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.

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.

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.