An international cross-laboratory survey on fish vitellogenin analysis: Methodological challenges and opportunities for best practice.

Vitellogenin (VTG) is a biomarker for possible endocrine activity of chemicals acting via the estrogen, androgen, or steroidogenesis pathways. VTG is assessed in standardised fish guideline studies conducted for regulatory safety assessment of chemicals. VTG data can be highly variable leading to concerns for potential equivocal, false positive and/or negative outcomes. Consequently, additional fish testing may be required to address uncertainties in the VTG response, and possibly erroneous/missed identification of endocrine activity. To better understand the technical challenges of VTG assessment and reporting for regulatory purposes, a survey was sent to 27 testing laboratories performing these analyses. The survey results from 16 respondents (6 from the UK, 3 from the USA, and 7 from the EU) were analysed and discussed in a follow-up webinar. High variability in background VTG concentrations was widely acknowledged and thought to be associated with fish batch, husbandry, laboratory practices, and several methodological aspects. These include sample collection and storage, VTG quantification, data handling, and the benchmarks used for data acceptability. Information gathered in the survey provides a basis for improving and harmonizing the measurement of VTG in fish, and an opportunity to reassess the suitability of current acceptability criteria in test guidelines.

Disinfection Byproducts Bind Human Estrogen Receptor-α.

Disinfection byproducts are formed during most drinking water treatment and presently number >800, some of which are implicated in human health outcomes including bladder cancer and infertility, with unknown mechanisms of action. In particular, it is not yet understood whether these compounds can disrupt the estrogen-signaling pathway through binding to the human estrogen receptor (ER). In the present study, 21 disinfection byproducts, selected for their predicted involvement in endocrine-related diseases and their structural diversity, were individually evaluated for their binding affinity to the human ER and in silico, and then a subset of these chemicals was studied in binary mixtures with the known weak estrogen, 4-n-nonylphenol. Individually, 9 of the 21 disinfection byproducts were able to weakly bind to the ER, with affinities ranging from log median inhibitory concentration values of -3.83 to -2.19 M. In binary mixtures, the chemicals followed concentration addition, with their weak binding affinities having little contribution to the overall mixture affinity. These results demonstrate the variety of small-molecule disinfection byproduct structures that are capable of binding to the ER, and that their weak binding can still be of importance when overall human exposure to mixtures of disinfection byproducts in disinfected drinking water is considered. Environ Toxicol Chem 2019;9999:1-9. © 2019 SETAC.

Identification of endocrine active disinfection by-products (DBPs) that bind to the androgen receptor.

The formation of disinfection by-products (DBPs) in drinking water occurs when chemical disinfectants such as chlorine and chloramine react with natural organic matter and anthropogenic pollutants. Some DBPs have been linked to bladder cancer and infertility; however, the underlying mechanism of action is unknown. One possibility is disruption of the endocrine system, with DBPs binding to the androgen receptor and subsequently altering gene expression. Using the androgen receptor-binding assay and in silico molecular docking, the binding affinity of 21 suspected and known DBPs were tested individually at concentrations over the range 0.1 nM-2 mM. 14 DBPs were found to bind at IC50 values ranging from 1.86 mM for 2,3-dichloropropionamide to 13.5 µM for 3,4,5,6-tetrachloro-benzoquinone as compared to the positive control, 4-n-nonylphenol which bound at 31.6 µM. Since DBPs are present in drinking waters as mixtures, the question of how IC50 values for individual DBPs might be affected by the presence of other chemicals is addressed. Seven of the chemicals with the strongest binding affinities and one chemical with no binding affinity were tested in binary mixtures with 4-n-nonylphenol, a known androgenic chemical found in some surface waters. In these binary mixtures, concentration additive binding was observed. While typical levels of individual androgenic DBPs in drinking water are below their measured IC50 values, their combined binding abilities in mixtures could be a source of androgen disruption.

Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH).

Two primary O(2)-sensors for humans are the HIF-hydroxylases, enzymes that hydroxylate specific residues of the hypoxia inducible factor-α (HIF). These enzymes are factor inhibiting HIF (FIH) and prolyl hydroxylase-2 (PHD2), each an α-ketoglutarate (αKG) dependent, non-heme Fe(II) dioxygenase. Although the two enzymes have similar active sites, FIH hydroxylates Asn(803) of HIF-1α while PHD2 hydroxylates Pro(402) and/or Pro(564) of HIF-1α. The similar structures but unique functions of FIH and PHD2 make them prime targets for selective inhibition leading to regulatory control of diseases such as cancer and stroke. Three classes of iron chelators were tested as inhibitors for FIH and PHD2: pyridines, hydroxypyrones/hydroxypyridinones and catechols. An initial screen of the ten small molecule inhibitors at varied [αKG] revealed a non-overlapping set of inhibitors for PHD2 and FIH. Dose response curves at moderate [αKG] ([αKG]~K(M)) showed that the hydroxypyrones/hydroxypyridinones were selective inhibitors, with IC(50) in the µM range, and that the catechols were generally strong inhibitors of both FIH and PHD2, with IC(50) in the low µM range. As support for binding at the active site of each enzyme as the mode of inhibition, electron paramagnetic resonance (EPR) spectroscopy were used to demonstrate inhibitor binding to the metal center of each enzyme. This work shows some selective inhibition between FIH and PHD2, primarily through the use of simple aromatic or pseudo-aromatic chelators, and suggests that hydroxypyrones and hydroxypyridones may be promising chelates for FIH or PHD2 inhibition.

The second coordination sphere of FIH controls hydroxylation.

The factor inhibiting HIF (FIH) is a proximate oxygen sensor for human cells, hydroxylating Asn(803) within the α-subunit of the hypoxia inducible factor (HIF). FIH is an α-ketoglutatrate (αKG)-dependent, non-heme Fe(II) dioxygenase, in which Fe(II) is coordinated by a (His(2)Asp) facial triad, αKG, and H(2)O. Hydrogen bonding among the facial triad, the HIF-Asn(803) side chain, and various second-sphere residues suggests a functional role for the second coordination sphere in tuning the chemistry of the Fe(II) center. Point mutants of FIH were prepared to test the functional role of the αKG-centered (Asn(205) and Asn(294)) or HIF-Asn(803)-centered (Arg(238) and Gln(239)) second-sphere residues. The second sphere was tested for local effects on priming Fe(II) to react with O(2), oxidative decarboxylation, and substrate positioning. Steady-sate kinetics were used to test for overall catalytic effects; autohydroxylation rates were used to test for priming and positioning, and electronic spectroscopy was used to assess the primary coordination sphere and the electrophilicity of αKG. Asn(205) → Ala and Asn(294) → Ala mutants exhibited diminished rates of steady-state turnover, while minimally affecting autohydroxylation, consistent with impaired oxidative decarboxylation. Blue-shifted metal to ligand charge transfer transitions for (Fe+αKG)FIH indicated that these point mutations destabilized the π* orbitals of αKG, further supporting a slowed rate of oxidative decarboxylation. The Arg(238) → Met mutant exhibited steady-state rates too low to measure and diminished product yields, suggesting impaired substrate positioning or priming; the Arg(238) → Met mutant was capable of O(2) activation for the autohydroxylation reaction. The Gln(239) → Asn mutant exhibited significantly slowed steady-state kinetics and diminished product yields, suggesting impaired substrate positioning or priming. As HIF binding to the Gln(239) → Asn mutant stimulated autohydroxylation, it is more likely that this point mutant simply mispositions the HIF-Asn(803) side chain. This work combines kinetics and spectroscopy to show that these second-sphere hydrogen bonds play roles in promoting oxidative decarboxylation, priming Fe(II) to bind O(2), and positioning HIF-Asn(803).