Michal Bittner

Yeast Biosensors for Detection of Environmental Pollutants: Current State and Limitations

Yeast biosensors have become suitable tools for the screening and detection of environmental pollutants because of their various advantages compared to other sensing technologies. On the other hand, many limitations remain with regard to their optimal performance and applicability in several contexts, such as low-concentration samples and on-site testing. This review summarizes the current state of yeast biosensors, with special focus on screening and assessment of environmental contaminants, discusses both pros and cons, and suggests steps towards their further development and effective use in the environmental assessment.

Enhancement of AhR-mediated activity of selected pollutants and their mixtures after interaction with dissolved organic matter.

Dissolved organic matter (DOM) in freshwaters is present at concentrations ranging from 0.5 to 50 mg L⁻¹, and consists of various organic compounds, including humic substances (HS). HS exert a variety of direct and indirect biological effects, including interaction with the aryl hydrocarbon receptor (AhR). AhR is a cytosolic receptor that binds various hydrophobic organic compounds (HOCs) and mediates some of their toxic effects. In vitro effects of binary mixtures of various DOM (mainly HS) with various HOCs on AhR-mediated responses were studied by use of H4IIE-luc cells. Six out of 12 DOM activated the AhR even at environmentally relevant concentrations (17 mg L⁻¹). In simultaneous exposures of H4IIE-luc cells to DOM (17 mg L⁻¹) and each of the model compounds, 2,3,7,8-TCDD, PCB126, PCB169, benzo[a]pyrene, benzo[a]anthracene, dibenz[a,h]anthracene, fluoranthene, a mixture of persistent organic pollutants (POPs), a mixture of polycyclic aromatic hydrocarbons (PAHs), and a mixture of all HOCs, either significant additive or facilitative effects were observed when compared to activities of single HOCs. No significant decrease of effects due to possible sorption of HOCs to DOM was observed, even in subsequent experiments when HOCs+DOM mixtures were preincubated for six days before exposure to H4IIE-luc. Thus, DOM does not seem to protect organisms against AhR-mediated toxic effects of HOCs (as usually predicted due to sorption of HOCs on DOM), but it can actually enhance their potency for AhR-mediated effects in some situations.

In vitro assessment of AhR-mediated activities of TCDD in mixture with humic substances.

Humic substances (HS) are ubiquitous natural products of decomposition of dead organic matter. HS is present in most freshwaters at concentrations ranging from 0.5 to 50mg L(-1). Organic carbon can represent 20% dry weight of sediments. Recently, the interaction of dissolved HS with the aryl hydrocarbon receptor (AhR) has been demonstrated. The AhR is a cytosolic receptor to which persistent organic pollutants (POPs) can bind and many of their toxic effects are mediated through interactions with this receptor. We describe in vitro effects (using H4IIE-luc cells) of binary mixtures of various HS with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), since in contaminated environments these compounds occur simultaneously. Six out of 12 HS samples activated AhR even at environmentally relevant concentrations (17 mg L(-1)), but did not reach the full AhR-activation even at excessive concentration. In simultaneous exposure of H4IIE-luc to HS (17 mg L(-1)) and TCDD (1.2 pM) without any preincubation prior to exposure, either significant additive or facilitative effects were observed. No negative interactions, due to possible sorption of TCDD to HS was observed. Nevertheless, if the HS-TCDD binary mixture was preincubated for 6 days prior to the exposure on H4IIE-luc cells, the additive and facilitative effects were less due to possible sorption of TCDD onto HS. Similar results were obtained from analogous experiments with greater concentrations of both TCDD and HS.

Interaction of temperature and an environmental stressor: Moina macrocopa responds with increased body size, increased lifespan, and increased offspring numbers slightly above its temperature optimum

For organisms, temperature is one of the most important environmental factors and gains increasing importance due to global warming, since increasing temperatures may pose organisms close to their environmental tolerance limits and, thus, they may become more vulnerable to environmental stressors. We analyzed the temperature-dependence of the water-soluble antioxidant capacity of the cladoceran Moina macrocopa and evaluated its life trait variables with temperature (15, 20, 25, 30°C) and humic substance (HS) concentrations (0, 0.18, 0.36, 0.90, 1.79 mM DOC) as stressors. Temperatures below and above the apparent optimum (20°C) reduced the antioxidative capacity. Additions of HSs increased body length, but decreased mean lifespan at 15 and 20°C. There was no clear HS-effect on offspring numbers at 15, 20, and 30°C. At 25°C with increasing HS-concentration, lifespan was extended and offspring numbers increased tremendously, reaching 250% of the control. Although the applied HS preparation possesses estrogenic and antiandrogenic activities, a xenohormone mechanism does not seem plausible for the reproductive increase, because comparable effects did not occur at other temperatures. A more convincing explanation appears to be the mitohormesis hypothesis which states that a certain increase of reactive oxygen production leads to improved health and longevity and, with Moina, also to increased offspring numbers. Our results suggest that at least with the eurythermic M. macrocopa, a temperature above the optimum can be beneficial for several life trait variables, even when combined with a chemical stressor. Temperatures approximately 10°C above its optimum appear to adversely affect the lifespan and reproduction of M. macrocopa. This indicates that this cladoceran species seems to be able to utilize temperature as an ecological resource in a range slightly above its thermal optimum.

Antiandrogenic activity of humic substances.

For long, natural organic matter (NOM) composed mainly of humic substances (HS) were regarded as inert in the ecosystems with respect to their possible chemical interaction with exposed organisms. However recently, NOM have been shown to elicit various adverse effects generally attributed to synthetic xenobiotics, including estrogenic effects translating into shifts of the gender ratios in populations. However, the anti/androgenic pathway was not yet evaluated. Here, we applied an anti/androgenic sensitive cell line MDA stably transfected with the firefly luciferase gene under transcriptional control of the androgen responsive element. With five out of twelve tested NOM preparations, particularly with soil and coal isolates, we identified a relatively high, concentration-dependent antiandrogenic effect. This appears to be the first study to show this endocrine disrupting pathway for a ubiquitous biogeochemical matrix, a potential activity which should not be neglected in forthcoming studies on synthetic endocrine disruptors in the environment.

Freeze-drying as suitable method to achieve ready-to-use yeast biosensors for androgenic and estrogenic compounds

Recombinant yeast assays (RYAs) have been proved to be a suitable tool for the fast screening of compounds with endocrine disrupting activities. However, ready-to-use versions more accessible to less equipped laboratories and field studies are scarce and far from optimal throughputs. Here, we have applied freeze-drying technology to optimize RYA for the fast assessment of environmental compounds with estrogenic and androgenic potencies. The effects of different cryoprotectants, initial optical density and long-term storage were evaluated. The study included detailed characterization of sensitivity, robustness and reproducibility of the new ready-to-use versions, as well as comparison with the standard assays. Freeze-dried RYAs showed similar dose-responses curves to their homolog standard assays, with Lowest Observed Effect Concentration (LOEC) and Median effective Concentration (EC50) of 1 nM and 7.5 nM for testosterone, and 0.05 nM and 0.5 nM for 17β-estradiol, respectively. Freeze-dried cells stored at 4 °C retained maximum sensitivity up to 2 months, while cells stored at -18 °C showed no decrease in sensitivity throughout the study (10 months). This ready-to-use RYA is easily accessible and may be potentially used for on-site applications.

Polymer-immobilized ready-to-use recombinant yeast assays for the detection of endocrine disruptive compounds

Recombinant yeast assays (RYAs) constitute a suitable tool for the environmental monitoring of compounds with endocrine disrupting activities, notably estrogenicity and androgenicity. Conventional procedures require yeast reconstitution from frozen stock, which usually takes several days and demands additional equipment. With the aim of applying such assays to field studies and making them more accessible to less well-equipped laboratories, we have optimized RYA by the immobilization of Saccharomyces cerevisiae cells in three different polymer matrices - gelatin, Bacto agar, and Yeast Extract Peptone Dextrose agar - to obtain a ready-to-use version for the fast assessment of estrogenic and androgenic potencies of compounds and environmental samples. Among the three matrices, gelatin showed the best results for both testosterone (androgen receptor yeast strain; AR-RYA) and 17β-estradiol (estrogen receptor yeast strain; ER-RYA). AR-RYA was characterized by a lowest observed effect concentration (LOEC), EC50 and induction factor (IF) of 1nM, 2.2nM and 51, respectively. The values characterizing ER-RYA were 0.4nM, 1.8nM, and 63, respectively. Gelatin immobilization retained yeast viability and sensitivity for more than 90d of storage at 4°C. The use of the immobilized yeast reduced the assay duration to only 3h without necessity of sterile conditions. Because immobilized RYA can be performed either in multiwell microplates or glass tubes, it allows multiple samples to be tested at once, and easy adaptation to existing portable devices for direct in-field applications.