Do cyanobacteria produce estrogenic compounds?

Transcription

1 Do cyanobacteria produce estrogenic compounds? Klára Hilscherová K. Nováková, E. Sychrová, T. Štěpánková, J.P.Giesy, L. Bláha Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Brno, Czech Republic Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

2 Bioactive compounds in cyanobacteria Extracts from aquatic water blooms greater effects than according to the cyanotoxin content (fish, amphibians, invertebrates, mammals) - cyanotoxins are produced in mixture with other cyanobacterial compounds - cyanobacterial blooms can accumulate various anthropogenic chemicals Effects of mixtures from cyanobacterial water bloom = result of interactions of many endogenous and accumulated compounds COOH CH 3 O NH N NH OCH 3 H 3 C O H O 2 C H 3 C O NH CH 3 NH CH 3 CH 3 NH NH CH 3 O O COOH O CH 3 NH HN NH 2 Known cyanotoxins (MCs, CYN, ATX, SXT,..) OH H H O 3 SO O??? Additivity Antagonism Synergism Other bioactive compounds produced by cyanobacteria N NH NH NH H 3 C H NH O Anthropogenic contaminants

3 In vivo studies: effects on fecundity, reproduction, development, sperm quality in mammals, male reproductive system in mammals and birds, survival, reproduction in invertebrates (Ding et al., 2006; Li et al., 2008, Kozlowsky-Suzuki et al., 2009; Trubetskova and Haney, 2006)? Specific mechanisms of effects? Endocrine disruption Possible consequences: disruption of homeostasis, reproduction, development, behavior, defective sexual development, low fecundity/fertility/immunity, malformations The aim of study: Find out if cyanobacteria can produce compounds that could act through important specific mechanisms and thus disrupt functioning of endocrine system

4 Disruption of hormonal regulation Synthesis Transport Inhibition Interaction with receptors Metabolization Stimulation

5 NUCLEAR RECEPTOR MEDIATED EFFECTS important mechanisms involved in chronic toxicity Dioxin-like activity Aryl hydrocarbon receptor (AhR) induction of detoxification systems hepatotoxicity, immunotoxicity affects reproduction, endocrine and nervous system, carcinogenesis, embryotoxicity cross-talk with signaling pathways of hormones Anti/estrogenicity Estrogen receptor (ER) sexual development, control of reproduction, carcinogenesis, affects cellular proliferation and differentiation, development and homeostasis Anti/androgenicity Androgen receptor (AR) sexual development, namely male sexual characteristics, control of reproduction, carcinogenesis, affects growth, spermatogenesis Glucocorticoid activity Glucocorticoid receptor (GR) affects growth, metabolisms, immune response, reactions to stress Anti/retinoid activity Retinoid acid receptor (RAR) regulates growth, morphogenesis, apoptosis and differentiation, affects nervous and immune system, vision and embryonal development

6 In vitro reporter gene trans-activation assays = cell lines with reporter gene luciferase under control: AhR dioxin-like toxicity ER anti/estrogenicity AR anti/androgenicity GR glucocorticoid activity RAR anti/retinoid activity

7 Complex cyanobacterial blooms from the environment Sample Species composition Date of sampling Locality Total microcystin content (µg/g dw) A Microcystis aeruginosa 8 October 2004 Brno - reservoir n.d. (90%), Microcystis wesenbergii (10%) B Microcystis aeruginosa 1 October 2007 Brno - reservoir 410 (30%), Microcystis wesenbergii (20%), Microcystis ichthyoblabe (25%), Aphanizomenon sp. (25%) C Anabaena spiroides 19 August 1998 Jedovnice - pond n.d. D Aphanizomenon flosaquae (70%), Microcystis viridis (30%) Olšovec 14 August 1997 Skalka - dam reservoir E Planktothrix agardhii 8 September 2004 Dubice - flooded pit

8 Complex cyanobacterial blooms from the environment Sample Species composition Samples Date of sampling Locality Total microcystin content (µg/g dw) A Microcystis aeruginosa 8 October 2004 Brno - reservoir n.d. Aqueous (90%), Microcystis extract from the biomass B Fraction wesenbergii 1 (10%) (SPE C18 permeate) polar Microcystis aeruginosa 1 October 2007 Brno - reservoir 410 Fraction (30%), Microcystis 2 (SPE eluate) less polar containing microcystins wesenbergii (20%), Microcystis Results ichthyoblabe (25%), Aphanizomenon sp. (25%) C No Anabaena significant spiroides effects at 19 noncytotoxic August 1998 concentrations: Jedovnice - pond n.d. AhR (dioxin-like toxicity) Olšovec D Aphanizomenon flosaquae 14 August 1997 Skalka - dam 715 GR (70%), (glucocorticoid Microcystis activity) reservoir viridis RAR (30%) (anti/retinoid activity) E Planktothrix agardhii 8 September 2004 Dubice - flooded pit 2290

9 ESTROGENIC ACTIVITY PRO-ANDROGENIC ACTIVITY % of E 2 max induction A A 1 A 2 B B 1 B % of E 2 max induction C C 1 C 2 D D 1 D 2 In both polar (without MC) and less 200 polar fraction (with MC) % of E 2 max induction E E 1 E 2 equivalent biomass concentration (g dw/l)

10 Model species cultured in laboratory Sample Species Collection Place of origin Country Water body CYANOBACTERIA 1 Anabaena flos-aquae UTEX USA Mississippi 2 Aphanizomenon flos-aquae PCC Netherland lake Brielse Meer 3 Aphanizomenon flos-aquae SAG Canada unspecified 4 Aphanizomenon gracile RCX Ireland lake LoughNeagh 5 Aphanizomenon gracile SAG Germany lake Plussee 6 Aphanizomenon klebahnii CCALA Great Britain reservoir Queen Elizabeth 7 Cylindrospermopsis raciborskii SAG Hungary lake Balaton 8 Microcystis aeruginosa PCC Netherland reservoir Braakman 9 Planktothrix agardhii CCALA Germany lake Plussee 10 Planktothrix agardhii SAG France Rendeau ALGAE 11 Chlorella kessleri CCALA USA 12 Scenedesmus quadricauda CCALA Germany Greifswald

11 Model species cultured in laboratory Sample Species Collection Place of origin Country Water body CYANOBACTERIA 1 Anabaena flos-aquae UTEX USA Mississippi 2 Aphanizomenon flos-aquae PCC Netherland lake Brielse Meer 3 Aphanizomenon flos-aquae SAG Canada unspecified 4 Aphanizomenon gracile RCX Ireland lake LoughNeagh 5 Aphanizomenon gracile SAG Germany lake Plussee Samples 6 Aphanizomenon klebahnii CCALA Great Britain reservoir Queen Elizabeth 7 Cylindrospermopsis raciborskii SAG Hungary lake Balaton Aqueous extracts from biomass 8 Microcystis aeruginosa PCC Netherland reservoir Braakman Extracellular spent media (Exudates) 9 Planktothrix agardhii CCALA Germany lake Plussee Preconcentration of organic compounds on tandem SPE (C18 OASIS HLB + ALLTECH CARBOGRAFF) 10 Planktothrix agardhii SAG France Rendeau ALGAE 11 Chlorella kessleri CCALA USA 12 Scenedesmus quadricauda CCALA Germany Greifswald

12 %E 2 max induction Estrogenity in model species cultured in laboratory Significant EEQs - in exudates of most species - in extracts of both algal species and in cyanobacteria Aphanizomenon gracile from two different collections % E 2 max induction E 2 calibration E 2 concentration (pm) %E 2 max induction concentration of exudate % E2max induction Aphanizomenon gracile CCALA Aphanizomenon gracile SAG Chlorella k essleri Scenedesmus quadricauda E2 calibration (pm) Anabaena flosaquae Microcystis aeruginosa Cylindrospermopsis raciborskii Chlorella kessleri Effects not related to the concentration of studied cyanotoxins (MC, CYN) concentration of exudate Aphanizomenon gracile CCALA Aphanizomenon gracile SAG Planktothrix agardhii UTEX Planktothrix agardhii SAG concentration (g/l)

13 Estrogen equivalents in biomass Environmental samples EEQ 20 (ng/g dw) Sample crude extract permeate eluate A B C n.s. n.s. n.s. D 760 * E n.c n.c. Laboratory cultures model species Sample Species Collection Maximal induction (% E 2 max) EEQ 50 (ng E 2 /g dw) EEQ 20 EEQ 80 (ng E 2 /g dw) 4 Aphanizomenon gracile RCX Aphanizomenon gracile SAG Chlorella kessleri CCALA Scenedesmus quadricauda CCALA l.i.

14 Estrogen equivalents in biomass Environmental samples EEQ 20 (ng/g dw) Sample crude extract permeate eluate A B C n.s. n.s. n.s. D 760 * E n.c n.c. Estimate for Brno lake At the density of cells/ml EEQ in biomass about 0.8 ng/l Laboratory cultures model species At the density of cels/ml EEQ 4.3 ng/l Sample Species Collection Maximal induction (% E 2 max) EEQ 50 (ng E 2 /g dw) EEQ 20 EEQ 80 (ng E 2 /g dw) 4 Aphanizomenon gracile RCX Aphanizomenon gracile SAG Chlorella kessleri CCALA Scenedesmus quadricauda CCALA l.i.

15 Exudates of model species cultured in laboratory Species CYANOBACTERIA Collection Toxin production (µg/g dw) EEQ (ng E 2 /L ) Anabaena flos-aquae UTEX n.d Aphanizomenon flos-aquae PCC CYN 3100 n.s. Aphanizomenon flos-aquae SAG n.d. n.s. Aphanizomenon gracile RCX n.d Aphanizomenon gracile SAG n.d Aphanizomenon klebahnii CCALA n.d Cylindrospermopsis raciborskii SAG n.d. 1.7 Microcystis aeruginosa PCC MC Planktothrix agardhii CCALA MC Planktothrix agardhii SAG MC ALGAE Chlorella kessleri CCALA 0.69 Scenedesmus quadricauda CCALA 2.7

16 Environmental relevance EEQ in ng/l levels can cause reproductive toxicity in aquatic animals Zha et al Complete inhibition of F-1 generation in multigeneration study with minnows (0.2 ng/l ethinylestradiol EE2, Gobiocypris rarus). Toxicol. Appl. Pharmacol. 226: Zha et al Adverse effects on fecundity and fertility, occurrence of ova-testes, increased plasma VTG (4 ng/l EE2, Gobiocypris rarus, 21 days). Ecotoxicol. Environ. Safety 71: Kidd et al Collapse of fish populations after exposure to estrogen (5 ng/l EE2, 7 years). Proceedings of the National Academy of Sciences 104(21): Controls +Ethinylestradiol

17 In search for cyano -estrogens C. raciborskii lab culture spent media - concentrated (C18/carbon SPE) - fractionated on HPLC / C18, slow methanol gradient - chromatogram nm 2 major estrogenic fractions Sum of fractions > whole sample (masking effects in the mixture)

18 Summary Compounds from cyanobacteria and algae can affect signaling of important nuclear receptors No significant dioxin-like, glucocorticoid or anti/retinoid activity Interference with signaling of androgen receptor Most important effect: ESTROGENICITY Both environmental and laboratory biomasses Both cyanobacteria and algae Both in extracts and exudates (! Effects for more species!) Both in polar and less polar fraction Unrelated to known cyanotoxins? cyano-phyto estrogens?

19 Acknowledgements..coworkers and students. Czech Science Foundation grant No. GACR P503/12/0553 project CETOCOEN (no. CZ.1.05/2.1.00/ ) from the European Regional Development Fund Thank you for your attention! contact: