Key learnings from performance of the U.S. EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 in vitro assays.

Tier 1 of the U.S. EPA Endocrine Disruptor Screening Program comprises 11 studies: five in vitro assays, four in vivo mammalian assays, and two in vivo nonmammalian assays. The battery is designed to detect compounds with the potential to interact with the estrogen, androgen, or thyroid signaling pathways. This article examines the procedures, results, and data interpretation for the five Tier 1 in vitro assays: estrogen receptor (ER) and androgen receptor binding assays, an ER transactivation assay, an aromatase assay, and a steroidogenesis assay. Data are presented from two laboratories that have evaluated approximately 11 compounds in the Tier 1 in vitro assays. Generally, the ER and androgen receptor binding assays and the aromatase assay showed good specificity and reproducibility. As described in the guideline for the ER transactivation assay, a result is considered positive when the test compound induces a reporter gene signal that reaches 10% of the response seen with 1 nM 17ß-estradiol (positive control). In the experience of these laboratories, this cutoff criterion may result in false-positive responses. For the steroidogenesis assay, there is variability in the basal and stimulated production of testosterone and estradiol by the H295R cells. This variability in responsiveness, coupled with potential cell stress at high concentrations of test compound, may make it difficult to discern whether hormone alterations are specific steroidogenesis alterations (i.e., endocrine active). Lastly, both laboratories had difficulty meeting some recommended performance criteria for each Tier 1 in vitro assay. Data with only minor deviations were deemed valid.

Relevance weighting of tier 1 endocrine screening endpoints by rank order.

Weight of evidence (WoE) approaches are recommended for interpreting various toxicological data, but few systematic and transparent procedures exist. A hypothesis-based WoE framework was recently published focusing on the U.S. EPA's Tier 1 Endocrine Screening Battery (ESB) as an example. The framework recommends weighting each experimental endpoint according to its relevance for deciding eight hypotheses addressed by the ESB. Here we present detailed rationale for weighting the ESB endpoints according to three rank ordered categories and an interpretive process for using the rankings to reach WoE determinations. Rank 1 was assigned to in vivo endpoints that characterize the fundamental physiological actions for androgen, estrogen, and thyroid activities. Rank 1 endpoints are specific and sensitive for the hypothesis, interpretable without ancillary data, and rarely confounded by artifacts or nonspecific activity. Rank 2 endpoints are specific and interpretable for the hypothesis but less informative than Rank 1, often due to oversensitivity, inclusion of narrowly context-dependent components of the hormonal system (e.g., in vitro endpoints), or confounding by nonspecific activity. Rank 3 endpoints are relevant for the hypothesis but only corroborative of Ranks 1 and 2 endpoints. Rank 3 includes many apical in vivo endpoints that can be affected by systemic toxicity and nonhormonal activity. Although these relevance weight rankings (WREL ) necessarily involve professional judgment, their a priori derivation enhances transparency and renders WoE determinations amenable to methodological scrutiny according to basic scientific premises, characteristics that cannot be assured by processes in which the rationale for decisions is provided post hoc.

Cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss): a validation study to support their application in bioaccumulation assessment.

Determination of biotransformation rates of xenobiotics in freshly isolated trout hepatocytes has been demonstrated to significantly improve the performance of bioaccumulation assessment models. In order to promote this in vitro approach, trout hepatocytes need to be cryopreserved to facilitate their availability while ensuring their metabolic competency. In the present study, we obtained basal level metabolic enzyme activities for cytochrome P450 (CYP) 1A, CYP3A, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase from trout hepatocytes cryopreserved for various periods of time up to three months and compared their values with those obtained from freshly isolated hepatocytes. Similarly, we compared intrinsic clearance (CL(int)) values determined in cryopreserved trout hepatocytes to those determined in freshly isolated hepatocytes for reference compounds molinate, michler's ketone, 4-nonylphenol, 2,4-ditert-butylphenol, benzo(a)pyrene, and pyrene. Our results show that cryopreserved trout hepatocytes maintained greater than 75% of their basal level enzyme activities and greater than 72% of xenobiotic biotransformation capabilities, regardless of the length of cryostorage. As a result, bioconcentration factors of the reference compounds were adequately predicted based on the CL(int) values. We simulated the condition for shipping cryopreserved trout hepatocytes and demonstrated that 24 h dry ice storage did not negatively affect the rates of xenobiotic biotransformation. We conclude that cryopreserved trout hepatocytes are suitable for biotransformation rate determination of xenobiotics in vitro, and therefore, are an acceptable alternative to freshly isolated trout hepatocytes in the application in bioaccumulation assessment.

Liver microsomes and S9 from rainbow trout (Oncorhynchus mykiss): comparison of basal-level enzyme activities with rat and determination of xenobiotic intrinsic clearance in support of bioaccumulation assessment.

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. The applicability of trout liver microsomes and S9 fraction in bioaccumulation assessment of xenobiotics in fish was investigated in the present study. Basal-level activities of 7-ethoxyresorufin-O-dealkylase, testosterone 6beta-hydroxylase, glutathione-S-transferase, and uridine 5'-diphospho-glucuronosyltransferase in trout liver microsomes and S9 were significantly lower than those in rat liver microsomes and S9. The in vitro-to- in vivo scaling factors, which are the values of liver microsomal and S9 protein contents per unit weight of trout liver, were determined to be 38.4 +/- 5.1 (mean +/- standard deviation throughout) and 95.9 +/- 11.9 mg/g, respectively. Intrinsic clearance (CL(int)) values for a number of reference compounds obtained from trout liver S9 were lower than those from trout liver microsomes. After correction with the scaling factors, trout liver microsomes and S9 provided equivalent prediction of trout hepatic clearance (CL(H)) using the well-stirred liver model, but their CL(H) values were significantly lower than those obtained from freshly isolated trout hepatocytes. Consequently, trout liver microsomes and S9 showed poorer prediction of the bioconcentration factors of the reference compounds compared with trout hepatocytes. Unit conversion revealed that CL(int) values obtained from trout liver microsomes and S9 were 6.3 to 22.4% of those from trout hepatocytes, which explained, to a large extent, the differences in their CL(H) and bioconcentration factor prediction.

Non-coplanar 2,2',3,3',4,4',5,5',6,6'-decachlorobiphenyl (PCB 209) did not induce cytochrome P450 enzyme activities in primary cultured rat hepatocytes, was not genotoxic, and did not exhibit endocrine-modulating activities.

2,2',3,3',4,4',5,5',6,6'-Decachlorobiphenyl (PCB 209) is a fully chlorinated, non-coplanar biphenyl. To demonstrate that PCB 209 is not likely to exhibit human health hazards common to coplanar PCBs it was tested for cytochrome P450 (P450) enzyme induction potentials, genetic toxicity, and endocrine-modulating activity. PCB 209 (dose from 0.005 to 5000 ng/mL) did not significantly induce P450 CYP1A, 2A, 2B, 3A, or 4A enzyme activities in primary cultured rat hepatocytes. In contrast, Aroclor 1260, a PCB mixture that contains approximately 60% chlorine by weight, showed significant induction of P450 CYP1A, 2A, 2B, and 3A within the same dose range. PCB 209 (dose from 100 to 5000 microg/plate) was negative in the bacterial mutagenicity (Ames) test in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 or in Eschericia coli strain WP2uvrA. PCB 209 (dose from 25 to 150 microg/mL) was also negative for forward mutations at the thymidine kinase (TK+/-) locus of L5178Y mouse lymphoma cells. The Ames and the mouse lymphoma assays were both conducted in the absence and presence of rat liver S9 fraction. PCB 209 (dose from 500 to 2000 mg/kg by single dose oral gavage) did not induce an increase in the frequency of micronuclei in polychromatic erythrocytes in mouse bone marrow in vivo. PCB 209 did not induce estrogenic effects when administered by gavage to ovariectomized adult female rats at 500 and 1000 mg/kg for 4 days, nor did it produce alterations consistent with endocrine-modulating activity in adult intact male rats when administered by gavage at 500 and 1000 mg/kg for 15 consecutive days.

Xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss): determination of trout hepatocellularity, optimization of cell concentrations and comparison of serum and serum-free incubations.

Metabolism plays an important role in bioaccumulation of xenobiotics in fish. In vitro determination of xenobiotic intrinsic clearance (CLint) in trout hepatocytes and subsequent extrapolation to in vivo hepatic clearance (CLH) using the "well-stirred" liver model greatly improved our current practice of bioaccumulation assessment [Han, X., Nabb, D.L., Mingoia, R.T., Yang, C.H., 2007. Determination of xenobiotic intrinsic clearance in freshly isolated hepatocytes from rainbow trout (Oncorhynchus mykiss) and rat and its application in bioaccumulation assessment. Environ. Sci. Technol. 41, 3269-3276]. In an effort to further optimize this approach, we experimentally obtained the value of trout hepatocellularity (HT), a key scaling factor in the "well-stirred" liver model. HT was determined to be (540+/-12)x10(6)cells/g liver for male trout. We also investigated the potential effect of different cell concentrations on the determination of CL(int) values of molinate, 4,4-bis(dimethylamino)benzophenone, 4-nonylphenol, 2,4-di-tert-butylphenol, and benzo(a)pyrene. Linear relationships were established between clearance rates and cell concentrations at 1x10(6), 2x10(6), 5x10(6), and 10x10(6)cells/mL. This suggests that under our experimental conditions, CLint determination was independent of hepatocyte concentrations. In order to better understand the "in vitro binding" effect in in vitro-to-in vivo scaling, we obtained CLint values for the above-mentioned compounds in trout hepatocytes that were suspended in trout serum. Incubations in serum, in general, resulted relatively larger prediction of CLH values. Our findings suggest that in bioaccumulation assessment, the traditional medium incubation method offers a conservative estimate on fish metabolism of xenobiotics and the serum incubation approach could be used for certain classes of compounds that are of challenge for in silico prediction of their plasma and in vitro binding properties.

Uptake of perfluorooctanoate in freshly isolated hepatocytes from male and female rats.

Liver is a primary target organ for perfluorooctanoate (PFO, the deprotonated form of perfluorooctanoic acid, PFOA) distribution in both male and female rats. We studied the uptake of PFO in freshly isolated hepatocytes from male and female rats. We identified a non-saturable cell partitioning process for PFO using on-ice incubations. At 37 degrees C, hepatic uptake of PFO was composed of the non-saturable partition as well as a saturable, active uptake process. The K(m) and V(max) values for the active uptake process were 88.0 +/- 9.1 microM and 5.61 +/- 0.88 nmol/(min 10(6)cells), respectively, for male rat hepatocytes, and 76.1 +/- 12.0 microM and 3.59 +/- 0.29 nmol/(min 10(6)cells), respectively, for female rat hepatocytes. The values of PFO clearance by active uptake were 64.8 +/- 15.7 and 47.6 +/- 4.7 microL/(min 10(6)cells) for male and female rat hepatocytes, respectively. The active uptake of PFO in rat hepatocytes was inhibited by sulfobromophthalein, a known substrate of organic anion transporting polypeptides, with apparent inhibition constants of 85.9 +/- 25.1 and 29.3 +/- 19.2 microM in male and female rat hepatocytes, respectively. When serum albumin was added to the incubations, PFO hepatic uptake rates were reduced, but were proportional to the unbound fractions of PFO.

Results of the negative control chemical allyl alcohol in the 15-day intact adult male rat screening assay for endocrine activity.

Development, standardization, and validation of methods to assess the potential of chemicals to disrupt hormonal homeostasis have been the focus of considerable research efforts over the past 10 years. As part of our validation effort, we evaluated the specificity of the 15-day intact adult male rat assay, using a negative control chemical, allyl alcohol, a known hepatotoxicant that was not expected to induce endocrine effects. Male rats were dosed for 15 days via oral gavage with 0, 10, 30, 40, or 50 mg/kg/day allyl alcohol. The endpoints evaluated included final body and organ weights, serum hormone concentrations, and a limited histopathology assessment. No mortality or adverse clinical signs were observed. Mean final body weight for rats in the 50-mg/kg/day dose group was decreased to 90% of control. Mean relative liver weights were increased at 40 and 50 mg/kg/day (115% and 117% of control, respectively). Serum testosterone and DHT concentrations were statistically significantly decreased at 50 mg/kg/day (72% of control). Serum prolactin concentrations were statistically significantly decreased at 40 mg/kg/day (58% of control), but not at 50 mg/kg/day. There were no effects on the other endpoints evaluated. Consistent with previous guidance for interpreting the 15-day intact adult male rat assay, histological and weight changes of target organs were given a higher weight-of-evidence than changes in serum hormone concentrations alone. Therefore, with only minimal changes in serum hormone concentrations and no effects on organ weights or microscopic alterations, the results of allyl alcohol in the 15-day intact adult male rat assay were considered negative and consistent with the predicted results.

Molecular characterization of thyroid toxicity: anchoring gene expression profiles to biochemical and pathologic end points.

Organic iodides have been shown to induce thyroid hypertrophy and increase alterations in colloid in rats, although the mechanism involved in this toxicity is unclear. To evaluate the effect that free iodide has on thyroid toxicity, we exposed rats for 2 weeks by daily gavage to sodium iodide (NaI). To compare the effects of compounds with alternative mechanisms (increased thyroid hormone metabolism and decreased thyroid hormone synthesis, respectively), we also examined phenobarbital (PB) and propylthiouracil (PTU) as model thyroid toxicants. Follicular cell hypertrophy and pale-staining colloid were present in thyroid glands from PB-treated rats, and more severe hypertrophy/colloid changes along with diffuse hyperplasia were present in thyroid glands from PTU-treated rats. In PB- and PTU-treated rats, thyroid-stimulating hormone (TSH) levels were significantly elevated, and both thyroxine and triiodothyronine hormone levels were significantly decreased. PB induced hepatic uridine diphosphate-glucuronyltransferase (UDPGT) activity almost 2-fold, whereas PTU reduced hepatic 5 -deiodinase I (5 -DI) activity to < 10% of control in support of previous reports regarding the mechanism of action of each chemical. NaI also significantly altered liver weights and UDPGT activity but did not affect thyroid hormone levels or thyroid pathology. Thyroid gene expression analyses using Affymetrix U34A GeneChips, a regularized t-test, and Gene Map Annotator and Pathway Profiler demonstrated significant changes in rhodopsin-like G-protein-coupled receptor transcripts from all chemicals tested. NaI demonstrated dose-dependent changes in multiple oxidative stress-related genes, as also determined by principal component and linear regression analyses. Differential transcript profiles, possibly relevant to rodent follicular cell tumor outcomes, were observed in rats exposed to PB and PTU, including genes involved in Wnt signaling and ribosomal protein expression.

Comparison of ELISAs for detecting vitellogenin in the fathead minnow (Pimephales promelas).

Measurement of vitellogenin (VTG) concentrations in the fathead minnow (Pimephales promelas) is currently being considered and evaluated for screening of endocrine active substances. One of the proposed methods, an enzyme-linked immunosorbent assay (ELISA) based on VTG from carp (Cyprinus carpio), was recently evaluated in an inter-laboratory ring test using whole body homogenates from juvenile fathead minnows. The objective of the current study was to compare the performance of three different ELISAs for measuring fathead minnow VTG: (1) a heterologous carp VTG (cVTG) ELISA used in the ring test, (2) a homologous fathead minnow VTG (fVTG) ELISA, and (3) a hybrid ELISA with the antibody developed for cVTG, but using fVTG for coating the plates and preparing standard curves. VTG was measured in whole body homogenates from juvenile fathead minnows exposed to 17alpha-ethynylestradiol (EE(2); 10 ng/l) and whole body homogenates and plasma from adult fathead minnows exposed to 17beta-estradiol (E(2); 5 mg/kg; i.p.). The cVTG assay showed lower specificity for fathead minnow VTG in whole body homogenates and plasma from treated fish, compared to the fVTG assay. VTG concentrations in juvenile fathead minnow homogenates from the EE(2)-exposed group were approximately 50-fold higher when measured using the fVTG method compared to the cVTG method. Use of the homologous fVTG in the hybrid cVTG assay yielded VTG concentrations in the range of the fVTG assay but the low specificity persisted. The homologous fVTG assay is recommended to achieve accurate quantification of VTG levels in fathead minnows.