Advertisement

Analytical and Bioanalytical Chemistry

, Volume 409, Issue 27, pp 6485–6494 | Cite as

A fluorescent, supramolecular chemosensor to follow steroid depletion in bacterial cultures

  • Antje Stahl
  • Alexandra I. Lazar
  • Veronica N. Muchemu
  • Werner M. Nau
  • Matthias S. Ullrich
  • Andreas Hennig
Research Paper

Abstract

Steroids have been identified as endocrine-disrupting agents, which are thought to impact the fertility of aquatic organisms and may even have direct effects on humans. The removal of steroids from wastewater is therefore essential, and this is most efficiently achieved by microbial treatment. We report herein a simple fluorescent method to identify microorganisms that are capable of steroid degradation and to optimize the conditions for steroid removal. The method is based on the supramolecular macrocycle cucurbit[8]uril (CB8), which can bind either the fluorescent dye berberine or a steroid in their inner cavity. In absence of steroid, the cavity is free to bind the dye, leading to a strong increase in fluorescence. In contrast, in the presence of steroid, the dye is displaced into the bulk solution. This principle affords a stable (no thermal or photodegradation was noted), fluorescent chemosensor (excitation ca. 450 nm, maximum emission at 525 nm), which can detect testosterone at concentrations > 0.7 μM. We show that this displacement principle can be applied to follow the removal of micromolar concentrations of the steroid testosterone from a bacterial culture of Buttiauxella sp. S19-1. The reliability of the chemosensor in screening applications is demonstrated by an excellent Z-factor, which was in the range of 0.52 to 0.74 for all experiments carried out with this assay.

Graphical abstract

Steroid depletion by bacterial cultures can be followed by fluorescence spectroscopy using a supramolecular chemosensor based on berberine and cucurbit[8]uril

Keywords

Fluorescence Supramolecular recognition Cucurbiturils Assay method Marine bacteria Steroids 

Notes

Acknowledgements

The authors would like to thank Prof. Dr. Edmund Maser and Dr. Guangming Xiong (Institute for Toxicology and Pharmacology for Natural Scientists, Christian-Albrechts-Universität Kiel, Germany) for providing Buttiauxella sp. S19. This project was funded by the Helmholtz Graduate School for Polar and Marine Research (POLMAR) and the Deutsche Forschungsgemeinschaft (grant numbers UL 169/6-1 for M.U., NA 686/11-1 for W.M.N., and HE 5967/4-1 for A.H.).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interests.

Supplementary material

216_2017_593_MOESM1_ESM.pdf (156 kb)
ESM 1 (PDF 156 kb)

References

  1. 1.
    Jobling S, Tyler CR. Endocrine disruption in wild freshwater fish. Pure Appl Chem. 2003;75:2219–34.CrossRefGoogle Scholar
  2. 2.
    Sumpter JP. Endocrine disrupters in the aquatic environment: an overview. Acta Hydrochim Hydrobiol. 2005;33:9–16.CrossRefGoogle Scholar
  3. 3.
    Gross-Sorokin MY, Roast SD, Brighty GC. Assessment of feminization of male fish in english rivers by the environment agency of England and Wales. Environ Health Perspect. 2006;114:147–51.CrossRefGoogle Scholar
  4. 4.
    Kidd KA, Blanchfield PJ, Mills KH, Palace VP, Evans RE, Lazorchak JM, et al. Collapse of a fish population after exposure to a synthetic estrogen. Proc Natl Acad Sci. U.S.A. 2007;104:8897–901.Google Scholar
  5. 5.
    Morthorst JE, Holbech H, Bjerregaard P. Trenbolone causes irreversible masculinization of zebrafish at environmentally relevant concentrations. Aquat Toxicol. 2010;98:336–43.CrossRefGoogle Scholar
  6. 6.
    Bjerregaard LB, Korsgaard B, Bjerregaard P. Intersex in wild roach (Rutilus rutilus) from Danish sewage effluent-receiving streams. Ecotoxicol Environ Saf. 2006;64:321–8.CrossRefGoogle Scholar
  7. 7.
    Viganò L, Arillo A, Bottero S, Massari A, Mandich A. First observation of intersex cyprinids in the Po River (Italy). Sci Total Environ. 2001;269:189–94.CrossRefGoogle Scholar
  8. 8.
    Guillette LJ, Jr, Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ Health Perspect. 1994;102:680–8.Google Scholar
  9. 9.
    Heistermann M, Möhle U, Vervaecke H, van Elsacker L, Hodges JK. Application of urinary and fecal steroid measurements for monitoring ovarian function and pregnancy in the bonobo (Pan paniscus) and evaluation of perineal swelling patterns in relation to endocrine events. Biol Reprod. 1996;55:844–53.CrossRefGoogle Scholar
  10. 10.
    Velle W. Endogenous anabolic agents in farm animals. Environ Qual Saf Suppl. 1976;5:159–70.Google Scholar
  11. 11.
    Zhang FS, Xie YF, Li XW, Wang DY, Yang LS, Nie ZQ. Accumulation of steroid hormones in soil and its adjacent aquatic environment from a typical intensive vegetable cultivation of North China. Sci Total Environ. 2015;538:423–30.CrossRefGoogle Scholar
  12. 12.
    Baynes A, Green C, Nicol E, Beresford N, Kanda R, Henshaw A, et al. Additional treatment of waste water reduces endocrine disruption in wild fish—a comparative study of tertiary and advanced treatments. Environ Sci Technol. 2012;46:5565–73.CrossRefGoogle Scholar
  13. 13.
    Huang B, Wang B, Ren D, Jin W, Liu J, Peng J, et al. Occurrence, removal, and bioaccumulation of steroid estrogens in Dianchi Lake catchment, China. Environ Int. 2013;59:262–73.CrossRefGoogle Scholar
  14. 14.
    Johnson AC, Williams RJ, Simpson P, Kanda R. What difference might sewage treatment performance make to endocrine disruption in rivers? Environ Pollut. 2007;147:194–202.CrossRefGoogle Scholar
  15. 15.
    Kolodziej EP, Gray JL, Sedlak DL. Quantification of steroid hormones with pheromonal properties in municipal wastewater effluent. J Environ Toxicol Chem. 2003;22:2622–9.Google Scholar
  16. 16.
    Jasinska EJ, Goss GG, Gillis PL, Van Der Kraak GJ, Matsumoto J, de Souza Machado AA, et al. Assessment of biomarkers for contaminants of emerging concern on aquatic organisms downstream of a municipal wastewater discharge. Sci Total Environ. 2015;530–531:140–53.CrossRefGoogle Scholar
  17. 17.
    Jobling S, Beresford N, Nolan M, Rodgers-Gray T, Brighty GC, Sumpter JP, et al. Altered sexual maturation and gamete production in wild roach (Rutilus rutilus) living in rivers that receive treated sewage effluents. Biol Reprod. 2002;66:272–81.CrossRefGoogle Scholar
  18. 18.
    Ciparis S, Iwanowicz LR, Voshell JR. Effects of watershed densities of animal feeding operations on nutrient concentrations and estrogenic activity in agricultural streams. Sci Total Environ. 2012;414:268–76.CrossRefGoogle Scholar
  19. 19.
    Lee LS, Carmosini N, Sassman SA, Dion HM, Sepúlveda MS. Agricultural contributions of antimicrobials and hormones on soil and water quality. Adv Agron. 2007;93:1–68.CrossRefGoogle Scholar
  20. 20.
    Durhan EJ, Lambright CS, Makynen EA, Lazorchak J, Hartig PC, Wilson VS, et al. Identification of metabolites of trenbolone acetate in androgenic runoff from a beef feedlot. Environ Health Perspect. 2006;114:65–8.CrossRefGoogle Scholar
  21. 21.
    McAdam EJ, Bagnall JP, Koh YKK, Chiu TY, Pollard S, Scrimshaw MD, et al. Removal of steroid estrogens in carbonaceous and nitrifying activated sludge processes. Chemosphere. 2010;81:1–6.CrossRefGoogle Scholar
  22. 22.
    Qiang Z, Dong H, Zhu B, Qu J, Nie Y. A comparison of various rural wastewater treatment processes for the removal of endocrine-disrupting chemicals (EDCs). Chemosphere. 2013;92:986–92.CrossRefGoogle Scholar
  23. 23.
    Xiong G, Draus E, Luo Y, Maser E. 3-Alpha-hydroxysteroid dehydrogenase/carbonyl reductase as a tool for isolation and characterization of a new marine steroid degrading bacterial strain. Chem Biol Interact. 2009;178:206–10.CrossRefGoogle Scholar
  24. 24.
    Yam KC, Okamoto S, Roberts JN, Eltis LD. Adventures in Rhodococcus—from steroids to explosives. Can J Microbiol. 2011;57:155–68.CrossRefGoogle Scholar
  25. 25.
    Horinouchi M, Hayashi T, Kudo T. Steroid degradation in Comamonas testosteroni. J Steroid Biochem Mol Biol. 2012;129:4–14.CrossRefGoogle Scholar
  26. 26.
    Philip B. Bacterial degradation of bile salts. Appl Microbiol Biotechnol. 2011;89:903–15.CrossRefGoogle Scholar
  27. 27.
    Angus RA, McNatt HB, Howell WM, Peoples SD. Gonopodium development in normal male and 11-ketotestosterone-treated female mosquitofish (Gambusia affinis): a quantitative study using computer image analysis. Gen Comp Endocrinol. 2001;123:222–34.CrossRefGoogle Scholar
  28. 28.
    Leusch FDL, Chapman HF, Kay GW, Gooneratne SR, Tremblay LA. Anal fin morphology and gonadal histopathology in mosquitofish (Gambusia holbrooki) exposed to treated municipal sewage effluent. Arch Environ Contam Toxicol. 2006;50:562–74.CrossRefGoogle Scholar
  29. 29.
    Zhang T, Xiong G, Maser E. Characterization of the steroid degrading bacterium S19-1 from the Baltic Sea at Kiel, Germany. Chem Biol Interact. 2011;191:83–8.CrossRefGoogle Scholar
  30. 30.
    Varriale A, Pennacchio A, Pinto G, Oliviero G, D'Errico SD, Majoli A, et al. A fluorescence polarization assay to detect steroid hormone traces in milk. J Agric Food Chem. 2015;63:9159–64.Google Scholar
  31. 31.
    Xiao L, Zhang Z, Wu C, Han L, Zhang H. Molecularly imprinted polymer grafted paper-based method for the detection of 17 β-estradiol. Food Chem. 2017;221:82–6.CrossRefGoogle Scholar
  32. 32.
    Bailey K, Yazdi T, Masharani U, Tyrrell B, Butch A, Schaufele F. Advantages and limitations of androgen receptor-based methods for detecting anabolic androgenic steroid abuse as performance enhancing drugs. PLoS One. 2016  https;//doi.org/10.1371/journal.pone.0151860
  33. 33.
    Chang C-C, Huang S-D. Determination of the steroid hormone levels in water samples by dispersive liquid-liquid microextraction with solidification of a floating organic drop followed by high-performance liquid chromatography. Anal Chim Acta. 2010;662:39–43.Google Scholar
  34. 34.
    Mascotti ML, Palazzolo MA, Bisogno FR, Kurina-Sanz M. Biotransformation of dehydro-epi-androsterone by Aspergillus parasiticus: metabolic evidences of BVMO activity. Steroids. 2016;109:44–9.CrossRefGoogle Scholar
  35. 35.
    Wang W, Ge F, Ma C, Li J, Ren Y, et al. Heterologous expression and characterization of a 3-ketosteroid-Δ1-dehydrogenase from Gordonia neofelifaecis and its utilization. 3 Biotech. 2017;7:19.Google Scholar
  36. 36.
    Sang Y, Xiong G, Maser E. Steroid degradation and two steroid-inducible enzymes in the marine bacterium H5. Chem Biol Interact. 2011;191:89–94.CrossRefGoogle Scholar
  37. 37.
    Yang Y-Y, Borch T, Young RB, Goodridge LD, Davis JG, Degradation kinetics of testosterone by manure-borne bacteria: influence of temperature, pH, glucose amendments, and dissolved oxygen. J Environ Qual. 2010;39:1153–60.Google Scholar
  38. 38.
    Holert J, Yücel O, Jagmann N, Prestel A, Möller HM, Philipp B (2016) Identification of bypass reactions leading to the formation of one central steroid degradation intermediate in metabolism of different bile salts in Pseudomonas sp. strain Chol1. Environ Microbiol. 2016;18:3373–89.Google Scholar
  39. 39.
    Ruprecht A, Maddox J, Stirling AJ, Visaggio N, Seah SYK. Characterization of novel acyl coenzyme A dehydrogenases involved in bacterial steroid degradation. J Bacteriol. 2015;197:1360–7.CrossRefGoogle Scholar
  40. 40.
    Drzyzga O, Fernández de la Heras L, Morales V, Navarro Llorens JM, Perera J. Cholesterol degradation by Gordonia cholesterolivorans. Appl Environ Microbiol. 2011;77:4802–10.Google Scholar
  41. 41.
    You L, Zha D, Anslyn EV. Recent advances in supramolecular analytical chemistry using optical sensing. Chem Rev. 2015;115:7840–92.CrossRefGoogle Scholar
  42. 42.
    Nilam M, Gribbon P, Reinshagen J, Cordts K, Schwedhelm E, Nau WM, et al. A label-free continuous fluorescence-based assay for monitoring ornithine decarboxylase activity with a synthetic putrescine receptor. SLAS Discov. 2017;22:906–14.Google Scholar
  43. 43.
    Dsouza RN, Hennig A, Nau WM. Supramolecular tandem enzyme assays. Chem – Eur J. 2012;18:3444–59.CrossRefGoogle Scholar
  44. 44.
    Lazar AI, Biedermann F, Mustafina KR, Assaf KI, Hennig A, Nau WM. Nanomolar binding of steroids to cucurbit[n]urils: selectivity and applications. J Am Chem Soc. 2016;138:13022–9.CrossRefGoogle Scholar
  45. 45.
    Schnurr M, Sloniec-Myszk J, Döpfert J, Schröder L, Hennig A. Supramolecular assays for mapping enzyme activity by displacement-triggered change in hyperpolarized 129Xe magnetization transfer NMR spectroscopy. Angew Chemie Int Ed. 2015;54:13444–7.CrossRefGoogle Scholar
  46. 46.
    Hennig A, Bakirci H, Nau WM. Label-free continuous enzyme assays with macrocycle-fluorescent dye complexes. Nat Methods. 2007;4:629–32.CrossRefGoogle Scholar
  47. 47.
    Uzunova VD, Cullinane C, Brix K, Nau WM, Day AI. Toxicity of cucurbit[7]uril and cucurbit[8]uril: an exploratory in vitro and in vivo study. Org Biomol Chem. 2010;8:2037–42.CrossRefGoogle Scholar
  48. 48.
    Miskolczy Z, Biczók L. Sequential inclusion of two berberine cations in cucurbit[8]uril cavity: kinetic and thermodynamic studies. Phys Chem Chem Phys. 2014;16:20147–56.CrossRefGoogle Scholar
  49. 49.
    Kaeppel EC, Gärdes A, Seebah S, Grossart HP, Ullrich MS. Marinobacter adhaerens sp. nov., isolated from marine aggregates formed with the diatom Thalassiosira weissflogii. Int J Syst Evol Microbiol. 2012;62:124–8.CrossRefGoogle Scholar
  50. 50.
    Handley KM, Lloyd JR. Biogeochemical implications of the ubiquitous colonization of marine habitats and redox gradients by Marinobacter species. Front Microbiol. 2013;4:136.CrossRefGoogle Scholar
  51. 51.
    Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30.CrossRefGoogle Scholar
  52. 52.
    Sonnenschein EC, Gärdes A, Seebah S, Torres-Monroy I, Grossart HP, Ullrich MS. Development of a genetic system for Marinobacter adhaerens HP15 involved in marine aggregate formation by interacting with diatom cells. J Microbiol Methods. 2011;87:176–83.CrossRefGoogle Scholar
  53. 53.
    Seymour JR, Ahmed T, Marcos, Stocker R. A microfluidic chemotaxis assay to study microbial behavior in diffusing nutrient patches. Limnol Oceanogr Methods. 2008;6:477–88.CrossRefGoogle Scholar
  54. 54.
    Zhang J-H, Chung TDY, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4(2):67–73.CrossRefGoogle Scholar
  55. 55.
    Atkinson SK, Marlatt VL, Kimpe LE, Lean DRS, Trudeau VL, Blais JM. The occurrence of steroidal estrogens in south-eastern Ontario wastewater treatment plants. Sci Total Environ. 2012;430:119–25.CrossRefGoogle Scholar
  56. 56.
    Liu Z-H, Lu G-N, Yin H, Dang Z, Rittmann B. Removal of natural estrogens and their conjugates in municipal wastewater treatment plants: a critical review. Environ Sci Technol. 2015;49:5288–300.CrossRefGoogle Scholar
  57. 57.
    Martinovic-Weigelt D, Minarik TA, Curran EM, Marchuk JS, Pazderka MJ, Smith EA, et al. Environmental estrogens in an urban aquatic ecosystem: I. Spatial and temporal occurrence of estrogenic activity in effluent-dominated systems. Environ Int. 2013;61:127–37.CrossRefGoogle Scholar
  58. 58.
    Assaf KI, Nau WM. Cucurbiturils: from synthesis to high-affinity binding and catalysis. Chem Soc Rev. 2015;44:394–418.CrossRefGoogle Scholar
  59. 59.
    Barrow SJ, Kasera S, Rowland MJ, del Barrio J, Scherman OA. Cucurbituril-based molecular recognition. Chem Rev. 2015;115:12320–406.CrossRefGoogle Scholar
  60. 60.
    Isaacs L. Stimuli responsive systems constructed using cucurbit[n]uril-type molecular containers. Acc Chem Res. 2014;47:2052–62.CrossRefGoogle Scholar
  61. 61.
    Lee JW, Samal S, Selvapalam N, Kim H, Kim K. Cucurbituril homologues and derivatives: new opportunities in supramolecular chemistry. Acc Chem Res. 2003;36:621–30.CrossRefGoogle Scholar
  62. 62.
    Das G, Matile S. Substrate-independent transduction of chromophore-free organic and biomolecular transformations into color. Chem – Eur J. 2006;12:2936–44.CrossRefGoogle Scholar
  63. 63.
    Bailey DM, Hennig A, Uzunova VD, Nau WM. Supramolecular tandem enzyme assays for multiparameter sensor arrays and enantiomeric excess determination of amino acids. Chem – Eur J. 2008;14:6069–77.CrossRefGoogle Scholar
  64. 64.
    Yoo H, Ahn K-H, Lee H-J, Lee K-H, Kwak Y-J, Song K-G. Nitrogen removal from synthetic wastewater by simultaneous nitrification and denitrification (SND) via nitrite in an intermittently-aerated reactor. Water Res. 1999;33:145–54.CrossRefGoogle Scholar
  65. 65.
    Ismail NS, Müller CE, Morgan RR, Luthy RG. Uptake of contaminants of emerging concern by the bivalves Anodonta californiensis and Corbicula fluminea. Environ Sci Technol. 2014;48:9211–9.CrossRefGoogle Scholar
  66. 66.
    McEneff G, Barron L, Kelleher B, Paull B, Quinn B. A year-long study of the spatial occurrence and relative distribution of pharmaceutical residues in sewage effluent, receiving marine waters and marine bivalves. Sci Total Environ. 2014;476–477:317–26.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Department of Life Sciences and ChemistryJacobs University BremenBremenGermany

Personalised recommendations