Marie Tremblay-Franco

Effects of Low Doses of Bisphenol A on the Metabolome of Perinatally Exposed CD-1 Mice

Background: Bisphenol A (BPA) is a well-known endocrine disruptor used to manufacture polycarbonate plastics and epoxy resins. Exposure of pregnant rodents to low doses of BPA results in pleiotropic effects in their offspring. Objective: We used metabolomics—a method for determining metabolic changes in response to nutritional, pharmacological, or toxic stimuli—to examine metabolic shifts induced in vivo by perinatal exposure to low doses of BPA in CD-1 mice. Methods: Male offspring born to pregnant CD-1 mice that were exposed to vehicle or to 0.025, 0.25, or 25 µg BPA/kg body weight/day, from gestation day 8 through day 16 of lactation, were examined on postnatal day (PND) 2 or PND21. Aqueous extracts of newborns (PND2, whole animal) and of livers, brains, and serum samples from PND21 pups were submitted to 1H nuclear magnetic resonance spectroscopy. Data were analyzed using partial least squares discriminant analysis. Results: Examination of endogenous metabolic fingerprints revealed remarkable discrimination in whole extracts of the four PND2 newborn treatment groups, strongly suggesting changes in the global metabolism. Furthermore, statistical analyses of liver, serum, and brain samples collected on PND21 successfully discriminated among treatment groups. Variations in glucose, pyruvate, some amino acids, and neurotransmitters (γ-aminobutyric acid and glutamate) were identified. Conclusions: Low doses of BPA disrupt global metabolism, including energy metabolism and brain function, in perinatally exposed CD-1 mouse pups. Metabolomics can be used to highlight the effects of low doses of endocrine disruptors by linking perinatal exposure to changes in global metabolism.

Metabolomics Tools for Describing Complex Pesticide Exposure in Pregnant Women in Brittany (France)

Background The use of pesticides and the related environmental contaminations can lead to human exposure to various molecules. In early-life, such exposures could be responsible for adverse developmental effects. However, human health risks associated with exposure to complex mixtures are currently under-explored. Objective This project aims at answering the following questions: What is the influence of exposures to multiple pesticides on the metabolome? What mechanistic pathways could be involved in the metabolic changes observed? Methods Based on the PELAGIE cohort (Brittany, France), 83 pregnant women who provided a urine sample in early pregnancy, were classified in 3 groups according to the surface of land dedicated to agricultural cereal activities in their town of residence. Nuclear magnetic resonance-based metabolomics analyses were performed on urine samples. Partial Least Squares Regression-Discriminant Analysis (PLS-DA) and polytomous regressions were used to separate the urinary metabolic profiles from the 3 exposure groups after adjusting for potential confounders. Results The 3 groups of exposure were correctly separated with a PLS-DA model after implementing an orthogonal signal correction with pareto standardizations (R2 = 90.7% and Q2 = 0.53). After adjusting for maternal age, parity, body mass index and smoking habits, the most statistically significant changes were observed for glycine, threonine, lactate and glycerophosphocholine (upward trend), and for citrate (downward trend). Conclusion This work suggests that an exposure to complex pesticide mixtures induces modifications of metabolic fingerprints. It can be hypothesized from identified discriminating metabolites that the pesticide mixtures could increase oxidative stress and disturb energy metabolism.

Hepatic circadian clock oscillators and nuclear receptors integrate microbiome-derived signals

The liver is a key organ of metabolic homeostasis with functions that oscillate in response to food intake. Although liver and gut microbiome crosstalk has been reported, microbiome-mediated effects on peripheral circadian clocks and their output genes are less well known. Here, we report that germ-free (GF) mice display altered daily oscillation of clock gene expression with a concomitant change in the expression of clock output regulators. Mice exposed to microbes typically exhibit characterized activities of nuclear receptors, some of which (PPARα, LXRβ) regulate specific liver gene expression networks, but these activities are profoundly changed in GF mice. These alterations in microbiome-sensitive gene expression patterns are associated with daily alterations in lipid, glucose, and xenobiotic metabolism, protein turnover, and redox balance, as revealed by hepatic metabolome analyses. Moreover, at the systemic level, daily changes in the abundance of biomarkers such as HDL cholesterol, free fatty acids, FGF21, bilirubin, and lactate depend on the microbiome. Altogether, our results indicate that the microbiome is required for integration of liver clock oscillations that tune output activators and their effectors, thereby regulating metabolic gene expression for optimal liver function.

Salivary markers of taste sensitivity to oleic acid: a combined proteomics and metabolomics approach

Saliva is a biological fluid playing numerous roles in the oral cavity and increasingly considered as a source of markers. The role of saliva in sensory perception has been known for years but it is only recently that its potential role in oral fatty acids (FA) perception has been suggested. The aim of the present work was to study the relationships between taste sensitivity to oleic acid and the salivary proteome (2D electrophoresis) and metabolome (1H NMR). This was achieved by comparing saliva from two groups of subjects, highly (sensitive+) and weakly sensitive (sensitive−) to the taste of oleic acid. Partial least squares-discriminant analyses (PLS-DA) were used to model the relationship between sensitivity to C18:1, and the proteome and metabolome data. The two groups could be discriminated by ten spots. In particular, cystatin SN, cystatin D, zinc-alpha-2-glycoprotein and carbonic anhydrase 6 were overexpressed in the sensitive+ group. The latter result was confirmed by ELISA. The overexpression of these proteins, which have been associated to taste perception, supports the argument that C18:1 is perceived by the taste system. The two groups could also be discriminated on the basis of eight metabolites, with FA, FA/proline, lysine and FA/pyruvate overexpressed in the sensitive+ group and acetate, leucine/isoleucine and butyrate overexpressed in the sensitive− group. The overexpression of these metabolites suggests a higher bacterial load in this group which could be implicated in perception of FAs.

Untargeted profiling of pesticide metabolites by LC–HRMS: an exposomics tool for human exposure evaluation

Human exposure to xenobiotics is usually estimated by indirect methods. Biological monitoring has emerged during the last decade to improve assessment of exposure. However, biomonitoring is still an analytical challenge, because the amounts of sample available are often very small yet analysis must be as thorough and sensitive as possible. The purpose of this work was to develop an untargeted “exposomics” approach by using ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC–HRMS), which was applied to the characterization of pesticide metabolites in urine from pregnant women from a French epidemiological cohort. An upgradable list of pesticides commonly used on different crops, with their metabolites (more than 400 substances) was produced. Raw MS data were then processed to extract signals from these substances. Metabolites were identified by tandem mass spectrometry; putative identifications were validated by comparison with standards and metabolites generated by experiments on animals. Finally, signals of identified compounds were statistically analyzed by use of multivariate methods. This enabled discrimination of exposure groups, defined by indirect methods, on the basis of four metabolites from two fungicides (azoxystrobin, fenpropimorph) used in cereal production. This original approach applied to pesticide exposure can be extended to a variety of contaminant families for upstream evaluation of exposure from food and the environment.

Relationships between the metabolome and the fatty acid composition of human saliva; effects of stimulation

Saliva is a biological fluid that is easy to collect and is of considerable interest as a source of biomarkers. To date, its protein composition has been the most extensively studied but its metabolic composition is also of real interest. However, the composition of saliva is complex and dependent on numerous factors, among which stimulation is source of many variations. The aim of this work was to study the effects of a stimulating condition (chewing) versus a resting condition on the human salivary metabolome. Saliva from 16 subjects was collected on three occasions and studied using nuclear magnetic resonance. The two conditions could be separated by PLS-DA analysis. Fatty acids, some organic acids and amino acids, probably arising from the degradation of prolin-rich proteins, were over-represented in stimulated saliva, whereas taurine, and propionate were over-represented in resting saliva. To clarify further the identification of fatty acids, the free and total fatty acid contents were studied by gas chromatography. The principal fatty acids identified were oleic, stearic and palmitic acids. It was also possible to separate the two conditions of stimulation by PLS-DA. These results show that the regulation of saliva and sampling conditions must be taken into account when studying markers in saliva.

Dynamic Metabolic Disruption in Rats Perinatally Exposed to Low Doses of Bisphenol-A

Along with the well-established effects on fertility and fecundity, perinatal exposure to endocrine disrupting chemicals, and notably to xeno-estrogens, is strongly suspected of modulating general metabolism. The metabolism of a perinatally exposed individual may be durably altered leading to a higher susceptibility of developing metabolic disorders such as obesity and diabetes; however, experimental designs involving the long term study of these dynamic changes in the metabolome raise novel challenges. 1H-NMR-based metabolomics was applied to study the effects of bisphenol-A (BPA, 0; 0.25; 2.5, 25 and 250 μg/kg BW/day) in rats exposed perinatally. Serum and liver samples of exposed animals were analyzed on days 21, 50, 90, 140 and 200 in order to explore whether maternal exposure to BPA alters metabolism. Partial Least Squares-Discriminant Analysis (PLS-DA) was independently applied to each time point, demonstrating a significant pair-wise discrimination for liver as well as serum samples at all time-points, and highlighting unequivocal metabolic shifts in rats perinatally exposed to BPA, including those exposed to lower doses. In BPA exposed animals, metabolism of glucose, lactate and fatty acids was modified over time. To further explore dynamic variation, ANOVA-Simultaneous Component Analysis (A-SCA) was used to separate data into blocks corresponding to the different sources of variation (Time, Dose and Time*Dose interaction). A-SCA enabled the demonstration of a dynamic, time/age dependent shift of serum metabolome throughout the rats’ lifetimes. Variables responsible for the discrimination between groups clearly indicate that BPA modulates energy metabolism, and suggest alterations of neurotransmitter signaling, the latter finding being compatible with the neurodevelopmental effect of this xenoestrogen. In conclusion, long lasting metabolic effects of BPA could be characterized over 200 days, despite physiological (and thus metabolic) changes connected with sexual maturation and aging.

Effect of obesity and metabolic syndrome on plasma oxysterols and fatty acids in human.

BACKGROUND Obesity and the related entity metabolic syndrome are characterized by altered lipid metabolism and associated with increased morbidity risk for cardiovascular disease and cancer. Oxysterols belong to a large family of cholesterol-derived molecules known to play crucial role in many signaling pathways underlying several diseases. Little is known on the potential effect of obesity and metabolic syndrome on oxysterols in human. OBJECTIVES In this work, we questioned whether circulating oxysterols might be significantly altered in obese patients and in patients with metabolic syndrome. We also tested the potential correlation between circulating oxysterols and fatty acids. METHODS 60 obese patients and 75 patients with metabolic syndrome were enrolled in the study along with 210 age- and sex-matched healthy subjects, used as control group. Plasma oxysterols were analyzed by isotope dilution GC/MS, and plasma fatty acids profiling was assessed by gas chromatography coupled with flame ionization detection. RESULTS We found considerable differences in oxysterols profiling in the two disease groups that were gender-related. Compared to controls, males showed significant differences only in 4α- and 4β-hydroxycholesterol levels in obese and metabolic syndrome patients. In contrast, females showed consistent differences in 7-oxocholesterol, 4α-hydroxycholesterol, 25-hydroxycholesterol and triol. Concerning fatty acids, we found minor differences in the levels of these variables in males of the three groups. Significant changes were observed in plasma fatty acid profile of female patients with obesity or metabolic syndrome. We found significant correlations between various oxysterols and fatty acids. In particular, 4β-hydroxycholesterol, which is reduced in obesity and metabolic syndrome, correlated with a number of saturated and mono-unsaturated fatty acids that are end-products of de novo lipogenesis. CONCLUSIONS Our data provide the first evidence that obesity and metabolic syndrome are associated with major, gender-specific, changes in circulating oxysterols and fatty acids. These findings suggest a metabolic link between oxysterols and fatty acids, and that oxysterols may contribute to the epidemic diseases associated with obesity and metabolic syndrome in female.

Potential Input From Metabolomics for Exploring and Understanding the Links Between Environment and Health

Humans may be exposed via their environment to multiple chemicals as a consequence of human activities and use of synthetic products. Little knowledge is routinely generated on the hazards of these chemical mixtures. The metabolomic approach is widely used to identify metabolic pathways modified by diseases, drugs, or exposures to toxicants. This review, based on the state of the art of the current applications of metabolomics in environmental health, attempts to determine whether metabolomics might constitute an original approach to the study of associations between multiple, low-dose environmental exposures in humans. Studying the biochemical consequences of complex environmental exposures is a challenge demanding the development of careful experimental and epidemiological designs, in order to take into account possible confounders associated with the high level of interindividual variability induced by different lifestyles. The choices of populations studied, sampling and storage procedures, statistical tools used, and system biology need to be considered. Suggestions for improved experimental and epidemiological designs are described. Evidence indicates that metabolomics may be a powerful tool in environmental health in the identification of both complex exposure biomarkers directly in human populations and modified metabolic pathways, in an attempt to improve understanding the underlying environmental causes of diseases. Nevertheless, the validity of biomarkers and relevancy of animal-to-human extrapolation remain key challenges that need to be properly explored.

Dietary exposure to a low dose of pesticides alone or as a mixture: The biological metabolic fingerprint and impact on hematopoiesis

Consumers are exposed to a mixture of pesticides through their food intake. These compounds are considered risk factors for human health, and the impact of dietary exposure to low doses of pesticide mixtures remains poorly understood. For this study we developed a mouse model to mimic consumer exposure in order to compare the effect of pesticides both alone or combined at doses corresponding to their Acceptable Daily Intake value. Female mice were exposed to pesticides throughout gestation and lactation. After weaning pups were fed the same pesticide-enriched diet their mothers had received for an additional 11 weeks. A metabonomic approach using (1)H NMR-based analysis of plasma showed that exposure to each pesticide produced a specific metabolic fingerprint in adult offspring. Discriminant metabolites between groups were glucose or lactate, choline, glycerophosphocholine and phosphocholine. Interestingly, metabolite differences were observed as early as weaned animals that had not yet been directly exposed themselves. Studies of the hematopoietic system revealed that dietary exposure to one particular pesticide, endosulfan, produced a significant decrease in red blood cell and hemoglobin levels, consistent with hemolytic anemia. Moreover, cell signaling profiles of bone marrow progenitors were also clearly affected. Expression of cell signaling proteins such as P35, CDC27, FAK, P38 MAP kinase, calcineurin and caspase as well as proteins involved in the stability or structure of the cytoskeleton (vinculin, MAP2) was changed upon dietary exposure to pesticides. Finally, we found that dietary exposure to a mixture of pesticides had effects that differed and were often lesser or equal to that of the most efficient pesticide (endosulfan), suggesting that the effect of pesticide mixtures cannot always be predicted from the combined effects of their constituent compounds.