Analytical challenges related to the measurement of chlorothalonil in serum in the perspective of human biomonitoring studies.

Chlorothalonil (CTN) is a popular fungicide widely used in the world. However, its determination in serum samples is highly challenging, preventing a reliable investigation of human CTN internal exposure. We first investigated CTN's behaviour all along this analytical process on spiked serum samples. We used a radiolabelled 14C-CTN standard to monitor CTN in spiked serum samples and observed (1) a complete degradation of CTN in deproteinised serum samples after 4 h of contact; (2) a strong interaction between serum proteins and CTN by-products, with only 20 % of the radioactivity found to be extractable after 24 h of contact and (3) a slightly improved stability of CTN in serum following a first step of acidification or EDTA addition to samples. Using liquid chromatography coupled to high resolution mass spectrometry, 4-hydroxy-2,5,6-trichloroisophthalonitrile (HCTN) was identified as the major serum by-product of CTN. A protocol was developed to monitor both extractable CTN and HCTN from serum. This method was implemented on 36 human adult serum samples from the French "Esteban" Cohort. No free CTN was identified in these serum samples. Conversely, HCTN was detected in all samples at concentrations around 15 ± 2 ng mL-1, corresponding to the extractable fraction of CTN. Thus, HCTN may constitute a relevant biomarker of human internal exposure. Of note, the potential CTN contamination during blood collection could also be a source of HCTN detection in serum samples. Finally, blood sampling in EDTA tubes would seem more appropriate than in dry tubes for any future internal exposure studies on CTN.

Non-targeted analysis of lipidic extracts by high-resolution mass spectrometry to characterise the chemical exposome: Comparison of four clean-up strategies applied to egg.

Biota samples are used to monitor chemical stressors and their impact on the ecosystem and to describe dietary chemical exposure. These complex matrices require an extraction step followed by clean-up to avoid damaging sensitive analytical instruments based on chromatography coupled to mass spectrometry. While interest for non-targeted analysis (NTA) is increasing, there is no versatile or generic sample preparation for a wide range of contaminants suitable for a diversity of biotic matrices. Among the contaminants' variety, persistent contaminants are mostly hydrophobic (mid- to non-polar) and bio-magnify through the lipidic fraction. During their extraction, lipids are generally co-extracted, which may cause matrix effect during the analysis such as hindering the acquired signal. The aim of this study was to evaluate the efficacy of four clean-up methods to selectively remove lipids from extracts prior to NTA. We evaluated (i) gel permeation chromatography (GPC), (ii) Captiva EMR-lipid cartridge (EMR), (iii) sulphuric acid degradation (H2SO4) and (iv) polydimethyl siloxane (PDMS) for their efficiency to remove lipids from hen egg extracts. Gas and liquid chromatography coupled with high-resolution mass spectrometry fitted with either electron ionisation or electrospray ionisation sources operating in positive and negative modes were used to determine the performances of the clean-up methods. A set of 102 chemicals with a wide range of physico-chemical properties that covers the chemical space of mid- to non-polar contaminants, was used to assess and compare recoveries and matrix effects. Matrix effects, that could hinder the mass spectrometer signal, were lower for extracts cleaned-up with H2SO4 than for the ones cleaned-up with PDMS, EMR and GPC. The recoveries were satisfactory for both GPC and EMR while those determined for PDMS and H2SO4 were low due to poor partitioning and degradation/dissociation of the compounds, respectively. The choice of the clean-up methods, among those assessed, should be a compromise that takes into account the matrix under consideration, the levels and the physico-chemical properties of the contaminants.

Persistent organochlorine pesticides in periprostatic adipose tissue from men with prostate cancer: Ethno-geographic variations, association with disease aggressiveness.

Although several studies have examined the relationship between organochlorine pesticides (OCPs) and prostate cancer (PCa) risk, no data are available concerning the association between OCPs concentrations in periprostatic adipose tissue (PPAT), which reflects cumulative exposure, and PCa aggressiveness. Moreover, no previous study has compared OCPs exposure in two distinct ethno-geographical populations. The objectives were to analyze OCPs in PPAT of PCa patients from either Mainland France or French West Indies in correlation with features of tumor aggressiveness, after adjusting for potential confounders such age, BMI, and polyunsaturated fatty acid (PUFA) content of PPAT. PPAT was analyzed in 160 patients (110 Caucasians and 50 African-Caribbeans), 80 with an indolent tumor (ISUP group 1 + pT2), and 80 with an aggressive tumor (ISUP group more than 3 + pT3). The concentrations of 29 OCPs were measured in PPAT concomitantly with the characterization of PUFA content. Exposure patterns of OCPs differed according to the ethno-geographical origin. Most OCPs were found at higher concentration in Caucasian patients, whereas pp'-DDE content was twice as high in African-Caribbeans. Chlordecone was only detected in PPAT from African-Caribbean patients. Most OCP concentrations were positively correlated with age, and some with BMI. After adjusting for age, BMI, and PUFA composition of PPAT, no significant association was found between OCPs content and risk of aggressive disease, except of mirex which appeared inversely associated with aggressive features of PCa in Caucasian patients. These results highlight a significant ethno-geographic variation in internal exposure to OCPs, which likely reflects differences in consumption patterns. The inverse relationship observed between mirex concentration and markers of PCa aggressiveness need to be further investigated.

Health risk assessment to polychlorinated naphthalenes dioxin-like compounds in French sea food consumers.

There has been a recent revival of interest in some historical contaminants such as polychlorinated naphthalenes (PCNs). However, occurrence data are still lacking in some countries although industrial production of PCNs has been reported. This observation led to the first ever assessment of their presence in fish and seafood products in France in the present work. Their analysis was integrated in an already validated method applied for polychlorinated dibenzodioxins/furans (PCDD/Fs) and polychlorinated biphenyls (PCBs), based on the structural similarity existing between these POPs. Performances of the method (LODs in the range 0.10-0.28 pg g-1 wet weight (ww), LOQs in the range 0.33-0.93 pg g-1 ww), enabled monitoring 69 di-to octachlorinated congeners in a large representative set of fish and seafood samples collected in 2005 in four coastal areas of the French mainland (n > 30). Their systematic presence was demonstrated in all the investigated seafood products, with levels (ΣPCNs in the range 2-440 pg g-1 wet weight) close to those already reported in other European fish and seafood sampled at a similar period. In addition, the robust measurement of almost all possible PCNs (69/75) allowed a fine interpretation of the observed profiles, highlighting in particular the specificities between species and fishing areas. Compared to the PCDD/Fs, PCBs, and polybrominated diphenylethers levels also measured for this set of samples, PCNs were observed as minor contributors to total concentrations (0.05-3.2%). The specific PCN related dietary dioxin-like exposure could be evaluated at 0.028-0.051 pg of toxic equivalent (TEQ) per kg of body weight per week for an adult, based on fish and seafood consumption only. Overall, this study provides the first baseline data on the occurrence of a large number of PCNs in France, which will allow future evaluation of temporal trends and associated risks.

Non-targeted screening methodology to characterise human internal chemical exposure: Application to halogenated compounds in human milk.

Suspect and non-targeted screening approaches are a matter of increasing interest notably with regard to the Exposome contextual framework, but their application to human samples still remains limited at this date. The aim of the present study was to develop a non-targeted workflow from sample preparation to data processing and method assessment to characterise the human internal chemical exposure at early life stage. The method was focused on human milk to investigate mother and newborn exposure to known organic contaminants and to extend the characterisation to unknown compounds. We specifically focused on halogenated biomarkers of exposure due to persistence and potential toxicological impact reasons. The newly developed approach was based on a simple and fast sample preparation followed by a comprehensive analysis by both liquid and gas phase chromatography coupled to high resolution mass spectrometry. Critical steps of the non-targeted workflow as the method assessment have been addressed with a reference mix of 30 chlorinated and brominated contaminants encompassing various substances groups and a statistical approach. Data processing until the identification of biomarkers of exposure was possible with homemade bioinformatics tools. On the other hand, the method was validated by the identification of historical chemicals as hexachlorobenzene and p,p'-DDE and emerging chemical as 4-hydroxychlorothalonil. This approach opens the door to further extensions and consolidations to offer new capabilities for exposomics and environmental health research.

Quantification of light polycyclic aromatic hydrocarbons in seafood samples using on-line dynamic headspace extraction, thermodesorption, gas chromatography tandem mass spectrometry, based on an isotope dilution approach.

The aim of our work was to develop an analytical strategy to quantify naphthalene, acenaphthylene, acenaphthene, fluorene, phenanthrene and anthracene in fish products by on-line dynamic headspace extraction, followed by thermodesorption injection and gas chromatography analysis coupled with tandem mass spectrometry using electron ionization mode (DHS-TD-GC-EI-MS/MS). The developed protocol used 1 g of freeze-dried or oil sample supplemented with perdeuterated light PAHs. The sample was heated at [90 -100 °C], the headspace of the sample was swept by nitrogen and the trapping of the PAHs was carried out on a Tenax-type adsorbent placed at 25 °C. Analytes were thermodesorbed at 300 °C from the dried adsorbant and then cryofocused on a cooled injection system (CIS) at -25 °C before injection (12 °C s-1 up to 300 °C). The chromatographic separation of PAHs was carried out on a 5-MS type column (30 m × 0.25 mm, 0.25 µm) and the acquisition of the signals was performed in SRM following the transitions, involving the loss of one or two hydrogen atoms from the molecular ion. In view of the principle of extraction, the calibration curve was performed on a representative matrix or using the standard addition method. Quantification limits were determined between 0.01 and 0.6 ng g-1 of matrix from the method blank results. The method was validated by a series of multi-level supplemented matrix assays and by the analysis of a reference material from an inter-laboratory test (mussels, IAEA-432). The average of the expanded measurement uncertainty was from 9 to 44% for the four lightest PAHs, except for fluorene when the sample incubation was set at 90 °C. Occurrence measurements were performed on almost two hundred samples of molluscs, echinoderms and fish. The results have shown a quantification frequency greater than 66% for naphthalene and fluorene, at concentrations below a few ng g-1 of dry matter of fishery products. With this methodology, the light PAHs occurrence can now be measured in a wider range of foodstuffs in order to better characterize their contamination trends and the associated risk simultaneously.

In vivo comparison of the proangiogenic properties of chlordecone and three of its dechlorinated derivatives formed by in situ chemical reduction.

In situ chemical reduction (ISCR) has been identified as a possible way for the remediation of soils contaminated by chlordecone (CLD). Evidences provided by the literature indicate an association between the development of prostate cancer and CLD exposure (Multigner et al. 2010). In a previous in vitro study, we demonstrated that the two main dechlorinated CLD derivatives formed by ISCR, CLD-1Cl, and CLD-3Cl have lower cytotoxicity and proangiogenic properties than CLD itself (Legeay et al. 2017). By contrast, nothing is known on the in vivo proangiogenic effect of these dechlorinated derivatives. Based on in vitro data, the aims of this study were therefore to evaluate the in vivo influence of CLD and three of its dechlorinated metabolites in the control of neovascularization in a mice model of prostate cancer. The proangiogenic effect of CLD and three of its dechlorinated derivatives, CLD-1Cl, CLD-3Cl, and CLD-4Cl, was evaluated on a murine model of human prostate tumor (PC-3) treated, at two exposure levels: 33 µg/kg and 1.7 µg/kg respectively reflecting acute and chronic toxic exposure in human. The results of serum measurements show that, for the same ingested dose, the three metabolite concentrations were significantly lower than that of CLD. Dechlorination of CLD lead therefore to molecules that are biologically absorbed or metabolized, or both, faster than the parent molecule. Prostate tumor growth was lower in the groups treated by the three metabolites compared to the one treated by CLD. The vascularization measured on the tumor sections was inversely proportional to the rate of dechlorination, the treatment with CLD-4Cl showing no difference with control animals treated with only the vehicle oil used for all substances tested. We can therefore conclude that the proangiogenic effect of CLD is significantly decreased following the ISCR-resulting dechlorination. Further investigations are needed to elucidate the molecular mechanisms by which dechlorination of CLD reduces proangiogenic effects in prostate tumor.

Assessment of Dechlorane Plus and related compounds in foodstuffs and estimates of daily intake from Lebanese population.

Dechlorane Related Compounds (DRCs), including Dechlorane Plus (syn/anti-DP or syn/anti-DDC-CO) and related compounds (Dec-601 or DDC-ID, Dec-602 or DDC-DBF, Dec-603 or DDC-Ant and Chlordene Plus or DDC-PDD), are a group of polychlorinated flame retardants of concern since they were first reported in various environmental and biota matrices about one decade ago. In this work, we investigated the dietary intake of the Lebanese population to these lipophilic environmental contaminants upon the evaluation of selected foodstuff contamination. Collected food samples (n = 58) were selected to be representative of the lipid fraction of the whole diet of the Beiruti population. The samples were analysed using pressurized liquid extraction, silica multilayer column followed by gel permeation chromatography for purification and GC-EI-HRMS for separation and detection. Detection frequency of at least one compound among Dechlorane Plus (syn-DP and anti-DP), Dechlorane 602, 603 and Chlordene Plus) was 91%. The mean concentrations of ∑6DRCs, by food group, ranged from 4.7 to 29.5 pg g-1 wet weight in lowerbound (LB) and from 6.7 to 76.9 pg g-1 wet weight in upperbound (UB). Based on food habits, the dietary intake of Beiruti adults was estimated to be between 3.71 (LB) and 5.61 (UB) ng day-1. Dechlorane Plus and Dechlorane 602 were the dominant compounds, contributing to 70 and 24% of the total intake (LB value), respectively. This study reports for the first time the occurrence of Dechloranes in Lebanese foods and proposes corresponding deterministic dietary exposure scenario.

Simultaneous analysis of historical, emerging and novel brominated flame retardants in food and feed using a common extraction and purification method.

Brominated Flame Retardants (BFRs) are still widely used for industrial purposes. These contaminants may enter the food chain where they mainly occur in food of animal origin. The aim of our work was to provide a unique method able to quantify the widest range of BFRs in feed and food items. After freeze-drying and grinding, a pressurized liquid extraction was carried out. The extract was purified on acidified silica, Florisil® and carbon columns, the four separated fractions were analyzed by gas and liquid chromatography coupled to high resolution and tandem mass spectrometry. Isotopic dilution was preferentially used when commercial labelled compounds were available. Analytical sensitivity was in accordance with the expectations of Recommendation 2014/118/EU for PBDEs, HBCDDs, TBBPA, TBBPA-bME, EHTBB, BEHTEBP and TBBPA-bME. Additional BFRs were included in this analytical method with the same level of performances (LOQs below 0.01 ng g-1 ww). These are PBBs, pTBX, TBCT, PBBz, PBT, PBEB, HBBz, BTBPE, OBIND and T23BPIC. However, some of the BFRs listed in Recommendation 2014/118/EU are not yet covered by our analytical method, i.e. TBBPA-bOHEE, TBBPA-bAE, TBBPA-bGE, TBBPA-bDiBPrE, TBBPS, TBBPS-bME, TDBPP, EBTEBPI, HBCYD and DBNPG. The uncertainty measurement was fully calculated for 21 of the 31 analytes monitored in the method. Reproducibility uncertainty was below 23% in isotopic dilution. Certified reference materials are now required to better characterize the trueness of this method, which was applied in the French National Control Plans.

Ultra-trace quantification method for chlordecone in human fluids and tissues.

Chlordecone is an organochlorine pesticide (OCP) considered as a Persistent Organic Pollutant (POP) as it persists in the environment, bio-accumulates through the food web, causes adverse effects to human health and the environment and transports across international boundaries far from its sources. The atypical physico-chemical properties of chlordecone make its inclusion in classical analytical approaches non applicable. The aim of our work was to include chlordecone in a multi organochlorine residue method preventing any degradation during the analytical process and thus allowing quantification at ppt (ngkg(-1) or ngL(-1)) levels for a wide range of OCPs in breast milk, human serum and adipose tissue. After GC-HRMS vs. MS/MS and EI vs. APCI comparisons, the major improvement in terms of sensitivity was found in decreasing the length and film thickness of the gas chromatography column. Thanks to a linear correlation between relative response and quantity of chlordecone injected, LC-(ESI-)-MS/MS was finally preferred. An acetonitrile based gradient optimized on a C30 coreshell HPLC column has led to reaching limits of quantification as low as 8ngL(-1), 25pgmL(-1) and 0.2ngg(-1) fat for breast milk, serum and adipose tissue, respectively, allowing multiresidue OCP quantification at concentration levels compatible with biomonitoring purposes and pre-requisites.

Pollutants in pet dogs: a model for environmental links to breast cancer.

PURPOSE: Invasive breast carcinoma is the most common cancer in women as in non-ovariectomised pet dogs, which are already identified as a valuable spontaneous preclinical model for that disease. Geographical and time trends suggest that environmental factors may play an important role in the etiology and pathogenesis of breast cancer. Persistent organic pollutants (POPs) fit perfectly with these trends and are known to interact with hormonal receptors implicated in breast cancer subtyping. The aim of this innovating study was to evaluate the interest of the companion dog model in assessing chemical exposure and breast cancer associations, in order to identify common etiological features with the human disease in a context of comparative oncology. METHODS: We monitored a hundred of molecules belonging to a large panel of POPs (dioxins, dioxin-like and non dioxin-like polychlorobisphenyls, organochlorine pesticides, brominated flame retardants, perfluorinated alkylated substances) in companion dogs diagnosed for mammary adenocarcinoma (n = 54) and non cancer controls (n = 47). RESULTS: All targeted chemical families were able to be detected in canine samples. We identified pollutants associated with mammary cancer belonging to the dioxin like-PCB family (notably PCB-118, -156, -105, -114) that were already pointed out in human epidemiological studies on breast cancer, and that fit with the fundamental role of the Aryl Hydrocarbon Receptor in the promotion of breast cancer. CONCLUSIONS: Similarities observed in the spontaneous dog model are very helpful to progress in interpretation of human breast cancer-environment relationships. This study provides a new insight focusing on this discrete but recurrent signature.