Liquid chromatography-mass spectrometry based metabolomics investigation of different tissues of Mytilus galloprovincialis.

The Mediterranean mussel (Mytilus galloprovincialis) is widely used in monitoring programs and in ecotoxicological studies to examine the biological effects of physicochemical parameter changes and the impact of chemical pollutants. Metabolomics has recently demonstrated high potential to gain further insight into the molecular effects of chemical exposure and the success of its application is dependent on the extent of prior metabolomics knowledge available on the target organism. Therefore, the purpose of this study was the investigation of the metabolites of five different functional tissues of male and female Mediterranean mussels (digestive gland, foot, gill and gonad tissues and in the remaining soft tissues) accessible to the analysis using the most common sample preparation recommended for tissue analysis (i.e. Bligh & Dyer). Metabolic fingerprints were acquired via liquid chromatography high-resolution mass spectrometry and the identification was based on an internal database developed in the laboratory. It led to the identification of 110 metabolites, among which amino acids, carboxylic acids, purine and pyrimidine metabolites were often the most abundant. The metabolic contents of the five tissues quantitatively and qualitatively differed, with a clear distinction between male and female contents observed in the gonads and digestive glands. These results underline the importance of selecting the most suitable tissue and sex to study the impact of contamination on metabolism and the need for further research to deeper characterize the metabolome of this organism.

Transcriptomic and metabolomic integration to assess the response of gilthead sea bream (Sparus aurata) exposed to the most used insect repellent: DEET.

DEET is one of the most frequently detected insect repellents in the environment reaching concentrations of several µg L-1 in surface water. There is scarce information available regarding its mode of action in non-target organisms. Here, we have used an integrated metabolomic and transcriptomic approach to elucidate the possible adverse effects of DEET exposure in the marine fish gilthead sea bream (Sparus aurata). Individuals were exposed at an environmentally relevant concentration of DEET (10 µg L-1) for 22 days in a continuous flow-through system. Transcriptomic analysis revealed 250 differentially expressed genes in liver, while metabolomic analysis identified 190 differentially modulated features in liver and 98 in plasma. Multi-omic data integration and visualization allowed elucidation of the modes of action of DEET exposure, including: energy depletion through the disruption of carbohydrate and amino acids metabolisms, oxidative stress leading to DNA damage, lipid peroxidation, and damage to cell membrane and apoptosis. Activation of xenobiotic pathway as well as the inmune-inflammatory reaction was evidenced in the present work.

Metabolism of the aquatic pollutant diclofenac in the Lymnaea stagnalis freshwater gastropod.

The metabolism of organic contaminants in Lymnaea stagnalis freshwater gastropod remains unknown. Yet, pharmaceuticals-like the NSAID diclofenac-are continuously released in the aquatic environment, thereby representing a risk to aquatic organisms. In addition, lower invertebrates may be affected by this pollution since they are likely to bioaccumulate contaminants. The metabolism of pharmaceuticals in L. stagnalis requires further investigation to understand their detoxification mechanisms and characterized the risk posed by contaminant exposure in this species. In this study, a non-targeted strategy using liquid chromatography combined with high-resolution mass spectrometry was applied to highlight metabolites formed in L. stagnalis freshwater snails exposed to 300 µg/L diclofenac for 3 and 7 days. Nineteen metabolites were revealed by this approach, 12 of which were observed for the first time in an aquatic organism exposed to diclofenac. Phase I metabolism involved hydroxylation, with detection of 3'-, 4'-, and 5-hydroxydiclofenac and three dihydroxylated metabolites, as well as cyclization, oxidative decarboxylation, and dehydrogenation, while phase II metabolism consisted of glucose and sulfate conjugation. Among these reactions, the two main DCF detoxification pathways detected in L. stagnalis were hydroxylation (phase I) and glucosidation (phase II).

Early Biological Modulations Resulting from 1-Week Venlafaxine Exposure of Marine Mussels Mytilus galloprovincialis Determined by a Metabolomic Approach.

There is growing evidence of the presence of pharmaceuticals in natural waters and their accumulation in aquatic organisms. While their mode of action on non-target organisms is still not clearly understood, their effects warrant assessment. The present study assessed the metabolome of the Mediterranean mussel (Mytilus galloprovincialis) exposed to a 10 µg/L nominal concentration of the antidepressant venlafaxine (VLF) at 3 time-points (1, 3, and 7 days). Over the exposure period, we observed up- or down-modulations of 113 metabolites, belonging to several metabolisms, e.g., amino acids (phenylalanine, tyrosine, tryptophan, etc.), purine and pyrimidine metabolisms (adenosine, cyclic AMP, thymidine, etc.), and several other metabolites involved in diverse functions. Serotonin showed the same time-course modulation pattern in both male and female mussels, which was consistent with its mode of action in humans, i.e., after a slight decrease on the first day of exposure, its levels increased at day 7 in exposed mussels. We found that the modulation pattern of impacted metabolites was not constant over time and it was gender-specific, as male and female mussels responded differently to VLF exposure.

Environmental Metabolomics Promises and Achievements in the Field of Aquatic Ecotoxicology: Viewed through the Pharmaceutical Lens.

Scientists often set ambitious targets using environmental metabolomics to address challenging ecotoxicological issues. This promising approach has a high potential to elucidate the mechanisms of action (MeOAs) of contaminants (in hazard assessments) and to develop biomarkers (in environmental biomonitoring). However, metabolomics fingerprints often involve a complex mixture of molecular effects that are hard to link to a specific MeOA (if detected in the analytical conditions used). Given these promises and limitations, here we propose an updated review on the achievements of this approach. Metabolomics-based studies conducted on the effects of pharmaceutical active compounds in aquatic organisms provide a relevant means to review the achievements of this approach, as prior knowledge about the MeOA of these molecules could help overcome some shortcomings. This review highlighted that current metabolomics advances have enabled more accurate MeOA assessment, especially when combined with other omics approaches. The combination of metabolomics with other measured biological endpoints has also turned out to be an efficient way to link molecular effects to (sub)-individual adverse outcomes, thereby paving the way to the construction of adverse outcome pathways (AOPs). Here, we also discuss the importance of determining MeOA as a key strategy in the identification of MeOA-specific biomarkers for biomonitoring. We have put forward some recommendations to take full advantage of environmental metabolomics and thus help fulfil these promises.

Multi-omic approach to evaluate the response of gilt-head sea bream (Sparus aurata) exposed to the UV filter sulisobenzone.

Sulisobenzone (BP-4) is one of the benzophenone type UV filters most frequently detected in aquatic ecosystems. As a suspected endocrine disrupting compound, scarce information is available yet about other molecular effects and its mechanism of action. Here, we used an integrated transcriptomic and metabolomic approach to improve the current understanding on the toxicity of BP-4 towards aquatic species. Gilt-head sea bream individuals were exposed at environmentally relevant concentrations (10 µg L-1) for 22 days. Transcriptomic analysis revealed 371 differentially expressed genes in liver while metabolomic analysis identified 123 differentially modulated features in plasma and 118 in liver. Integration of transcriptomic and metabolomic data showed disruption of the energy metabolism (>10 pathways related to the metabolism of amino acids and carbohydrates were impacted) and lipid metabolism (5 glycerophospholipids and the expression of 3 enzymes were affected), suggesting oxidative stress. We also observed, for the first time in vivo and at environmental relevant concentrations, the disruption of several enzymes involved in the steroid and thyroid hormones biosynthesis. DNA and RNA synthesis was also impacted by changes in the purine and pyrimidine metabolisms. Overall, the multiomic workflow presented here increases the evidence on suspected effects of BP-4 exposure and identifies additional modes of action of the compounds that could have been overlooked by using single omic approaches.

An integrated metabolomics and proteogenomics approach reveals molecular alterations following carbamazepine exposure in the male mussel Mytilus galloprovincialis.

Carbamazepine is one of the most abundant pharmaceutical active compounds detected in aquatic systems. Based on laboratory exposures, carbamazepine has been proven to adversely affect aquatic organisms. However, the underlying molecular events remain poorly understood. This study aims to investigate the molecular mechanisms potentially associated with toxicological effects of carbamazepine on the mussel Mytilus galloprovincialis exposed for 3 days at realistic concentrations encountered in coastal environments (80 ng/L and 8 µg/L). An integrated metabolomics and proteogenomics approach, including data fusion strategy, was applied to gain more insight in molecular events and cellular processes triggered by carbamazepine exposure. Consistent metabolic and protein signatures revealed a metabolic rewiring and cellular stress at both concentrations (e.g. intensification of protein synthesis, transport and catabolism processes, disruption of lipid and amino acid metabolisms). These highlighted molecular signatures point to the induction of autophagy, closely related with carbamazepine mechanism of action, as well as a destabilization of the lysosomal membranes and an enzymatic overactivity of the peroxisomes. Induction of programmed cell death was highlighted by the modulation of apoptotic cognate proteins. The proposed integrative omics data analysis was shown to be highly relevant to identify the modulations of the two molecular levels, i.e. metabolites and proteins. Multi-omics approach is able to explain the resulting complex biological system, and document stronger toxicological pieces of evidence on pharmaceutical active compounds at environmental concentrations in sentinel organisms.

Long-term exposure to environmental diclofenac concentrations impairs growth and induces molecular changes in Lymnaea stagnalis freshwater snails.

As pharmaceutical substances are highly used in human and veterinary medicine and subsequently released in the environment, they represent emerging contaminants in the aquatic compartment. Diclofenac (DCF) is one of the most commonly detected pharmaceuticals in water and little research has been focused on its long-term effects on freshwater invertebrates. In this study, we assessed the chronic impacts of DCF on the freshwater gastropod Lymnaea stagnalis using life history, behavioral and molecular approaches. These organisms were exposed from the embryo to the adult stage to three environmentally relevant DCF concentrations (0.1, 2 and 10 µg/L). The results indicated that DCF impaired shell growth and feeding behavior at the juvenile stage, yet no impacts on hatching, locomotion and response to light stress were noted. The molecular findings (metabolomics and transcriptomic) suggested that DCF may disturb the immune system, energy metabolism, osmoregulation and redox balance. In addition, prostaglandin synthesis could potentially be inhibited by DCF exposure. The molecular findings revealed signs of reproduction impairment but this trend was not confirmed by the physiological tests. Combined omics tools provided complementary information and enabled us to gain further insight into DCF effects in freshwater organisms.

In vivo exposure of marine mussels to venlafaxine: bioconcentration and metabolization.

Pharmaceuticals are present in natural waters, thus contributing to the general exposure of aquatic organisms, but few data are available on the accumulation of these substances in marine organisms. The present study evaluated the in vivo bioconcentration of an antidepressant-venlafaxine (VLF)-in marine mussels (Mytilus galloprovincialis) during 7 days of exposure at nominal 10 µg/L concentration, followed by a 7-day depuration period. The bioconcentration factor (BCF) was 265 mL/g dry weight (dw). VLF accumulation reached an average tissue concentration of 2146 ± 156 ng/g dw within 7 days, showing a first-order kinetics process. N-desmethylvenlafaxine (N-VLF) and O-desmethylvenlafaxine (O-VLF) metabolites were quantified in mussel tissues, whereas N,N-didesmethylvenlafaxine (NN-VLF) was only recorded as being detected. These three metabolites were also quantified in water, indicating an active metabolism and VLF excretion in Mediterranean mussels. Complementary experiments conducted at nominal concentrations of 1, 10, and 100 µg/L for 7 days confirmed the VLF bioconcentration and metabolism and allowed us to quantify a supplementary metabolite, i.e., N,O-didesmethylvenlafaxine (NO-VLF), in mussel tissues. These results encourage further research on a more complete characterization of metabolism and on any disturbances linked to bioconcentration of VLF on bivalves.

Multifactorial Analysis of Environmental Metabolomic Data in Ecotoxicology: Wild Marine Mussel Exposed to WWTP Effluent as a Case Study.

Environmental metabolomics is a powerful approach to investigate the response of organisms to contaminant exposure at a molecular scale. However, metabolomic responses to realistic environmental conditions can be hindered by factors intrinsic to the environment and the organism. Hence, a well-designed experimental exposure associated with adequate statistical analysis could be helpful to better characterize and relate the observed variability to its different origins. In the current study, we applied a multifactorial experiment combined to Analysis of variance Multiblock Orthogonal Partial Least Squares (AMOPLS), to assess the metabolic response of wild marine mussels, Mytilus galloprovincialis, exposed to a wastewater treatment plant effluent, considering gender as an experimental factor. First, the total observed variability was decomposed to highlight the contribution of each effect related to the experimental factors. Both the exposure and the interaction gender × exposure had a statistically significant impact on the observed metabolic alteration. Then, these metabolic patterns were further characterized by analyzing the individual variable contributions to each effect. A main change in glycerophospholipid levels was highlighted in both males and females as a common response, possibly caused by oxidative stress, which could lead to reproductive disorders, whereas metabolic alterations in some polar lipids and kynurenine pathway were rather gender-specific. This may indicate a disturbance in the energy metabolism and immune system only in males. Finally, AMOPLS is a useful tool facilitating the interpretation of complex metabolomic data and is expected to have a broad application in the field of ecotoxicology.

Metabolomics approach reveals disruption of metabolic pathways in the marine bivalve Mytilus galloprovincialis exposed to a WWTP effluent extract.

Conventional wastewater treatment plants (WWTPs) discharge a highly diverse range of organic contaminants in aquatic environments, including marine waters. The health of marine ecosystems could be threatened by contaminants release. Environmental metabolomics can be helpful to assess the effects of multi-contamination on marine organisms without any a priori information since it is able to provide meaningful information on the biochemical response of organisms to a stress. The aim of the present study was to evaluate the potential of metabolomics to highlight key metabolites disrupted by a WWTP effluent extract exposure and then elucidate the biological effects of such exposure on Mediterranean mussels (Mytilus galloprovincialis). Exposed male mussels showed numerous metabolites altered in response to WWTP effluent exposure. The highlighted metabolites belong mainly to amino acids metabolism (e.g. tyrosine, phenylalanine, leucine, proline, etc.), neurohormones (dopamine and a serotonin metabolite), purine and pyrimidine metabolism (e.g. adenosine, adenine, guanine, uracil etc.), citric acid cycle intermediates (e.g. malate, fumarate), and a component involved in oxidative stress defense (oxidized glutathione). Modulation of these metabolites could reflect the alteration of several biological processes such as energy metabolism, DNA and RNA synthesis, immune system, osmoregulation, byssus formation and reproduction, which may lead to a negative impact of organism fitness. Our study provided further insight into the effects of WWTP effluents on marine organisms.

Urinary excretion of sex steroid hormone metabolites after consumption of cow milk: a randomized crossover intervention trial.

Background: Current cow milk production practices introduce considerable levels of pregnancy hormones into the milk. Humans are exposed to these hormones when cow milk is consumed, and this may explain the observed association between cow milk consumption and several hormone-sensitive cancers. Objectives: The aim of the study was to evaluate whether cow milk consumption is associated with an increase in urinary excretion of sex steroid hormones and their metabolites in humans. Methods: We conducted a randomized crossover intervention feeding experiment. A total of 109 postmenopausal women consumed 1 L of semiskimmed milk (1.5% fat) per day for 4 d and 1 L of whole milk (3.5% fat) per day for 4 d, intersected by 4-d wash-out periods. Sex steroid hormone levels were measured in 24-h urine samples collected at the end of each intervention and wash-out period. Results: Estrogens, androgens, and progesterone were detected in the examined milk samples used for our intervention. Although a very high proportion of the estrogens were conjugated, only small proportions of the androgens and progesterone were conjugated. Milk consumption resulted in a significant increase in urinary estrone (E1) excretion, whereas estradiol (E2), estriol (E3), and 16ketoE2 excretion only increased after semiskimmed milk consumption. Urinary pregnanediol glucuronide excretion was not significantly affected. Conclusion: Cow milk consumption increases urinary excretion of E1 in humans. Ingestion of semiskimmed milk appears also to raise E2, E3, and 16ketoE2 excretion, but future studies need to confirm these associations. This trial was registered at https://www.drks.de as DRKS00003377.

Diclofenac in the marine environment: A review of its occurrence and effects.

Interest in the presence and effects of diclofenac (DCF) and other pharmaceutical products (PPs) in the aquatic environment has been growing over the last 20 years. DCF has been included in the First Watch List of the EU Water Framework Directive in order to gather monitoring data in surface waters. Despite PP input in water bodies, few studies have been conducted to determine the extent of DCF occurrence and effects on marine ecosystems, which is usually the final recipient of surface waters. The present article reviews available published data on DCF occurrence in marine water, sediment and organisms, and its effects on marine organisms. The findings highlight the scarcity of available data on the occurrence and effects of DCF in marine ecosystems, and the need for further data acquisition to assess the risks associated with the presence of this compound in the environment.

Metabolomics assessment of the effects of diclofenac exposure on Mytilus galloprovincialis: Potential effects on osmoregulation and reproduction.

The presence of pharmaceutically active compounds in aquatic environments has become a major concern over the past 20years. Elucidation of their mode of action and effects in non-target organisms is thus now a major ecotoxicological challenge. Diclofenac (DCF) is among the pharmaceutical compounds of interest based on its inclusion in the European Union Water Framework Directive Watch List. In this study, our goal was to investigate the potential of a metabolomic approach to acquire information without any a priori hypothesis about diclofenac effects on marine mussels. For this purpose, mussel's profiles were generated by liquid chromatography combined with high resolution mass spectrometry. Two main metabolic pathways were found to be impacted by diclofenac exposure. The tyrosine metabolism was mostly down-modulated and the tryptophan metabolism was mostly up-modulated following exposure. To our knowledge, such DCF effects on mussels have never been described despite being of concern for these organisms: catecholamines and serotonin may be involved in osmoregulation, and in gamete release in mollusks. Our results suggest potential impairment of mussel osmoregulation and reproduction following a DCF exposure.

Exposure of marine mussels to diclofenac: modulation of prostaglandin biosynthesis.

Human pharmaceuticals, such as nonsteroidal anti-inflammatory drugs (NSAIDs), are an emerging threat to marine organisms. NSAIDs act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid into prostaglandins. One experiment was carried out whereby marine mussels were exposed for 72 h to 1 and 100 µg/L diclofenac (DCF). A specific and sensitive method using liquid chromatography high-resolution tandem mass spectrometry was developed to quantify DCF in mussel tissues. The developed method could also clearly identify and quantify COX products, i.e., prostaglandin levels, and be used to assess their modulation following DCF exposure. Prostaglandin-D2 (PGD2) was always found below the detection limit (20 µg/kg dry weight (dw)). Basal prostaglandin-E2 (PGE2) concentrations ranged from below the detection limit to 202 µg/kg dw. Exposure of 100 µg/L resulted in a significant reduction in PGE2 levels, whereas a downward trend was observed at 1 µg/L exposure. No difference was observed for prostaglandin-F2α (PGF2α) levels between controls and exposed organisms.

Metabolic profiling identification of metabolites formed in Mediterranean mussels (Mytilus galloprovincialis) after diclofenac exposure.

Despite the growing concern on the presence of pharmaceutically active compounds in the environment, few studies have been conducted on their metabolism in marine organisms. In this study, a non-targeted strategy based on the generation of chemical profiles generated by liquid chromatography combined with high resolution mass spectrometry was used to highlight metabolite production by the Mediterranean mussel (Mytilus galloprovincialis) after diclofenac exposure. This method allowed revealing the production of 13 metabolites in mussel tissues. Three of them were phase I metabolites, including 4'-hydroxy-diclofenac and 5-hydroxy-diclofenac. The remaining 10 were phase II metabolites, including sulfate and amino acids conjugates. Among all of the metabolites highlighted, 5 were reported for the first time in an aquatic organism exposed to diclofenac.

Development of a molecular recognition based approach for multi-residue extraction of estrogenic endocrine disruptors from biological fluids coupled to liquid chromatography-tandem mass spectrometry measurement.

Multi-residue methods permitting the high-throughput and affordable simultaneous determination of an extended range of endocrine disrupting chemicals (EDCs) with reduced time and cost of analysis is of prime interest in order to characterize a whole set of bioactive compounds. Such a method based on UHPLC-MS/MS measurement and dedicated to 13 estrogenic EDCs was developed and applied to biological matrices. Two molecular recognition-based strategies, either molecular imprinted polymer (MIP) with phenolic template or estrogen receptors (ERα) immobilized on a sorbent, were assessed in terms of recovery and purification efficiency. Both approaches demonstrated their suitability to measure ultra-trace levels of estrogenic EDCs in aqueous samples. Applicability of the MIP procedure to urine and serum samples has also been demonstrated.

Can we trust untargeted metabolomics? Results of the metabo-ring initiative, a large-scale, multi-instrument inter-laboratory study.

The metabo-ring initiative brought together five nuclear magnetic resonance instruments (NMR) and 11 different mass spectrometers with the objective of assessing the reliability of untargeted metabolomics approaches in obtaining comparable metabolomics profiles. This was estimated by measuring the proportion of common spectral information extracted from the different LCMS and NMR platforms. Biological samples obtained from 2 different conditions were analysed by the partners using their own in-house protocols. Test #1 examined urine samples from adult volunteers either spiked or not spiked with 32 metabolite standards. Test #2 involved a low biological contrast situation comparing the plasma of rats fed a diet either supplemented or not with vitamin D. The spectral information from each instrument was assembled into separate statistical blocks. Correlations between blocks (e.g., instruments) were examined (RV coefficients) along with the structure of the common spectral information (common components and specific weights analysis). In addition, in Test #1, an outlier individual was blindly introduced, and its identification by the various platforms was evaluated. Despite large differences in the number of spectral features produced after post-processing and the heterogeneity of the analytical conditions and the data treatment, the spectral information both within (NMR and LCMS) and across methods (NMR vs. LCMS) was highly convergent (from 64 to 91 % on average). No effect of the LCMS instrumentation (TOF, QTOF, LTQ-Orbitrap) was noted. The outlier individual was best detected and characterised by LCMS instruments. In conclusion, untargeted metabolomics analyses report consistent information within and across instruments of various technologies, even without prior standardisation.

Basics of mass spectrometry based metabolomics.

The emerging field of metabolomics, aiming to characterize small molecule metabolites present in biological systems, promises immense potential for different areas such as medicine, environmental sciences, agronomy, etc. The purpose of this article is to guide the reader through the history of the field, then through the main steps of the metabolomics workflow, from study design to structure elucidation, and help the reader to understand the key phases of a metabolomics investigation and the rationale underlying the protocols and techniques used. This article is not intended to give standard operating procedures as several papers related to this topic were already provided, but is designed as a tutorial aiming to help beginners understand the concept and challenges of MS-based metabolomics. A real case example is taken from the literature to illustrate the application of the metabolomics approach in the field of doping analysis. Challenges and limitations of the approach are then discussed along with future directions in research to cope with these limitations. This tutorial is part of the International Proteomics Tutorial Programme (IPTP18).

In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental signalling.

STUDY QUESTION: How does maternal cigarette smoking disturb development of the human fetal ovary? SUMMARY ANSWER: Maternal smoking increases fetal estrogen titres and dysregulates several developmental processes in the fetal ovary. WHAT IS KNOWN ALREADY: Exposure to maternal cigarette smoking during gestation reduces human fetal ovarian cell numbers, germ cell proliferation and subsequent adult fecundity. STUDY DESIGN, SIZE, DURATION: The effects of maternal cigarette smoking on the second trimester human fetal ovary, fetal endocrine signalling and fetal chemical burden were studied. A total of 105 fetuses were studied, 56 from mothers who smoked during pregnancy and 49 from those who did not. PARTICIPANTS/MATERIALS, SETTING METHODS: Ovary, liver and plasma samples were collected from electively terminated, normally progressing, second trimester human fetuses. Circulating fetal hormones, levels of 73 fetal ovarian transcripts, protein localization, density of oocytes/primordial follicles and levels of 16 polycyclic aromatic hydrocarbons (PAHs) in the fetal liver were determined. MAIN RESULTS AND THE ROLE OF CHANCE: Circulating fetal estrogen levels were very high and were increased by maternal smoking (ANOVA, P = 0.055-0.004 versus control). Smoke exposure also dysregulated (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.046-0.023) four fetal ovarian genes (cytochrome P450 scc [CYP11A1], NOBOX oogenesis homeobox [NOBOX], activator of apoptosis harakiri [HRK], nuclear receptor subfamily 2, group E, member 1 [NR2E1]), shifted the ovarian Inhibin ßA/inhibin α ratio (NHBA/INHA) transcript ratio in favour of activin (ANOVA, P = 0.049 versus control) and reduced the proportion of dominant-negative estrogen receptor 2 (ERß: ESR2) isoforms in half the exposed fetuses. PAHs, ligands for the aryl hydrocarbon receptor (AHR), were increased nearly 6-fold by maternal smoking (ANOVA, P = 0.011 versus control). A fifth transcript, COUP transcription factor 1 (nuclear receptor subfamily 2, group F, member 1: NR2F1, which contains multiple AHR-binding sites), was both significantly increased (ANOVA, P = 0.026 versus control) and dysregulated by (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.021) maternal smoking. NR2F1 is associated with repression of FSHR expression and smoke-exposed ovaries failed to show the normal increase in FSHR expression during the second trimester. There was a significantly higher number of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) VASA-positive (ANOVA, P = 0.016 versus control), but not POU domain, class 1, transcription factor 1 (POU5F1) OCT3/4-positive, oocytes in smoke-exposed fetuses and this matched with a significantly higher number of primordial follicles (ANOVA, P = 0.024 versus control). LIMITATIONS, REASONS FOR CAUTION: The effects of maternal smoking on establishment of the maximum fetal primordial follicle pool cannot be reliably studied in our population since the process is not completed until 28 weeks of gestation and normal fetuses older than 21 weeks of gestation are not available for study. Our data suggest that some fetal ovaries are affected by smoke exposure while others are not, indicating that additional studies, with larger numbers, may show more significant effects. WIDER IMPLICATIONS OF THE FINDINGS: Fetal exposure to chemicals in cigarette smoke is known to lead to reduced fecundity in women. Our study suggests, for the first time, that this occurs via mechanisms involving activation of AHR, disruption of inhibin/activin and estrogen signalling, increased exposure to estrogen and dysregulation of multiple molecular pathways in the exposed human fetal ovary. Our data also suggest that alterations in the ESR2 positive and dominant negative isoforms may be associated with reduced sensitivity of some fetuses to increased estrogens and maternal smoking. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by grants from the Chief Scientist Office (Scottish Executive, CZG/1/109, and CZG/4/742), NHS Grampian Endowments (08/02), the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 212885, a Society for Reproduction & Fertility summer studentship, Medical Research Scotland (research grant 354 FRG) and the Medical Research Council (WBS: U.1276.00.002.00001 and G1100357). The authors declare they have no competing interests, be it financial, personal or professional.