Standardized application of yeast bioluminescent reporters as endocrine disruptor screen for comparative analysis of wastewater effluents from membrane bioreactor and traditional activated sludge.

A standardized protocol is demonstrated for bioluminescent strains Saccharomyces cerevisiae BLYES, BLYAS and BLYR as high-throughput screening tools to monitor the estrogenic, androgenic and toxic potencies in wastewater. The sensitivity and reproducibility of the assay in wastewater monitoring was evaluated for 7 day semi-continuous batch reactor using activated sludge with hormones spiked raw sewage. Yeast bioluminescent assay successfully captured the rapid removal of estrogenic and androgenic activities in the bioreactors, and demonstrated rapid response (≤4 h) with good reproducibility. This standardized protocol was then applied in a 12 months monitoring of the effluent of a WWTP located at Powell, TN, USA featuring parallel-operated full-scale membrane bioreactor (MBR) and traditional activated sludge (TAS) treatment. Monitoring results showed that estrogenic activity was persistent in all TAS and most MBR effluent samples, while residual androgenic activity was non-detectable throughout the monitored period. The estrogenic equivalents (EEQ) in TAS effluent ranged from 21.61 ng/L to 0.04 pg/L and averaged 3.25 ng/L. The EEQ in MBR effluent ranged from 2.88 ng/L to 0.0134 pg/L and averaged ~10 fold less (0.32 ng/L) than TAS. Despite the large temporal variation, MBR effluent EEQ was consistently lower than TAS on any given sampling date. Most MBR effluent samples also exhibited less cytotoxicity than TAS. Further analysis did not demonstrate significant correlation between effluent EEQ level and WWTP operational parameters including MLSS, SRT, HRT and BOD.

Integrated metagenomics and metatranscriptomics analyses of root-associated soil from transgenic switchgrass.

The benefits of using transgenic switchgrass with decreased levels of caffeic acid 3-O-methyltransferase (COMT) as biomass feedstock have been clearly demonstrated. However, its effect on the soil microbial community has not been assessed. Here we report metagenomic and metatranscriptomic analyses of root-associated soil from COMT switchgrass compared with nontransgenic counterparts.

A computational approach predicting CYP450 metabolism and estrogenic activity of an endocrine disrupting compound (PCB-30).

Endocrine disrupting chemicals influence growth and development through interactions with the hormone system, often through binding to hormone receptors such as the estrogen receptor. Computational methods can predict endocrine disrupting chemical activity of unmodified compounds, but approaches predicting activity following metabolism are lacking. The present study uses a well-known environmental contaminant, PCB-30 (2,4,6-trichlorobiphenyl), as a prototype endocrine disrupting chemical and integrates predictive (computational) and experimental methods to determine its metabolic transformation by cytochrome P450 3A4 (CYP3A4) and cytochrome P450 2D6 (CYP2D6) into estrogenic byproducts. Computational predictions suggest that hydroxylation of PCB-30 occurs at the 3- or 4-phenol positions and leads to metabolites that bind more strongly than the parent molecule to the human estrogen receptor alpha (hER-α). Gas chromatography-mass spectrometry experiments confirmed that the primary metabolite for CYP3A4 and CYP2D6 is 4-hydroxy-PCB-30, and the secondary metabolite is 3-hydroxy-PCB-30. Cell-based bioassays (bioluminescent yeast expressing hER-α) confirmed that hydroxylated metabolites are more estrogenic than PCB-30. These experimental results support the applied model's ability to predict the metabolic and estrogenic fate of PCB-30, which could be used to identify other endocrine disrupting chemicals involved in similar pathways.

Application of a yeast estrogen reporter system for screening zearalenone degrading microbes.

The aim of this study was to screen microbes for their zearalenone degrading potential and to select microbes whose activities do not create toxic or endocrine disrupting metabolites. Bioluminescent bioreporters (Saccharomyces cerevisiae BLYES and BLYR) were successfully used to monitor toxin degradation; the results of zearalenone biodegradation experiments were confirmed by parallel chemical analysis (HPLC-FLD) and immunoanalytical (ELISA) tests. Using the BLYES/BLYR bioreporters, the most appropriate microbes (ones that produced minimal toxic products and products with lower estrogenic potential) could be selected. The most promising strains belong to Streptomyces and Rhodococcus genera. Our findings demonstrate the benefit of using biological tests beside the analytical method, since bioreporters were able to monitor the samples for toxicity and estrogenic potential even after substantial degradation. We conclude that the BLYES/BLYR bioreporter system is a cost effective, fast and reliable tool for screening zearalenone-degrading microbes.

Bats track and exploit changes in insect pest populations.

The role of bats or any generalist predator in suppressing prey populations depends on the predator's ability to track and exploit available prey. Using a qPCR fecal DNA assay, we document significant association between numbers of Brazilian free-tailed bats (Tadarida brasiliensis) consuming corn earworm (CEW) moths (Helicoverpa zea) and seasonal fluctuations in CEW populations. This result is consistent with earlier research linking the bats' diet to patterns of migration, abundance, and crop infestation by important insect pests. Here we confirm opportunistic feeding on one of the world's most destructive insects and support model estimates of the bats' ecosystem services. Regression analysis of CEW consumption versus the moth's abundance at four insect trapping sites further indicates that bats track local abundance of CEW within the regional landscape. Estimates of CEW gene copies in the feces of bats are not associated with seasonal or local patterns of CEW abundance, and results of captive feeding experiments indicate that our qPCR assay does not provide a direct measure of numbers or biomass of prey consumed. Our results support growing evidence for the role of generalist predators, and bats specifically, as agents for biological control and speak to the value of conserving indigenous generalist predators.

Lack of androgenicity and estrogenicity of the three monomers used in Eastman's Tritan™ copolyesters.

Eastman Tritan™ copolyester, a novel plastic from Eastman is manufactured utilizing three monomers, di-methylterephthalate (DMT), 1,4-cyclohexanedimethanol (CHDM), and 2,2,4,4-tetramethyl-1,3-cyclobutanediol (TMCD) in various ratios. As with most any polymer, the monomers along with the high molecular weight oligomers, whose toxicity is most commonly represented by the monomers, make up the predominate amount of free chemicals available for leaching into the environment and/or foods. In light of the high level of public concern about the presence of endocrine (primarily estrogenic) activity ascribed to certain plastics and chemicals in the environment, Tritan's™ monomers were evaluated using QSAR for binding to the androgen receptor and estrogen receptors (alpha and beta) as well as a battery of in vitro and in vivo techniques to determine their potential androgenicity or estrogenicity. The findings were universally negative. When these data are coupled with other in vivo data developed to assess systemic toxicity and developmental and reproductive toxicity, the data clearly indicate that these monomers do not pose an androgenic or estrogenic risk to humans. Additional data presented also support such a conclusion for terephthalic acid (TPA). TPA is also a common polyester monomer and is the main mammalian metabolite formed from DMT.

Bioluminescent yeast estrogen assay (BLYES) as a sensitive tool to monitor surface and drinking water for estrogenicity.

Estrogenic Endocrine Disrupting Chemicals (EDCs) are a concern due to their ubiquity and recognized adverse effects to humans and wildlife. Methods to assess exposure to and associated risks of their presence in aquatic environment are still under development. The aim of this work is to assess estrogenicity of raw and treated waters with different degrees of pollution. Chemical analyses of selected EDCs were performed by liquid chromatography-tandem mass spectrometry, and estrogenic activity was evaluated using in vitro bioluminescent yeast estrogen assay (BLYES). Most raw water samples (18/20) presented at least one EDC and 16 rendered positive in BLYES. When EDCs were detected, the bioassay usually provided a positive response, except when only bisphenol A was detected at low concentrations. The highest values of estrogenic activity were detected in the most polluted sites. The maximum estrogenic activity observed was 8.7 ng equiv. of E2 L(-1). We compared potencies observed in the bioassay to the relative potency of target compounds and their concentrations failed to fully explain the biological response. This indicates that bioassay is more sensitive than the chemical approach either detecting estrogenic target compounds at lower concentrations, other non-target compounds or even synergistic effects, which should be considered on further investigations. We have not detected either estrogenic activity or estrogenic compounds in drinking water. BLYES showed good sensitivity with a detection limit of 0.1 ng equiv. E2 L(-1) and it seems to be a suitable tool for water monitoring.

Screening of potentially hormonally active chemicals using bioluminescent yeast bioreporters.

Saccharomyces cerevisiae bioluminescent bioreporter assays were developed previously to assess a chemical's estrogenic or androgenic disrupting potential. S. cerevisiae BLYES, S. cerevisiae BLYAS, S. cerevisiae BLYR, were used to assess their reproducibility and utility in screening 68, 69, and 71 chemicals for estrogenic, androgenic, and toxic effects, respectively. EC(50) values were 6.3 +/- 2.4 x 10(-10)M (n = 18) and 1.1 +/- 0.5 x 10(-8)M (n = 13) for BLYES and BLYAS, using 17beta-estradiol and 5alpha-dihydrotestosterone over concentration ranges of 2.5 x 10(-12) through 1.0 x 10(-6)M, respectively. Based on analysis of replicate standard curves and comparison to background controls, a set of quantitative rules have been formulated to interpret data and determine if a chemical is potentially hormonally active, toxic, both, or neither. The results demonstrated that these assays are applicable for Tier I chemical screening in Environmental Protection Agency's Endocrine Disruptor Screening and Testing Program as well as for monitoring endocrine-disrupting activity of unknown chemicals in water.

Morphological and chemical optimization of microcantilever surfaces for thyroid system biosensing and beyond.

The development of biosensors is vital in many areas of biotechnology and biomedical research. A prominent new class of label-free biosensors are those based on ligand-induced nanomechanical responses of microcantilevers (MCs). The interaction between biologically significant ligands with bioreceptors (e.g., antibodies or nuclear receptor proteins) immobilized on one side of the MC surface causes an apparent surface stress, resulting in static bending of the MC, which can be detected by an optical beam bending technique. The three key performance metrics of sensitivity, selectivity, and reversibility are foci of the work reported herein. The nature of the MC surface and the method by which the bioreceptor is immobilized influence these performance metrics and, hence, optimization studies involving these were conducted. In our work, the gold surface on one side of the MC is first activated via self-assembled monolayer formation with amino ethane thiol (AET) then reacted with glutaraldehyde (GA) as a crosslinker before finally functionalizing with the protein receptor. We report the effect of concentration, reaction time, and pH for these reagents on the magnitude of the nanomechanical responses using an anti-immunoglobulin G (anti-IgG) receptor: IgG ligand test system. By vapor depositing an alloy of silver and gold and then etching away the former, a nanostructured "dealloyed" MC surface is created that outperforms a smooth gold MC in terms of nanomechanical responses. Optimization of the dealloying parameters (thickness, metal ratio) is also reported herein using the aforementioned anti-IgG-IgG system. Maximum response was obtained with these conditions: 150 nm dealloyed surface, 1 mM aqueous solution of AET-incubation time 1h, 1% GA solution in 10mM pH 8 phosphate buffered saline (PBS)-incubation time 3h, and 0.5 mg mL(-1) of receptor protein solution in 10mM pH 7 PBS-incubation time 1h. Additionally, surprising results are reported when Protein A is immobilized first to properly orient the bioreceptor IgG molecules. We also report the application of optimum and non-optimum conditions to detect thyroid disrupting chemicals (TDCs) using MCs functionalized with the transport protein thyroxine-binding globulin. Selectivity patterns are reported for several TDCs and sensitive detection of thyroxin at sub-nM levels is demonstrated.

Saccharomyces cerevisiae BLYAS, a new bioluminescent bioreporter for detection of androgenic compounds.

A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 +/- 4.6) x 10(-9) M, respectively. The lower limit of detection in response to DHT was 2.5 x 10(-9) M, and in response to testosterone it was 2.5 x 10(-10) M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.

Marine and freshwater cyanophages in a Laurentian Great Lake: evidence from infectivity assays and molecular analyses of g20 genes.

While it is well established that viruses play an important role in the structure of marine microbial food webs, few studies have directly addressed their role in large lake systems. As part of an ongoing study of the microbial ecology of Lake Erie, we have examined the distribution and diversity of viruses in this system. One surprising result has been the pervasive distribution of cyanophages that infect the marine cyanobacterial isolate Synechococcus sp. strain WH7803. Viruses that lytically infect this cyanobacterium were identified throughout the western basin of Lake Erie, as well as in locations within the central and eastern basins. Analyses of the gene encoding the g20 viral capsid assembly protein (a conservative phylogenetic marker for the cyanophage) indicate that these viruses, as well as amplicons from natural populations and the ballast of commercial ships, are related to marine cyanophages but in some cases form a unique clade, leaving questions concerning the native hosts of these viruses. The results suggest that cyanophages may be as important in freshwater systems as they are known to be in marine systems.