Fungal tools for the degradation of endocrine disrupting compounds

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http://urn.fi/URN:ISBN:978-952-10-9025-7
Title: Fungal tools for the degradation of endocrine disrupting compounds
Author: Kabiersch, Grit
Contributor: University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2013-08-23
Language: en
URI: http://urn.fi/URN:ISBN:978-952-10-9025-7
http://hdl.handle.net/10138/40137
Thesis level: Doctoral dissertation (article-based)
Abstract: Endocrine disrupting compounds are synthetic or natural compounds that mimic the action of hormones and thus disrupt or alter functions of the endocrine system usually through direct interactions with nuclear receptors. The main objective of this work was to develop strategies of how to degrade endocrine disrupting compounds and how to monitor the removal of the endocrine disrupting effect with focus on the estrogenic compound bisphenol A and on the virilizing compound tributyltin. Bisphenol A is used as plasticizer of polycarbonate plastics, which are used for food packaging, coatings of metal cans and baby bottles. It can leach out from these materials during washing and sterilization processes or after landfilling. The endocrine disrupting effects of bisphenol A are exerted through binding and activating the estrogen receptor, a nuclear receptor. As a consequence, bisphenol A acts as a potential risk factor for the development of prostate and breast cancer and is suspected to reduce the number of sperm cells in men. Tributyltin has been widely used in antifouling paints and is found in high concentrations in the vicinity of shipping routes and in harbor sediments. Through binding and activation of the retinoic X receptor, also a nuclear receptor, tributyltin induces a phenomenon called imposex in female gastropods, which means that in addition to female sex organs a penis and vas deferens are formed. The occurrence of both compounds, bisphenol A and tributyltin, can be monitored and quantified by the use of analytical methods such as HPLC, LC-MS and GC-MS. However, they are time-consuming, require high amounts of environmentally hazardous solvents and eluents as well as specific technical equipment and competence. In contrast, microbial bioassays offer inexpensive, easy-to-use and small-scale measurements. Furthermore, they can be employed to assess bioavailability. In this work, a receptor-based bioluminescent yeast assay was developed that has the advantage of qualitatively monitoring the endocrine disrupting effect of the compound as well as its metabolites. Furthermore, the lignin-decomposing abilities of litter-degrading fungi were exploited. These fungi produce highly active extracellular oxidative enzymes such as laccase and manganese peroxidase. These enzymes degrade recalcitrant substances such as plant lignin but also persistent environmental chemicals. A novel bioluminescent yeast assay was constructed and characterized to detect organotin compounds such as tributyltin, triphenyltin and their metabolites. A chimeric human retinoic X receptor alpha is expressed from a plasmid. Upon binding of tributyltin or another ligand, expression of a luciferase reporter gene is triggered. After addition of D-luciferin, light is emitted and detected luminometrically. The assay has proven to be highly specific towards organotin compounds and natural ligands of the retinoic X receptor. Tributyltin was detected in nanomolar concentrations. At these low concentrations, tributyltin already exerts an endocrine disrupting effect in nature. Experiments were performed in small-scale and high-throughput manner and results of one analysis were obtained within one working day. Next, a novel sampling method to determine extracellular fungal enzymes in agar was developed. Small pieces of growing solid-state fungal cultures were placed in a centrifugation tube filter, containing a cellulose acetate membrane. Centrifugation recovered water-soluble material including many enzymes. The recovery of two added model enzymes, laccase and manganese peroxidase, was in the range of 50 to 75 %. This method allowed the collection of spatial data from very small and defined areas of solid fungal cultures. It is also very well suitable for screening approaches, i.e. it can combine toxicity tests and the investigation of the influence of toxic/endocrine disrupting compounds on enzyme production of the fungus under study. Finally, the degradation of the endocrine disrupting compound bisphenol A was studied in detail. Cultures of the litter-degrading fungi Stropharia rugosoannulata and Stropharia coronilla as well as a partially purified neutral manganese peroxidase from the latter were used to successfully degrade the estrogenic compound bisphenol A in culture medium or cell-free reaction solution, respectively. A bioluminescent yeast assay, expressing the estrogen receptor alpha, was employed to follow the removal of estrogenic activity. S. coronilla was shown to be the most efficient fungus; the estrogenic activity was reduced by 100 % in liquid cultures as well as during treatment with manganese peroxidase. In cultures of S. rugosoannulata, the estrogenic activity declined as well but temporarily re-appeared. Selected samples from this cultivation were additionally investigated for potential metabolites using LC-MS analysis and a pathway of bisphenol A conversion was hypothesized.Hormonihäiritsijöiksi kutsutaan yhdisteitä, jotka ovat hormonien kaltaisia, ja jotka voivat häiritä elimistön normaalia toimintaa. Hormonit toimivat elimistössä avain lukko-periaatteella: hormoni on avain , joka sitoutuu vastaanottavaan molekyyliin, reseptoriin eli lukkoon . Hormonihäiritsijä bisfenoli A:ta käytetään polykarbonaattimuovien pehmittimenä, joita taas käytetään elintarvike-pakkauksissa, säilykepurkkien pinnoitteissa ja tuttipulloissa. Muoveja pestessä, steriloidessa tai kaatopaikkasijoituksessa bisfenoli A:ta voi huuhtoutua pesuveteen. Altistuminen bisfenoli A:lle voi aiheuttaa eturauhas- tai rintasyöpää ja sen on myös epäilty vähentävän siittiöiden määrää. Hormonihäiritsijä tributyylitina on ollut yleisesti käytössä veneenpohjamaalien tehoaineena, ja sitä on päätynyt suuria määriä laivaväylien läheisyyteen ja satamien pohjasedimentteihin. Maaleilla on pyritty estämään kotiloiden kiinnittyminen laivojen runkoon, sillä kotiloiden massa lisää laivan polttoaineenkulutusta. Tributyylitina aiheuttaa kotiloille, kuten merirokolle ja etanalle maskulinisaatiota , ts. tributyylitinalle altistuneelle naaraskotilolle saattaa kehittyä penis tai muita uroskotiloiden elimiä. Hormonihäiritsijöihin voidaan vaikututtaa kahdella tavalla: ongelmaa valvomalla, jolloin tilanteesta saadaan kokonaiskuva, ja poistamalla ongelma. Tässä työssä kehitettiin bioluminesenssiin perustuva hiivatesti, jossa geneettisesti muunneltu leivinhiiva tuottaa valosignaalin tributyylitinan läsnä ollessa. Testin avulla tributyylitina ja sen hormonaalisesti aktiiviset hajoamistuotteet voidaan havaita näytteestä nopeasti ja tarkasti. Lisäksi näytettä tarvitaan vain pieni määrä, ja tulokset saadaan yhden työpäivän aikana. Karikkeenlahottajasienten orgaanisten aineiden hajotuskykyä voidaan hyödyntää hormonihäiritsijöiden hajottamiseen eli ongelman poistamiseen. Luonnossa nämä sienet hajottavat kasvimateriaalia, kuten lehtiä ja oksia tuottamillaan solunulkoisilla, voimakkaasti hapettavilla entsyymeillä. Tässä työssä kehitetyn toisen menetelmän avulla voidaan helposti löytää sienilajeja, joilla on tehokas entsyymintuotto. Agarmaljalla kasvatetusta sienirihmasta erotetaan entsyymit yhdistetyn suodatuksen ja sentrifugoinnin avulla, ja entsyymiaktiivisuus määritetään suodoksesta. Valittuja sieniä voidaan hyödyntää prosessissa, jossa sieni tuottaa hapettavia entsyymeitä ja hajottaa hormonihäiritsijät. Karikkeenlahottajasienet Stropharia rugosoannulata (viljelykaulussieni) ja Stropharia coronilla (nurmikaulussieni) valittiin bisfenoli A:n hajotuskokeeseen. Seurattaessa yhdisteen hajoamista hiivatestillä, havaittiin, että sekä S. coronilla sieni että sienen tuottama mangaaniperoksidaasi-entsyymi yksin pystyivät hajottamaan bisfenoli A:n täydellisesti. S. rugosoannulata sienen kasvatusliemessä bisfenoli A:n hormonaalinen aktiivisuus myös hävisi, mutta myöhemmin aktiivisuus palautui osittain. Kasvatusliemen analyysi paljasti, että vaikka suurin osa bisfenoli A:sta hajosi harmittomiksi yhdisteiksi, pieni osa muuntui bisfenoli A:n kaltaiseksi hormonihäiritsijäksi. Työssä kehitettyjen menetelmien avulla hormonihäiritsijöiden hajotus on mahdollista ja hajoamisen seuraaminen on helppoa. Seurantatestin avulla selviää myös milloin tarkentavat kemialliset analyysit ovat tarpeen.
Subject: biotechnology
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