Frandsen, Henrik Lauritz

Hadrup, Niels

Skov, Kasper

Smedsgaard, Jørn

Vestergaard, Anne Marie

Endocrine disrupting chemicals are compounds which interfere with normal hormone homeostasis. So far the main concern has been the effect on the reproduction and development. This study has been conducted to find the effect of EDC on the human metabolome, using LC high resolution mass spectrometry and chemical separation of plasma metabolites it is possible to find the difference in the plasma metabolome affected by EDC. The study was conducted given a group of rats an EDC and another group a combination of EDC’s and CYP inhibitors

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Downloaded from orbit.dtu.dk on: Dec 19, 2017Effects of perfluorononanoic acid (PFNA) on the metabolic profiling of rat serum byUHPLC-ESI-Q-TOF MSMSSkov, Kasper; Hadrup, Niels; Vestergaard, Anne Marie; Smedsgaard, Jørn; Frandsen, Henrik LauritzPublication date:2013Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Skov, K., Hadrup, N., Vestergaard, A. M., Smedsgaard, J., & Frandsen, H. L. (2013). Effects ofperfluorononanoic acid (PFNA) on the metabolic profiling of rat serum by UHPLC-ESI-Q-TOF MSMS. Postersession presented at 9th International Conference of the Metabolomics Society, Glasgow, United Kingdom.Results• The phospholipid phase shows some effect at the middle concentration. The maineffect shown is a cocktail effect but there is also a trend of an additive effect.• The analysis also reveals a compound verified by MS/MS to be oleamide. Oleamideis a slippery agent but also an endogenous compound and would therefore normallybe discarded as an interesting compound. As the animals has been treated alikeand as all blood taken from the animals it is interesting that there is a difference inoleamide between control and dosed animals.• The un-target analysis shows primarily an effect from the cocktail. The main effectshown is a lowered amount of a given metabolite when the cocktail is givencompared to control, though in some cases an overexpression is also shown.BackgroundEndocrine disrupting chemicals (EDC) are chemicals disturbing the hormones of the body. Many chemical compounds are undersuspicion of being an endocrine disruptor. The effect of a variety of EDC has been tested for changes in male and female hormonecomposition.In order to understand the effect of EDC on the metabolome an analytical platform has been established. The method focusesseparating the compounds from the plasma into three groups: phospholipids, lipids and a fraction containing hormones, organicacids etc. thereby avoiding ion supression.The main goal of the present study is to identify if a human relevant dose of EDC will have an effect on the rat metabolome. Ahuman relevant dose of a possible EDC, perflournonanoic acid, was given to a group of rats. To another group PFNA and 12 otherEDCs were given. These two groups were compared to a group given only the 12 EDC’s and a control group.By separating the metabolites into three fractions and using high resolution mass spectrometry it is possible to achieve high levelof information of how EDC affects the rat metabolome.Name Ratio mg/L mg/400 mLBisphenol A 0,005 5 2Butyl paraben 0,257 257 103DBP d=1.05 0,030 30 12DDE 0,003 3 1DEHP d=0.98 0,043 43 17Epoxiconazole 0,025 25 10Linuron 0,002 2 1MBC 0,194 194 78OMC d=1.01 0,340 340 136Prochloraz 0,031 31 12Procymidone 0,044 44 18Vinclozolin 0,026 26 11Changes in phospholipid concentrationChanges in bucket intensity based on a anova test. Asshown on the figure are the a lower level of phospholipidsin the rats dose with both PFNA and cocktail than theother groups. This support the general idea than moreEDC will effect the metabolome more than a single EDC.OleamideOleamide contance from the animal study. Eventough,oleamide is a slippery agent it is interesting that there is alower level of this compound in the control compared tothe other 3 groupsHuman relevant dose of endocrine disrupting chemicals effect on the rat plasma metabolomeAnalytical setupDionex 3000 series UHPLC system combined with a Bruker Daltonics maxis qTOF instrument.A: Water with 5 mM NH4OH and 0.1 % formic acidB: Acetonitrile with 0.1 % formic acidHydrophilic gradient system0 min, 0 % B – 1 min, 0 % B – 3 min, 5 % B – 10 min, 100 % B – 12, 100 % B – 12.1, 0 % B – 14, 0 % BHydrophobic gradient system0 min, 70 % B – 1 min, 70 % B – 3 min, 75 % B – 8 min, 100 % B – 10, 100 % B – 10.1, 70 % B – 12, 70 % BThe column used was a poreshell EC-C8 column from supleco (Agienlent Technologies, MO, USA)Plasma analysis• 100 µl plasma is extracted with 300 µl icecold acetonitrile and left inthe freezer for 20 min• The sample is centrifuged at 10000 g and supernatant removed• A SPE hybrid column (Supleco, Sigma-Aldrich, USA) is activated atthe supernatant added• The throughput (T) is collected and the phospholipid eluate with 300µl 10 % NH4OH in methanol and collected in another fraction• The T is evaporated by a gentle stream of nitrogen and the driedcompound extracted with three different solvents• Firstly, in 200 µl heptane• Secondly, in 200 µl methanol• Lastly, in 200 µl 5 % acetonitrile• The heptane is evaporated using a gentle stream of nitrogen and thedried compound ressolved in 200 µl 50:50 acetonitrile:isopropanol• The method separates the plasma sample into four different fractions,analyzed by two different LC-MS methods - a hydrophilic andhydrophobic LC system.Cocktail of EDCBergamottinGlabridinKasper Skov1, Niels Hadrup2, Anne Marie Vestergaard2, Jørn Smedsgaard1 and Henrik Frandsen11National Food Institute, Department of Food Chemistry,  Technical University of Denmark, contact: Kasko@food.dtu.dk2National Food Institute, Department of Toxicology and Risk Assessment, Technical University of DenmarkConclusion• The phospholipid are significantly down-regulated when the rats are given PFNA and cocktail. Furthermore, is there a trend that the animals given both PFNA and cocktail have a larger down regulation that cocktail alone. • The heptane phase shows a ‘cocktail effect’ – meaning that the animals are effectedby a low dose cocktail. These compounds are believed to be mono- and di-glycerides but this is not yet varified by MS/MS• The methanol also show a cocktail effect , though the metabolites are not yetidentified. Perflourononanoic acid is a compound primarily used in packing materials. The compound is a surfactant used to lower the water tension within packing material. PFNA have been shown to have an endocrine disrupting effect cellularassays and on zebra fish.  Un-targed metabolomics. The top 3 graphs shows unidentified metabolites from themethanol phase. The button three graphs are metabolites from the heptane phase. Themain overall effect is an effect coursed by the cocktail.Human relavant dose of EDC’s effect on the metabolomePossible mechanism of cocktail The analysis of thephospholipids show asignificantly decrease insignal when cocktail ispresent. Furthermore,compounds with a masscorresponding toarachidonic acid and someof the prostagladins whichtoo are down regulated.

Effects of perfluorononanoic acid (PFNA) on the metabolic profiling of rat serum by UHPLC-ESI-Q-TOF MSMS

http://orbit.dtu.dk/ws/files/70645749/Poster%20SoM.pdf

Effects of perfluorononanoic acid (PFNA) on the metabolic profiling of rat serum by UHPLC-ESI-Q-TOF MSMS

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