014.

The European Regulations laying down methods of sampling and analysis for the EU official control of levels of polychlorodibenzo-p-dioxins (PCDDs), polychloro-dibenzofurans (PCDFs), dioxin-like (DL) and non dioxin-like (NDL) PCBs in food and feed have been recently amended by EU Regulation Nos. 589/2014 and 709/2014. A major update is the recognition of gas chromatography (GC) triple quadrupole mass spectrometry (GC-QQQMS/MS) as a confirmatory tool for checking compliance with maximum levels (ML). These revisions have been initiated since this technology now exhibits similar performances to GC (magnetic sector) high resolution mass spectrometry (GC-HRMS). In this paper, we show a fully validated method for PCDD/Fs and DL-PCBs analysis in feed material of plant origin (vegetable oil) using GC-QQQMS/MS following the dedicated EU Regulation 709/2014. We show that individual analytical criteria (selectivity, linearity, quant/qual MRM transitions, accuracy around ML of 1.50ng WHO2005TEQ/kg, within-lab reproducibility, robustness, and background subtraction) meet the strict requirements set by the EU Regulation. We also propose a clear interpretation of instrumental limit of quantitation (iLOQ) as a 'performance-LOQ', defined in a specific way for GC-QQQMS/MS, and method limit of quantitation (mLOQ) as 'real-LOQ' that is used to report bound results. Eventually, the evaluation of measurement uncertainty, following a top-down approach and data produced with our method, demonstrates similar results than with GC-HRMS, thus offering a reliable alternative to the standard method for vegetable oil.

Combined intracellular nitrate and NIT2 effects on storage carbohydrate metabolism in Chlamydomonas.

Microalgae are receiving increasing attention as alternative production systems for renewable energy such as biofuel. The photosynthetic alga Chlamydomonas reinhardtii is widely recognized as the model system to study all aspects of algal physiology, including the molecular mechanisms underlying the accumulation of starch and triacylglycerol (TAG), which are the precursors of biofuel. All of these pathways not only require a carbon (C) supply but also are strongly dependent on a source of nitrogen (N) to sustain optimal growth rate and biomass production. In order to gain a better understanding of the regulation of C and N metabolisms and the accumulation of storage carbohydrates, the effect of different N sources (NH4NO3 and ) on primary metabolism using various mutants impaired in either NIA1, NIT2 or both loci was performed by metabolic analyses. The data demonstrated that, using NH4NO3, nia1 strain displayed the most striking phenotype, including an inhibition of growth, accumulation of intracellular nitrate, and strong starch and TAG accumulation. The measurements of the different C and N intermediate levels (amino, organic, and fatty acids), together with the determination of acetate and remaining in the medium, clearly excluded the hypothesis of a slower and acetate assimilation in this mutant in the presence of NH4NO3. The results provide evidence of the implication of intracellular nitrate and NIT2 in the control of C partitioning into different storage carbohydrates under mixotrophic conditions in Chlamydomonas. The underlying mechanisms and implications for strategies to increase biomass yield and storage product composition in oleaginous algae are discussed.

Risk assessment of Belgian adults for furan contamination through the food chain.

Risk assessment is an interdisciplinary process used to quantify the risk linked to a hazard. In the present paper it is applied to quantify the risk linked to furan ingestion through the food chain for the Belgian adult population. Two approaches, deterministic and probabilistic, were carried out in parallel. The deterministic method relied on a case study, whereas the probabilistic approach involved statistical distributions of contamination and consumption data to calculate a statistical distribution of the daily intake. First, the deterministic method revealed a low estimated daily intake (EDI) for the average population (380 ng*(kg(bw)*day)⁻¹) and a huge contribution of coffee consumption to the EDI (55%). Increasing or decreasing the daily coffee consumption by one cup can affect the EDI by about 22%. Afterwards, the probabilistic approach showed that the average population has a low EDI (494 ng*(kg(bw)*day)⁻¹), and that high contamination levels were only registered in a small proportion of the population. Finally, a comparison of the RfD(chronic oral) showed that less than 10% of the Belgian population had an EDI above the reference dose proposed by the USEPA; the majority of the population had an EDI 20% below the reference dose. The margin of exposure (MoE) approach indicated that the level of risk related to furan intake through ingestion is low, with a MoE > 10,000 for more than 10% of the population and no result < 100.

Estimation of furan contamination across the Belgian food chain.

This paper provides an estimate of the furan content of Belgian foods. The objective of the study was to achieve the best food chain coverage with a restricted number of samples (n = 496). The geographic distribution, different market chains and labels, and consumption frequencies were taken into account in the construction of the sampling plan. Weighting factors such as contamination levels, consumption frequency and the diversity of food items were applied to set up the model. The very low detection capabilities (CC(ß)) of the analytical methods used (sub-ppb) allowed reporting of 78.2% of the overall dataset above CC(ß) and, in particular, 96.7% for the baby food category. The highest furan levels were found in powdered roasted bean coffee (1912 µg kg(-1)) with a mean of 756 µg kg(-1) for this category. Prepared meat, pasta and rice, breakfast cereals, soups, and baby food also showed high mean furan contents ranging from 16 to 43 µg kg(-1). Comparisons with contamination surveys carried out in other countries pointed out differences for the same food group and therefore contamination levels are related to the geographical origin of food items.

Reproduction of European eel jeopardised by high levels of dioxins and dioxin-like PCBs?

Dioxins, furans and dioxin-like polychlorinated biphenyls (PCBs) were analysed in muscle tissue from yellow phased European eel (Anguilla anguilla) from 38 sites in Belgium. Dioxin concentrations in eel vary considerably between sampling locations, indicating that yellow eel is a good indicator of local pollution levels. Measured levels of dioxin-like PCBs are much higher than those of the dioxins and furans. In the majority of the sites, eel has levels considered to be detrimental for their reproduction. Field levels of dioxin and dioxin-like PCBs are therefore suggested as an additional causal factor contributing to the decline of the European eel. 42% of the sampling sites show especially dioxin-like PCB levels exceeding the European consumption level (with a factor 3 on average). Human consumption of eel, especially in these highly contaminated sites, seems unjustified.

Dietary intake of PCDD/Fs and dioxin-like PCBs of the Belgian population.

The World Health Organization recommends assessing human exposure to contaminants on a regular basis. In order to assess the current dietary exposure of the Belgian adult population to PCDD/Fs and dioxin-like PCBs and to update exposure estimates of 2000-2001, a total diet study was designed. The mean dietary intake of PCDD/Fs and dioxin-like PCBs in the Belgian adult population in 2008 was estimated to be 0.72pgTEQkgbw(-1)d(-1) (middle bound concentrations, TEF of 1998) based on occurrence data of 2008 and national food consumption data of 2004. This value is clearly below the Tolerable Weekly Intake (TWI) of 14pgTEQkgbw(-1)week(-1) set by the Scientific Committee on Food of the European Commission and below the provisional tolerable monthly intake of 70pgTEQkgbw(-1)month(-1) set by the Joint FAO/WHO Expert Committee on Food Additives. Considering the cumulative distribution, the intake was less than 1pgTEQkgbw(-1)d(-1) for more than 80% of the population, and less than 2pgTEQkgbw(-1)d(-1) for the entire population. When using the 2005 TEF instead of the 1998 TEF, the mean dietary intake in the Belgian adult population was estimated to be 0.61pgTEQkgbw(-1)d(-1).

PCDD/Fs and dioxin-like PCBs in home-produced eggs from Belgium: levels, contamination sources and health risks.

This paper discusses the dioxin TEQ levels as determined by the chemically activated luciferase gene expression assay (CALUX) and by HRGC-HRMS in eggs, soils, faeces and kitchen waste samples obtained in the CONTEGG study. The samples were collected in each Belgian province at private homes and in small gardens where chickens are held. The CALUX levels for eggs sampled in autumn were higher than the levels in eggs obtained at the same locations in spring (median values of 5.86 and 4.08 pg CALUX TEQ/g fat, respectively). The total WHO-TEQ levels in eggs, determined by HRGC-HRMS, ranged from 3.29 to 95.35 pg TEQ/g fat in autumn and from 1.50 to 64.79 pg TEQ/g fat in spring. In the soils on which the chickens forage, levels of 2.51-11.35 pg I-TEQ/g in autumn and 2.00-7.86 pg I-TEQ/g in spring were found. The congener pattern of PCDD/Fs in the eggs, soils and faeces was dominated by OCDD, in addition to 1,2,3,4,6,7,8-HeptaCDD, OCDF and 1,2,3,4,6,7,8-HeptaCDF. The predominant dioxin-like PCBs were PCB118, PCB 105 and PCB 156. The dioxin-like PCBs contributed on average 47%, 14% and 20% to the total WHO-TEQ in eggs, soils and faeces, respectively. Kitchen waste samples were very low-contaminated with dioxin-like compounds. The present results showed a good agreement between egg and soil TEQ levels for PCDD/Fs but not for dioxin-like PCBs. This study showed that current soil levels found in some private gardens do not lead to egg levels below the current EU maximal level of 6 pg total TEQ/g fat for dioxins and dioxin-like PCBs. The consumers of the analysed eggs attained 5-79% of the tolerable weekly intake (TWI) of 14 pg TEQ/kg bw for dioxins and dioxin-like PCBs by exposure to their home-produced eggs only.

Assessment of the chemical contamination in home-produced eggs in Belgium: general overview of the CONTEGG study.

This overview paper describes a study conducted for the Belgian Federal Public Service of Health, Food Chain Safety and Environment during 2006-2007. Home-produced eggs from Belgian private owners of hens were included in a large study aiming to determine concentration levels of various environmental contaminants. By means of the analyses of soil samples and of kitchen waste samples, obtained from the same locations, an investigation towards the possible sources of contaminants was possible. Eggs, soils, faeces and kitchen waste samples were checked for the presence of dioxins, PCBs (including dioxin-like PCBs), organochlorine pesticides, trace elements, PAHs, brominated flame retardants and mycotoxins. The study design, sampling methodology and primary conclusions of the study are given. It was found that in some cases dioxin-like compounds were present at levels that are of concern for the health of the egg consumers. Therefore, measures to limit their contamination in eggs, produced by hens of private owners, were proposed and deserve further attention.

Gender dependent accumulation of dioxins in smokers.

AIMS: To evaluate the contribution of tobacco smoking to dioxin accumulation. METHODS: Dioxin (17 PCDD/F) concentrations in fasting blood from 251 subjects (161 never smokers, 54 past smokers, and 36 current smokers) were quantified. RESULTS: Whereas serum dioxin concentrations of male smokers were on average 40% higher than those of non-smokers, in women, smoking was associated with significantly lower serum dioxin levels. A synergistic potentiation of dioxin metabolism by tobacco smoke in women is postulated to explain these paradoxical findings. CONCLUSIONS: Current smoking is associated with gender dependent effects on dioxin body burden and is a potential source of confounding in human studies using blood dioxins as indicators of exposure.

Validation of the CALUX bioassay for PCDD/F analyses in human blood plasma and comparison with GC-HRMS.

Following the dioxin crisis of 1999, several studies were conducted to assess the impact of this crisis on the dioxin body burden in the Belgian population. The Scientific Institute of Public Health identified a population from whom plasma samples were available and from whom, during the follow up survey, plasma samples were obtained in 2000. In total, 496 samples were collected for GC-HRMS and CALUX analyses to verify statistical assessment conclusions. This study was seen as an opportunity to validate the CALUX bioassay for biological sample analysis and to compare toxic equivalency (TEQ) values obtained by the reference GC-HRMS technique and by the screening method. This article focuses on the validation results of the CALUX bioassay for the analyses of the dioxin fractions of blood plasma. The sample preparation is based on a liquid-liquid extraction, followed by an acid silica in series with an activated carbon clean-up. A good recovery (82%) and reproducibility (coefficient of variation less than 25%) were found for this method. Based on 341 plasma samples, a significant correlation was established between the bioassay and chemical method (R = 0.64). However, a proportional systematic error was observed when the results obtained with the CALUX bioassay were regressed with the results from the GC-HRMS analyses. The limit of quantification (LOQ) used to calculate TEQ values from the GC-HRMS determinations, the use of the relative potency values instead of the toxic equivalent factor and the potential of CALUX bioassay to measure all compounds with affinity for the AhR may partly explain this proportional systematic error. Nevertheless, the present results suggest that the CALUX bioassay could be a promising valid screening method for human blood plasma analyses.

Importance of clean-up for comparison of TEQ-values obtained by CALUX and chemo-analysis.

This paper presents Chemically Activated LUciferine gene eXpression (CALUX) TEQ-values obtained for nine plasma samples following two different purification procedures, one of them involving fractionation. CALUX results obtained for the dioxin (DX) and dioxin + PCB (DX + PCB) fractions were then compared to the GC-HRMS TEQ-values calculated for the 17 polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (17 PCDD/F) and 17 PCDD/F + 4 cPCB congeners, respectively. The overestimation of the CALUX (DX fraction) TEQ-values in comparison with the chemo-analyses of the 17 PCDD/F is mainly explained by the presence of other AhR agonists, like brominated compounds. Otherwise, the constancy of the CALUX (DX + PCB fraction) TEQ-value which compares to increasing the GC-HRMS (17 PCDD/F + 4 cPCB) TEQ results raises questions concerning (1) the significance of CALUX results obtained without fractionation as well as (2) the toxicological effect of a cocktail of contaminants on the human health.

Levels and congener distributions of PCDDs, PCDFs and non-ortho PCBs in Belgian foodstuffs--assessment of dietary intake.

Congener-specific analyses of 7 polychlorinated dibenzo-p-dioxins (PCDDs), 10 polychlorinated dibenzofurans (PCDFs) and 4 non-ortho (coplanar) polychlorinated biphenyls (cPCBs) were performed on 197 foodstuffs samples of animal origin from Belgium during years 2000 and 2001. All investigated matrices (except horse) present background levels lower than the Belgian non-commercialization value of 5 pg TEQ/g fat. Pork was the meat containing the lowest concentration of both PCDD/Fs and cPCBs. The mean background concentration of 2,3,7,8-TCDD toxicity equivalent in milk was 1.1 pg/g of fat, with a congener distribution typical of non-contaminated milk. The relative contribution of 2,3,7,8-TCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD and 2,3,4,7,8-PeCDF to the PCDD/Fs TEQ was 85+/-7.9% for all investigated matrices. The cPCBs contribution to the total TEQ was 47+/-19.0% for products of terrestrial species and 69+/-20.0% for aquatic species. Once the contribution of cPCBs was added to the TEQ, few foodstuffs such as horse, sheep, beef, eggs and cheese presented levels above the future European guidelines that currently only include PCDD/Fs but will be re-evaluated later in order to include 'dioxin-like' PCBs. Based on levels measured in the samples, the estimation of the dietary intake was 65.3 pg WHO-TEQ/day for PCDD/Fs only (1.00 pg WHO-TEQ/kg bw/day, for a 65 kg person) and 132.9 pg WHO-TEQ/day if cPCBs were included (2.04 pg WHO-TEQ/kg bw/day, for a 65 kg person). Meat (mainly beef), dairy products, and fish each account for roughly one third of the intake.

Fast clean-up for polychlorinated dibenzo-p-dioxins, dibenzofurans and coplanar polychlorinated biphenyls analysis of high-fat-content biological samples.

A fast clean-up procedure for the low level analysis of polychlorinated dibenzo-p-dioxins. polychlorinated dibenzofurans and coplanar polychlorinated biphenyls in highly fatty biological matrices using high capacity disposable multi-layer silica columns is presented. Results were compared with gel permeation chromatography for removal of lipids. Analytical criteria such as recovery rates, repeatability, reproducibility and robustness are evaluated through a broad range of biological matrices and reference materials analysis. The final proposed procedure for the complete analysis, including pressurized liquid extraction, Power-Prep system clean-up and GC-high-resolution MS analysis requires only 48 h, and allows the simultaneous preparation of up to 10 samples.

Optimisation and use of tandem-in-time mass spectrometry in comparison with immunoassay and HRGC/HRMS for PCDD/F screening.

Rapid screening of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans using quadrupole ion storage tandem-in-time mass spectrometry (QISTMS) conjointly with polyclonal antibody immunoassay has been considered. The optimisation of the fragmentation of the parent ion in the trap has been completed. The analysis of fly ashes from a municipal waste incinerator contaminated at different levels has then been realised. Results obtained using QISTMS, HRMS and immunoassay are compared.