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Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian species differ in sensitivity to the toxic effects of dioxin-like compounds (DLCs) and recent reports have provided insight into the molecular mechanisms underlying this variability. The sensitivity of avian species to DLCs is associated with the identity of amino acids at positions 324 and 380 within the ligand-binding domain (LBD) of the aryl hydrocarbon receptor 1 (AHR1). 6-formylindolo [3,2-b] carbazole (FICZ), a naturally produced photo-oxidation product of tryptophan, is a highly potent AHR ligand. Few studies have attempted to determine if there are species differences in AHR activation by FICZ in a systematic manner. Here we describe results from an in vitro assay that measures AHR1-mediated luciferase reporter gene activity to determine concentration-dependent effects of FICZ and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in COS-7 cells transfected with AHR1 constructs from chicken (Gallus gallus domesticus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica) and common tern (Sterna hirundo), and three mutant AHR1 constructs. Data were used to (a) compare the potency of FICZ and TCDD for each AHR1 construct (relative potency; ReP) and (b) the sensitivity of each construct to AHR1 activation by FICZ and TCDD (relative sensitivity; ReS). The results show that (1) FICZ was considerably more potent than TCDD in cells transfected with chicken AHR1 (RePavg=41), ring-necked pheasant AHR1 (RePavg=93), Japanese quail AHR1 (RePavg=1392) and common tern AHR1 (RePavg=1534), (2) there were no significant differences in sensitivity to FICZ in cells expressing chicken, pheasant, quail and tern AHR1, but there were significant differences in sensitivity to TCDD, (3) alteration of amino acids at positions 324 and 380 had no effect on avian AHR1 activity in response to FICZ, (4) there was no time-dependent change in the relative potency of FICZ in COS-7 cells, and (5) neither FICZ nor TCDD induced ethoxyresorufin O-deethylase (EROD activity) in COS-7 cells. Our results suggest that FICZ and TCDD activate avian AHR1 by different modes of interaction with AHR1., (Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.)

Some currently used brominated flame retardants (BFRs), such as hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO), bis(2-ethylhexyl)tetrabromophthalate (BEHTBP), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE), are persistent organic contaminants detected in various environmental matrices, including wild birds. Data on potential toxicological and molecular responses to exposure of these BFRs are lacking for avian species. A combined in vitro/in ovo approach was used to determine the concentration-dependent effects of these BFRs on overt toxicity and hepatic messenger RNA (mRNA) expression levels of 11 transcripts in (1) primary cultures of chicken embryonic hepatocytes (CEH; all four BFRs) and (2) chicken embryos (HCDBCO and BTBPE only). Neither hepatocyte viability nor embryonic pipping success were affected by the BFRs at any of the administered concentrations (CEH: 0.001-30 μM, egg injection: 0.1-10 μg/g nominal dose). In CEH, 10 μM HCDBCO induced cytochrome P450 2H1 (CYP2H1) and CYP3A37, while CYP1A4/5 were down-regulated at all tested concentrations. In contrast, only transthyretin was down-regulated by HCDBCO in embryonic liver. There was concordance between the BTBPE-induced transcriptional responses in vitro and in ovo for CYP1A4/5 (up-regulated) and type III iodothyronine 5'-deiodinase (DIO3; down-regulated). DBDPE induced CYP1A4/5 29- and 59-fold at 0.2 μM in CEH and increased DIO1. None of the gene targets were responsive to BEHTBP exposure in CEH. The multi-tiered in vitro/in ovo screening approach was effective for assessing toxicological and molecular biological effects of these BFRs in an avian species., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Several perfluoroalkyl compounds (PFCs) are ubiquitous environmental contaminants that can biomagnify in species at high trophic levels including wild birds. Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) have been detected in wild birds and are known to reduce hatching success of laboratory-exposed chicken embryos at environmentally relevant concentrations. Limited toxicity data are available regarding avian exposure to PFCs of chain lengths greater than C(8), which are of increasing environmental relevance following the recent phase-out of PFOS and PFOA. In this study, linear PFOA, perfluoroundecanoic acid (PFUdA) and perfluorodecane sulfonate (PFDS) were injected into the air cell of white leghorn chicken eggs (Gallus gallus domesticus) prior to incubation to determine effects on embryo pipping success. Furthermore, mRNA expression of key genes involved in pathways implicated in PFC toxicity was monitored in liver tissue. PFOA, PFUdA or PFDS had no effect on embryonic pipping success at concentrations up to 10 microg/g. All PFCs accumulated in the liver to concentrations greater than the initial whole-egg concentration as determined by HPLC/MS/MS. Hepatic accumulation was highest for PFOA (4.5 times) compared to PFUdA and PFDS. Cytochrome P450 1A4 and liver fatty acid binding protein mRNA expression increased after exposure to PFUdA but was only statistically significant at 10 microg/g; several orders of magnitude higher than levels found in wild bird eggs. Based on the present results for white leghorn chickens, current environmental concentrations of PFOA, PFUdA and PFDS are unlikely to affect the hatching success of wild birds.

Inducibility of the cytochrome P4501A4 (CYP1A4) enzyme, measured as ethoxyresorufin-O-deethylase (EROD) activity, has been used as a biomarker for sensitivity to the effects of dioxin-like compounds in avian species. Here, we present a quantitative reverse transcriptase-PCR (Q-PCR) method for assessing this biomarker response at the level of messenger RNA (mRNA) expression. The method was validated for use in fresh samples as well as samples that have been analyzed for EROD activity previously. Concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on CYP1A4 and CYP1A5 mRNA abundance were detected in fresh and post-EROD hepatocyte cultures. Although the quality of the RNA obtained from post-EROD samples was low, quantification of the CYP1A mRNA response to TCDD was not compromised. Several benefits of evaluating CYP1A mRNA expression in addition to EROD activity were noted. The CYP1A mRNA bioassay may provide more accurate estimates for the potency of environmental mixtures of contaminants and has a very low detection limit. When working with hepatocytes cultured from wild or endangered species, our approach can help to circumvent the problem of small sample size by maximizing the amount of data obtained from each sample.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.

An efficient method for measuring polychlorinated biphenyl-mediated induction of cytochrome P4501A, using the ethoxyresorufin-O-deethylase (EROD) assay, and total porphyrins in chicken embryo hepatocyte cultures is described. Hepatocytes were cultured in 48-well plates, assays were carried out within the wells, and concentrations of the product of the EROD reaction (resorufin), porphyrins, and total proteins were measured in the same wells with a fluorescence plate reader. The method differs from previous methods developed in this laboratory in that three fluorophors were measured in the same well rather than two.

A method for simultaneous measurement of the cytochrome P4501A-associated enzyme, ethoxyresorufin-O-deethylase (EROD), and total protein concentration in liver microsomes is described. EROD assays were carried out in multiwell plates, and the fluorescent product (resorufin) and total proteins were quantified within the same wells with a fluorescence plate reader. Fluorescamine was used for protein assays.

A new method to extract and concentrate porphyrins from liver tissue for analysis by high-performance liquid chromatography is described. Porphyrins were extracted with acetonitrile-hydrochloric acid, concentrated on disposable octadecylsilyl columns, brought to dryness, and reconstituted in 1.2 M hydrochloric acid. Dried porphyrins were stable at -80 degrees C for at least three weeks, and porphyrins reconstituted in hydrochloric acid were stable for at least 20 h. Yields of uro-, heptacarboxyl-, hexacarboxyl-, pentacarboxyl- and coproporphyrin were > or = 90%. The method allows determination of porphyrin concentrations in small (10-100 mg) samples of liver tissue.

Rapid and sensitive methods for measuring ethoxyresorufin-O-deethylase (EROD) induction and total intracellular porphyrin accumulation in cultured chicken embryo hepatocytes exposed to polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are described. Cells were cultured in 48-well cell culture plates, and EROD assays were carried out directly within the wells. EROD activity and porphyrin concentrations were determined with a computer-operated fluorescence multiwell plate reader. Porphyrin and EROD assays could be carried out on the same plates or on separate plates. The assays allow one to obtain dose-response curves at rates that are approximately 100 times faster than conventional methods.

A high-speed reversed-phase high-performance liquid chromatographic method using an octadecylsilyl 3 cm long (3 microns particle size) column to separate the free acids of uroporphyrins I and III and coproporphyrins I and III from each other, and from the type I isomers of several other porphyrin carboxylic acids, is described. Separation of the porphyrins was achieved in less than 8 min, and injections were possible every 12 min. The detection limits of uroporphyrin, coproporphyrin, and mesoporphyrin were 75, 45, and 35 fmol (at a signal-to-noise ratio of 2), respectively. Application of the method to the determination of urinary and liver porphyrin patterns is shown.

Porphyria development in female Wistar rats has been followed by dosing the animals with hexachlorobenzene (HCB) either dissolved in corn oil or as a solid mixed with the diet. It was found that the corn oil preparation resulted in much faster uptake of HCB into the liver, and much faster accumulation of liver porphyrins. Diet preparation is thus shown to be a major factor in determining whether the development of porphyria is associated with the delay phenomenon. Evidence is also presented suggesting that the neurological symptoms of porphyria are not caused by high porphyrin levels.

A rapid and sensitive method for the determination of porphyrin carboxylic acids in liver, kidney, and spleen by high-speed high-performance liquid chromatography is described. Porphyrins were extracted with recoveries greater than or equal to 98%, concentrated on disposable octadecylsilyl cartridges, and analyzed with a liquid chromatograph equipped with a 3 microns X 3 cm octadecylsilyl column and a fluorescence detector. Separation of di-, tetra-, penta-, hexa-, hepta-, and octacarboxylic acids was achieved within 5 min. The detection limits for uro, copro, and protoporphyrin were 20, 10, and 20 fmol, respectively.

Hepatic porphyria was induced in female Wistar rats exposed to dietary hexachlorobenzene (HCB) for 56 days. The well-documented several-week delay before liver total porphyrins became elevated was observed using conventional methods. However, a newly developed high-performance liquid chromatography (HPLC) technique revealed a much earlier response. Highly carboxylated porphyrins were found to increase soon after exposure to the toxicant. The long delay observed by total porphyrin analysis is shown to be due to the relatively small contribution of highly carboxylated porphyrins to the total porphyrin pool. It is concluded that the concept of a latent period is largely a methodological artefact which has confused the search for a fundamental understanding of chemically induced porphyria.

Four mature, cyclic ewes were given injections (I.M.) of a conjugate of 1,3,5 (10)-estratrien-3-ol-6,17-dione, 6 carboxyoxime bovine serum albumin (immunized ewes) on day 3 after estrus, and at days 10, 20, 40, 58, 91 and 134 after this initial treatment. Six control ewes treated with carrier emulsion alone continued to cycle normally. Three of the immunized ewes failed to exhibit estrus, an associated preovulatory surge of LH and ovulation. One ewe showed 1 abnormally short estrous period and then became anestrus. Injection of an estrone-protein-conjugate at days 3 and 13 after estrus did not appear to interfere with the rate of structural luteolysis of the corpus luteum present, but plasma concentrations of progesterone reached abnormally high luteal phase levels and in 2 ewes failed, subsequently to decline to normal follicular phase levels. Estrone binding capacity rose as early as day 9 after first treatment, and concentrations of LH rose as early as day 14. Subsequently, plasma levels of LH, estrone and progesterone and antisera titer rose; the only significant cross reaction of the antisera was with estradiol 17beta (11.32 +/- 2.80%).