Endocrine Disrupters in Human Blood and Breast Milk: Extraction Methodologies, Cellular Uptake and Effect on Key Nuclear Receptor Functions

• Main Author: Hjelmborg, Philip Sebastian
• Format: Book
• Language: English
• Published: 2010
• Subjects: Rå; inuit; Prøven
• Online Access: https://pure.au.dk/portal/da/publications/endocrine-disrupters-in-human-blood-and-breast-milk-extraction-methodologies-cellular-uptake-and-effect-on-key-nuclear-receptor-functions(b8274450-d6b5-11df-a891-000ea68e967b).html; https://pure.au.dk/ws/files/22176400/PhD_Dissertation__Philip_Hjelmborg__without_Papers_attached_

Indledning: Miljøet er under et konstant pres fra en lang række menneskeskabte fedtopløselige stoffer, hvoraf mange nedbrydes meget langsomt og som følge heraf kan de ophobes i organismer. Disse stoffer stammer fra mange forskellige kilder, hvoraf de største bidragydere er forbrændingsprodukter fra forbrændingsanlæg, tilsætningsstoffer fra plastik, tekniske industriprodukter, pesticider fra landbruget og nedbrydningsprodukter fra vaskemidler og detergenter. Den fælles betegnelse for disse stoffer er hormonforstyrrende stoffer (forkortet på engelsk til EDCs). Nogle EDCs er svært nedbrydelige i miljøet, og kaldes også for persistente organiske miljøgifte (forkortet på engelsk til POPs). Hormonforstyrrende stoffer er forbindelser der kan forstyrre en organismes hormonsystem ved at interagere med organismens hormonreceptorer. Mange af en organismes kropsfunktioner styres af samspillet mellem hormoner og hormonreceptorer, og forstyrrelser af dette samspil kan resultere i sygdomme og fejlfunktioner. Stoffer, som udviser hormonforstyrrende egenskaber er blevet forbundet med mange sygdomme, herunder misdannelser af kønsorganer, neurologiske lidelser, reproduktive problemer, insulinresistens og kræft. Alle levende organismer er udsat for hormonforstyrrende stoffer i komplekse blandinger, og der er derfor et behov for metoder der kan vurdere den kombinerede effekt af disse akkumulerede hormonforstyrrende stoffer på organismens kerne-hormonreceptorer såsom på østrogenreceptoren (ER), androgenreceptoren (AR) og thyroidhormonreceptoren (TR). Formål: Formålet med forskningen var 1) at isolere lav-densitet-lipoprotein (LDL) fra human serum og 2) at undersøge LDL-faciliteret optag af pesticidet p,p’-dichlorodiphenyl trichloroethane (DDT) i celler med og uden LDL-receptorer tilstede i cellemembranen; 3) at ekstrahere hormonforstyrrende stoffer fra humant serum og rense disse ekstrakter for østrogener og androgener; 4) at måle, ex vivo den kombinerede aktivitet af hormonforstyrrende stoffer ekstraheret fra serumprøver indsamlet i tre internationale studiegrupper, og endelig 5) at ekstrahere EDCs og østrogener fra brystmælk og at reducere indholdet af lipider i ekstraktet forud for analyser i et ER-reportergen assay. Metoder: Artikel I. Lipoprotein blev isoleret fra humant serum ved hjælp af ultracentrifugering. Massefylden blev justeret ved at tilsætte fast kaliumklorid til serumprøven før centrifugering, således at LDL kunne isoleres. Undersøgelser af DDT-optag blev udført ved hjælp af muse-embryonale fibroblastceller (MEF), der var genetisk modificeret således, at den ene cellelinie havde begge sine lipoproteinreceptorer i cellemembranen aktive, vild-typen (MEF-1 celler), mens den anden cellelinje havde begge sine lipoproteinreceptorer inaktiveret (MEF-4 celler). MEF-4 celler mangler derfor lav-densitet-lipoprotein receptor-relateret protein (LRP) og lav-densitet-lipoprotein receptor (LDLR), henholdsvis. Optaget blev undersøgt ved hjælp af [C-14]-DDT, der forud for eksponeringen var blevet inkuberet med forskellige koncentrationer af LDL. Til at studere receptorfunktion blev det receptor-associerede protein (RAP) inkuberet med DDT-LDL opløsningen sideløbende. RAP er et 40 kDa protein, der blokerer lipoproteinreceptorer. Artikel II. Ekstraktion af hormonforstyrrende stoffer fra serum blev gennemført med fastfase ekstraktion (SPE) sammen med væskekromatografi (HPLC). Det udnyttes, at retentionstiden for EDCs er mindre end retentionstiden for hormoner, og det muliggør fremstilling af serumekstrakter, der indeholder EDCs uden spor af endogene hormoner. Artikel III. Disse hormonfri ekstrakter blev efterfølgende analyseret i et ER transactivation assay, der er et reportergen assay, der er baseret på en stabil-transfekteret MVLN cellelinje. MVLN celler reagerer på eksponering af østrogen eller østrogenlignende kemikalier ved at udskille enzymet luciferase proportionalt med graden af receptoraktivering. Ved at måle koncentrationen af luciferase, er det derefter muligt at kvantificere graden af receptoraktivering og dermed bestemme omfanget af den hormonforstyrrende aktivitet i prøven. Artikel IV. Modermælk blev ekstraheret ved hjælp af SPE og disse ekstrakter blev yderligere behandlet ved hjælp af polyethylen membraner i et dialytisk setup (MDE ekstraktion). Formålet med dialyse var at reducere indholdet af lipider i det rå SPE ekstrakt fordi MVLN celler er sårbare over for prøver med højt lipidindhold. Østrogener nedbrydes ved ekstraktionsmetoder der inkluderer brugen af koncentreret svovlsyre og lipidreduktionen skulle derfor være skånsom. Resultater: Artikel I: Optaget af DDT i MEF celler blev kun delvist medieret af lipoproteinreceptorer. En forøgelse af LDL koncentrationen i vækstmediet resulterede i faldende optag af DDT i cellerne. Artikel II: Serum ekstrakter indeholdende EDCs og renset for endogene hormoner kunne opnås ved at anvende en kombineret SPE og HPLC procedure. Ekstrakter fra Inuit serum og PCB-spiket kontrolserum viste anti-østrogen aktivitet i de hormonfrie fraktioner, mens de efterfølgende fraktioner der indeholder endogene hormoner viste østrogen aktivitet. Artikel III: Agonistisk østrogenaktivitet kunne hovedsageligt måles i de europæiske serumekstrakter. I modsætning til dette var den største del af Inuit prøverne anti-østrogene. For Inuitter sås en invers korrelation mellem serum xenoestrogenicitet og p,p’-DDE og borderline invers til PCB-153 kunne observeres, hvorimod de europæiske studiegrupper fremkaldte en positiv og en negativ korrelation mellem xenoestrogenicitet og p,p’-DDE og PCB-153, henholdsvis. Selvom der ikke var nogen stærk korrelation for de poolede data mellem xenoestrogeniciteten og de to POP-biomarkører PCB-153 og p,p'-DDE. Artikel IV: Der kunne opnås et godt recovery af DDT ved hjælp af MDE ekstraktion, men for østrogener var recovery ikke tilstrækkeligt. Lipider i ekstraktet blev reduceret med ca. 88% efter 24 timers dialyse. Konklusioner: Artikel I: Optagelse af DDT i MEF celler foregår ved både passiv diffusion og i mindre grad via lipoprotein receptorer. I vækstmediet har DDT en præference for LDL partikler over celler, og ved eksperimentelt cellearbejde skal LDL koncentrationen være sammenlignelig for at man kan være i stand til at sammenligne forskellige undersøgelser med hensyn til optag af lipofile forbindelser i celler. Artikel II: Inuit EDC-fraktionerne indeholder høje niveauer af PCB, som er anti-østrogent. Disse resultater er sammenlignelige med resultater fra PCB spikede kontrolprøver, der understøtter dette resultat. Ved analyse af hormon-fraktionerne sås en østrogenaktivitet der viste store forskelle mellem prøver fra mænd og kvinder, hvilket antyder at østradiol var fjernet i EDC fraktion. Artikel III: De valgte POP-biomarkører for eksponerings- og receptoreffekter, isoleret set, kan ikke bruges til at beskrive en organismes EDC-byrde, fordi serum blandingsprofilen varierer geografisk. Artikel IV: Modermælksekstraktionen er egnet for EDCs, hvorimod østrogener ikke kunne ekstraheres i acceptable mængder og metoden er derfor ikke egnet til analyser, hvor østrogener skal være til stede. Lipid reduktionen var ikke tilfredsstillende for undersøgelser hvor MVLN celler skal benyttes, fordi det tilbageværende fedt i ekstrakterne dræber cellerne. Metoden er derfor ikke egnet til yderligere analyser baseret på MVLN celler. Introduction: The environment is under a constant pressure from a multitude of manmade lipophilic chemicals, many of which degrade very slowly and because of that they can accumulate in organisms. These chemicals originate from many sources, but the major contributors are combustion by-products from incineration plants, plastic additives, technical industry products, pesticides from the farming industry and detergent degradation products. Many of these substances can interfere with the hormonal system in organisms. The common name for these compounds is endocrine disrupters (EDCs). Some EDCs are persistent to degradation and are also called persistent organic pollutants (POPs). Endocrine disrupters are compounds that can interfere with an organism’s hormone system by interacting with the hormone receptors. Many of an organism’s body functions are controlled by interactions between hormones and hormone receptors and disturbance of these interactions can result in diseases and malfunctions. Compounds that exhibit endocrine disrupting properties have been linked to many diseases including genital malformations, neurological disorders, reproductive problems, insulin resistance and cancers. All living organisms are exposed to EDCs in complex mixtures and there is a need for methods with which to estimate the combined action of accumulated EDCs on nuclear hormone receptors such as the estrogen receptor (ER), androgen receptor (AR) and the thyroid hormone receptor (TR). Objectives: The objectives of the research were to 1) isolate low density lipoproteins (LDL) from human serum and 2) to study the uptake of the pesticide p,p’-dichlorodiphenyl trichloroethane (DDT) mediated by lipoproteins into cells with and without the LDL receptors present; 3) to extract EDCs from human serum and to clear these extracts from endogenous estrogens and androgens; 4) to determine ex vivo the combined activity of EDCs extracted from serum samples collected in three international study groups; and finally 5) to extract EDCs and estrogens from human breast milk and to reduce the lipid content in the extracts before analysis in an ER reporter gene assay. Methods: Paper 1. Lipoproteins were isolated from human serum using ultracentrifugation. The density was adjusted by adding solid potassium chloride to the serum prior to centrifugation enabling the isolation of low density lipoproteins (LDL). The uptake studies were carried out using mouse embryonic fibroblast (MEF) cells that had been genetically modified so that one cell line had both its lipoprotein receptors present in the cell membrane, the wild-type (MEF-1 cells) while the other cell line had both lipoprotein receptors inactivated (MEF-4 cells). The MEF-4 cells lacked the low density lipoprotein receptor-related protein (LRP) and the low density lipoprotein receptor (LDLR), respectively. The uptake was studied using [C-14]-DDT that was previously incubated with different concentrations of LDL. To study the receptor function, the receptor-associated protein (RAP) was incubated in parallel with the DDT-LDL mixture. RAP is a 40-kDa protein that blocks the lipoprotein receptors. Paper II. Extraction of EDCs from serum was achieved using solid phase extraction (SPE) together with high performance liquid chromatography (HPLC) that utilises that the retention of EDCs are less than the retention for hormones, and it is therefore possible to produce serum extracts that contains EDCs with no traces of endogenous hormones. Paper III. These hormone-free extracts were subsequently analysed in an ER transactivation assay that is a reporter gene assay based on the stable transfected MVLN cell line. MVLN cells respond to exposure by estrogens or estrogen-like chemicals by excreting the enzyme luciferase proportionally to the degree of receptor activation. By measuring the concentration of luciferase, it is then possible to quantify the degree of receptor activation and thus the level of endocrine disrupting activity in the sample. Paper IV. Breast milk was extracted using SPE and these extracts were further processed using polyethylene membranes in a dialytic setup (MDE extraction). The purpose of the dialysis was to reduce the content of lipids in the raw SPE extracts because MVLN cells in the reporter gene assay is vulnerable to samples with high lipid content. Estrogens are inactivated by extraction methods employing concentrated sulphuric acid and the lipid removal therefore had to be gentle. Results: Paper I: The uptake of DDT into MEF cells was only partially mediated by lipoprotein receptors and if the concentration of LDL in the media was increased, the uptake decreased. Paper II: Serum extracts rich in EDCs were obtained free of endogenous hormones by employing a combined SPE and HPLC procedure. Extracts from Inuit serum and PCB-spiked control serum both showed antiestrogenic activity in the hormone free fractions, while the subsequent fractions containing endogenous hormones showed estrogenic activity. Paper III: Agonistic estrogenic activity was predominantly seen in European serum extracts. Contrary to this elicited the major part of Inuit samples antiestrogenic activity. For the Inuit an inverse correlation between serum xenoestrogenicity and p,p’-DDE and borderline inverse to PCB-153 was observed, whereas the European study groups elicited a positive and negative correlation between xenoestrogenicity and p,p’-DDE and PCB-153, respectively. Although no strong correlations were found for the pooled data between xenoestrogenicity and the two POP-biomarkers PCB-153 and p,p’-DDE. Paper IV: Good recoveries of DDT could be obtained using MDE extraction, but recoveries of estrogens were not adequate. Lipids in the extracts were reduced by about 88% after 24 hours of dialysis. Conclusions: Paper I: Uptake of DDT in MEF cells is mediated both by passive diffusion and to a lesser extent by lipoprotein receptors. DDT prefers LDL particles to cells and for experimental work LDL concentration has to be controlled to be able to compare different studies with respect to uptake of lipophilic compounds. Paper II: Inuit EDC fractions contain high levels of PCBs that are antiestrogenic. These results were comparable with PCB spiked control samples supporting those results. Upon analysis of the hormone fractions, estrogenic activity was seen with large differences between men and women indicating that estradiol was not present in the EDC fraction. Paper III: The chosen POP-biomarkers of exposure and receptor effects alone cannot be used to describe body burden of EDCs because the serum mixture profile differs geographically. Paper IV: The breast milk extraction method is suitable for EDCs, whereas estrogens were not extracted in acceptable quantities and the method is therefore not suitable for analyses where estrogens must be present. Lipid reduction was not satisfactory for studies where MVLN cells are employed because the remaining content of lipids in the extracts kills the cells. The method is not suitable for further analysis using MVLN cells.