A Comparison of Environment Classification Among Premium Hearing Instruments.

Hearing aids classify acoustic environments into multiple, generic classes for the purposes of guiding signal processing. Information about environmental classification is made available to the clinician for fitting, counseling, and troubleshooting purposes. The goal of this study was to better inform scientists and clinicians about the nature of that information by comparing the classification schemes among five premium hearing instruments in a wide range of acoustic scenes including those that vary in signal-to-noise ratio and overall level (dB SPL). Twenty-eight acoustic scenes representing various prototypical environments were presented to five premium devices mounted on an acoustic manikin. Classification measures were recorded from the brand-specific fitting software then recategorized to generic labels to conceal the device company, including (a) Speech in Quiet, (b) Speech in Noise, (c) Noise, and (d) Music. Twelve normal-hearing listeners also classified each scene. The results revealed a variety of similarities and differences among the five devices and the human subjects. Where some devices were highly dependent on input overall level, others were influenced markedly by signal-to-noise ratio. Differences between human and hearing aid classification were evident for several speech and music scenes. Environmental classification is the heart of the signal processing strategy for any given device, providing key input to subsequent decision-making. Comprehensive assessment of environmental classification is essential when considering the cost of signal processing errors, the potential impact for typical wearers, and the information that is available for use by clinicians. The magnitude of differences among devices is remarkable and to be noted.

Dioxin male rat reproductive toxicity mode of action and relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran characterized by fetal pituitary and testis transcriptome profiling.

Fetal rat exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces epididymal sperm number involving altered pituitary-testicular hormonal signaling as the proposed mode-of-action (MOA). To evaluate this MOA and compare TCDD to 2,3,7,8-tetrachlorodibenzofuran (TCDF), an in utero rat exposure and study was conducted. Endpoints included congener tissue levels and transcriptomes of maternal liver and fetal liver, testis, and pituitary. Decreased gonadotropin subunit mRNAs levels (Lhb and Fshb) and enriched signaling pathways including GNRH Signaling and Calcium Signaling were observed in fetal pituitary after TCDD (but not TCDF) exposure. TCDD (but not TCDF) decreased fetal testis cholesterologenic and steroidogenic pathway genes. TCDD tissue concentrations in dam liver, dam adipose, and whole fetus were approximately 3- to 6-fold higher than TCDF. These results support a MOA for dioxin-induced rat male reproductive toxicity involving key events in both the fetal pituitary (e.g., reduced gonadotropin production) and fetal testis (e.g., reduced Leydig cell cholesterologenesis and steroidogenesis).

Trichloroethylene in drinking water throughout gestation did not produce congenital heart defects in Sprague Dawley rats.

BACKGROUND: Trichloroethylene (TCE) was negative for developmental toxicity after inhalation and oral gavage exposure of pregnant rats but fetal cardiac defects were reported following drinking water exposure throughout gestation. Because of the deficiencies in this latter study, we performed another drinking water study to evaluate whether TCE causes heart defects. METHODS: Groups of 25 mated Sprague Dawley rats consumed water containing 0, 0.25, 1.5, 500, or 1,000 ppm TCE from gestational day 1-21. TCE concentrations were measured at daily formulation, when placed into water bottles each day and when water bottles were removed from cages. Four additional mated rats per group were used for plasma measurements. At termination, fetal hearts were carefully dissected fresh and examined. RESULTS: All TCE concentrations were >90% of target when initially placed in water bottles and when bottles were placed on cages. All dams survived with no clinical signs. Rats in the two higher dose groups consumed less water/day than other groups but showed no changes in maternal or fetal weights. The only fetal cardiac observation was small (<1 mm) membranous ventricular septal defect occurring in all treated and water control groups; incidences were within the range of published findings for naive animals. TCE was not detected in maternal blood, but systemic exposure was confirmed by detecting its primary oxidative metabolite, trichloroacetic acid, although only at levels above the quantitation limit in the two higher dose groups. CONCLUSIONS: Ingesting TCE in drinking water ≤1,000 ppm throughout gestation does not cause cardiac defects in rat offspring.

Aryl hydrocarbon receptor knockout rats are insensitive to the pathological effects of repeated oral exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Sustained activation of the aryl hydrocarbon receptor (AHR) is believed to be the initial key event in AHR receptor-mediated tumorigenesis in the rat liver. The role of AHR in mediating pathological changes in the liver prior to tumor formation was investigated in a 4-week, repeated-dose study using adult female wild-type (WT) and AHR knockout (AHR-KO) rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Beginning at 8 weeks of age, AHR-KO and WT rats were dosed by oral gavage with varying concentrations of TCDD (0, 3, 22, 100, 300 and 1000 ng kg(-1) day(-1) ). Lung, liver and thymus histopathology, hematology, serum chemistry and the distribution of TCDD in liver and adipose tissue were examined. Treatment-related increases in the severity of liver and thymus pathology were observed in WT, but not AHR-KO rats. In the liver, these included hepatocellular hypertrophy, bile duct hyperplasia, multinucleated hepatocytes and inflammatory cell foci. A loss of cellularity in the thymic cortex and thymic atrophy was observed. Treatment-related changes in serum chemistry parameters were also observed in WT, but not AHR-KO rats. Finally, dose-dependent accumulation of TCDD was observed primarily in the liver of WT rats and primarily in the adipose tissue of AHR-KO rats. The results suggest that AHR activation is the initial key event underlying the progression of histological effects leading to liver tumorigenesis following TCDD treatment. Copyright © 2015 John Wiley & Sons, Ltd.

Quantitative analysis of unconjugated and total bisphenol A in human urine using solid-phase extraction and UPLC-MS/MS: method implementation, method qualification and troubleshooting.

The aim of the presented investigation was to document challenges encountered during implementation and qualification of a method for bisphenol A (BPA) analysis and to develop and discuss precautions taken to avoid and to monitor contamination with BPA during sample handling and analysis. Previously developed and published HPLC-MS/MS methods for the determination of unconjugated BPA (Markham et al. Journal of Analytical Toxicology, 34 (2010) 293-303) [17] and total BPA (Markham et al. Journal of Analytical Toxicology, 38 (2014) 194-203) [20] in human urine were combined and transferred into another laboratory. The initial method for unconjugated BPA was developed and evaluated in two independent laboratories simultaneously. The second method for total BPA was developed and evaluated in one of these laboratories to conserve resources. Accurate analysis of BPA at sub-ppb levels is a challenging task as BPA is a widely used material and is ubiquitous in the environment at trace concentrations. Propensity for contamination of biological samples with BPA is reported in the literature during sample collection, storage, and/or analysis. Contamination by trace levels of BPA is so pervasive that even with extraordinary care, it is difficult to completely exclude the introduction of BPA into biological samples and, consequently, contamination might have an impact on BPA biomonitoring data. The applied UPLC-MS/MS method was calibrated from 0.05 to 25ng/ml. The limit of quantification was 0.1ng/ml for unconjugated BPA and 0.2ng/ml for total BPA, respectively, in human urine. Finally, the method was applied to urine samples derived from 20 volunteers. Overall, BPA can be analyzed in human urine with acceptable recovery and repeatability if sufficient measures are taken to avoid contamination throughout the procedure from sample collection until UPLC-MS/MS analysis.

The adverse outcome pathway for rodent liver tumor promotion by sustained activation of the aryl hydrocarbon receptor.

An Adverse Outcome Pathway (AOP) represents the existing knowledge of a biological pathway leading from initial molecular interactions of a toxicant and progressing through a series of key events (KEs), culminating with an apical adverse outcome (AO) that has to be of regulatory relevance. An AOP based on the mode of action (MOA) of rodent liver tumor promotion by dioxin-like compounds (DLCs) has been developed and the weight of evidence (WoE) of key event relationships (KERs) evaluated using evolved Bradford Hill considerations. Dioxins and DLCs are potent aryl hydrocarbon receptor (AHR) ligands that cause a range of species-specific adverse outcomes. The occurrence of KEs is necessary for inducing downstream biological responses and KEs may occur at the molecular, cellular, tissue and organ levels. The common convention is that an AOP begins with the toxicant interaction with a biological response element; for this AOP, this initial event is binding of a DLC ligand to the AHR. Data from mechanistic studies, lifetime bioassays and approximately thirty initiation-promotion studies have established dioxin and DLCs as rat liver tumor promoters. Such studies clearly show that sustained AHR activation, weeks or months in duration, is necessary to induce rodent liver tumor promotion--hence, sustained AHR activation is deemed the molecular initiating event (MIE). After this MIE, subsequent KEs are 1) changes in cellular growth homeostasis likely associated with expression changes in a number of genes and observed as development of hepatic foci and decreases in apoptosis within foci; 2) extensive liver toxicity observed as the constellation of effects called toxic hepatopathy; 3) cellular proliferation and hyperplasia in several hepatic cell types. This progression of KEs culminates in the AO, the development of hepatocellular adenomas and carcinomas and cholangiolar carcinomas. A rich data set provides both qualitative and quantitative knowledge of the progression of this AOP through KEs and the KERs. Thus, the WoE for this AOP is judged to be strong. Species-specific effects of dioxins and DLCs are well known--humans are less responsive than rodents and rodent species differ in sensitivity between strains. Consequently, application of this AOP to evaluate potential human health risks must take these differences into account.

A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR) by 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and subsequent binding the activated AHR to xenobiotic response elements (XREs) on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT). In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs) at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism's ability to respond on a phenotypic level to various stimuli within an inconstant environment.

Extensive metabolism and route-dependent pharmacokinetics of bisphenol A (BPA) in neonatal mice following oral or subcutaneous administration.

Orally administered bisphenol A (BPA) undergoes efficient first-pass metabolism to produce the inactive conjugates BPA-glucuronide (BPA-G) and BPA-sulfate (BPA-S). This study was conducted to evaluate the pharmacokinetics of BPA, BPA-G and BPA-S in neonatal mice following the administration of a single oral or subcutaneous (SC) dose. This study consisted of 3 phases: (1) mass-balance phase in which effective dose delivery procedures for oral or SC administration of (3)H-BPA to postnatal day three (PND3) mice were developed; (2) pharmacokinetic phase during which systemic exposure to total (3)H-BPA-derived radioactivity in female PND3 mice was established; and (3) metabolite profiling phase in which 50 female PND3 pups received either a single oral or SC dose of (3)H-BPA. Blood was collected from 5 pups/route/time-point at various times post-dosing, the blood plasma samples were pooled by group, and time-point and samples were profiled by HPLC with fraction collection. Fractions were analyzed for total radioactivity and data used to reconstruct radiochromatograms and to integrate individual peaks. The identity of the BPA, BPA-G, and BPA-S peaks was confirmed using authentic standards and LC-MS/MS analysis. The result of this study revealed that female PND3 mice have the capacity to metabolize BPA to BPA-G, BPA-S and other metabolites after both routes of administration. Systemic exposure to free BPA is route-dependent as the plasma concentrations were lower following oral administration compared to SC injection.

Proposing a scientific confidence framework to help support the application of adverse outcome pathways for regulatory purposes.

An adverse outcome pathway (AOP) describes the causal linkage between initial molecular events and an adverse outcome at individual or population levels. Whilst there has been considerable momentum in AOP development, far less attention has been paid to how AOPs might be practically applied for different regulatory purposes. This paper proposes a scientific confidence framework (SCF) for evaluating and applying a given AOP for different regulatory purposes ranging from prioritizing chemicals for further evaluation, to hazard prediction, and ultimately, risk assessment. The framework is illustrated using three different AOPs for several typical regulatory applications. The AOPs chosen are ones that have been recently developed and/or published, namely those for estrogenic effects, skin sensitisation, and rodent liver tumor promotion. The examples confirm how critical the data-richness of an AOP is for driving its practical application. In terms of performing risk assessment, human dosimetry methods are necessary to inform meaningful comparisons with human exposures; dosimetry is applied to effect levels based on non-testing approaches and in vitro data. Such a comparison is presented in the form of an exposure:activity ratio (EAR) to interpret biological activity in the context of exposure and to provide a basis for product stewardship and regulatory decision making.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Knockout of the aryl hydrocarbon receptor results in distinct hepatic and renal phenotypes in rats and mice.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which plays a role in the development of multiple tissues and is activated by a large number of ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In order to examine the roles of the AHR in both normal biological development and response to environmental chemicals, an AHR knockout (AHR-KO) rat model was created and compared with an existing AHR-KO mouse. AHR-KO rats harboring either 2-bp or 29-bp deletion mutation in exon 2 of the AHR were created on the Sprague-Dawley genetic background using zinc-finger nuclease (ZFN) technology. Rats harboring either mutation type lacked expression of AHR protein in the liver. AHR-KO rats were also insensitive to thymic involution, increased hepatic weight and the induction of AHR-responsive genes (Cyp1a1, Cyp1a2, Cyp1b1, Ahrr) following acute exposure to 25 µg/kg TCDD. AHR-KO rats had lower basal expression of transcripts for these genes and also accumulated ~30-45-fold less TCDD in the liver at 7 days post-exposure. In untreated animals, AHR-KO mice, but not AHR-KO rats, had alterations in serum analytes indicative of compromised hepatic function, patent ductus venosus of the liver and persistent hyaloid arteries in the eye. AHR-KO rats, but not AHR-KO mice, displayed pathological alterations to the urinary tract: bilateral renal dilation (hydronephrosis), secondary medullary tubular and uroepithelial degenerative changes and bilateral ureter dilation (hydroureter). The present data indicate that the AHR may play significantly different roles in tissue development and homeostasis and toxicity across rodent species.

Incidence of jaw lesions and activity and gene expression of hepatic P4501A enzymes in mink (Mustela vison) exposed to dietary 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 2,3,4,7,8-pentachlorodibenzofuran.

This study assessed the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8 tetrachlorodibenzofuran (TCDF) on the incidence of jaw lesions and on hepatic cytochrome P4501A (CYP1A) endpoints in mink (Mustela vison). Adult female mink were assigned randomly to one of 13 dietary treatments (control and four increasing doses of TCDD, PeCDF, or TCDF) and provided spiked feed for approximately 150 d (60 d prior to breeding through weaning of offspring at 42 d post-parturition). Offspring were maintained on their respective diets for an additional 150 d. Activity of hepatic CYP1A enzymes in adult and juvenile mink exposed to TCDD, PeCDF, or TCDD was generally greater compared with controls, but changes in other CYP1A endpoints were less consistent. Histopathology of the mandible and maxilla of juvenile mink suggested a dose-related increase in the incidence of jaw lesions. The dietary effective doses (ED) for jaw lesions in 50% of the population (ED50) were estimated to be 6.6, 14, and 149 ng/kg body weight (bw)/d for TCDD, PeCDF, and TCDF, respectively. The relative potencies of PeCDF and TCDF compared with TCDD based on ED10, ED20, and ED50 values ranged from 0.5 to 1.9 and 0.04 to 0.09, respectively. These values are within an order of magnitude of the World Health Organization toxic equivalency factor (TEF(WHO)) values of 0.3 and 0.1 for PeCDF and TCDF, respectively.

Cross-species comparisons of transcriptomic alterations in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

A toxicogenomics approach was used to qualitatively and quantitatively compare the gene expression changes in human and rat primary hepatocytes exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Hepatocytes from five individual rats and five individual humans were exposed for 24 h to 11 concentrations of TCDD ranging from 0.00001 to 100nM and a vehicle control. Gene expression changes were analyzed using whole-genome microarrays containing 13,002 orthologs. Significant changes in expression of individual orthologs at any concentration (fold change [FC] ± 1.5 and false discovery rate < 0.05) were higher in the rat (1547) compared with human hepatocytes (475). Only 158 differentially expressed orthologs were common between rats and humans. Enrichment analysis was performed on the differentially expressed orthologs in each species with 49 and 34 enriched human and rat pathways, respectively. Only 12 enriched pathways were shared between the two species. The results demonstrate significant cross-species differences in expression at both the gene and pathway level. Benchmark dose analysis of gene expression changes showed an average 18-fold cross-species difference in potency among differentially expressed orthologs with the rat more sensitive than the human. Similar cross-species differences in potency were observed for signaling pathways. Using the maximum FC in gene expression as a measure of efficacy, the human hepatocytes showed on average a 20% lower efficacy among the individual orthologs showing differential expression. The results provide evidence for divergent cross-species gene expression changes in response to TCDD and are consistent with epidemiological and clinical evidence showing humans to be less sensitive to TCDD-induced hepatotoxicity.

An evaluation of single nucleotide polymorphisms in the human heat shock protein 90 kDa α and ß isoforms.

Single nucleotide polymorphisms (SNPs) in genes coding for proteins that maintain the cytosolic aryl hydrocarbon receptor (AHR) complex may affect individual susceptibility to dioxin-like compound (DLC)-induced toxicity. The cytosolic 90 kDa heat shock proteins (HSP90s) are ubiquitous chaperone proteins that bind to and stabilize numerous client proteins, including non-ligand-bound AHR. The objective of this study was to characterize SNPs in the human cytosolic HSP90 genes (HSP90AA1 and HSP90AB1). DNA sequencing of 101 human samples detected eight and seven unique SNPs at the HSP90AA1 and HSP90AB1 loci, respectively. For HSP90AA1, two non-synonymous (L71M and E554D) and one rare early termination (Q107X) SNP were observed. One SNP (E554D) was a rare novel polymorphism located in the middle substrate binding region. All SNPs detected in the HSP90AB1 gene were synonymous. With the exception of Q107X, in silico analyses predicted all HSP90 SNPs would have very low to medium risk of affecting the regulation of alternative splicing in gene transcription or protein function. Overall, a very limited presence of SNPs with predicted functional consequence in key domains of the human HSP90 proteins was observed in this study.

Single nucleotide polymorphisms in the human aryl hydrocarbon receptor nuclear translocator (ARNT) gene.

Species' variation(s) in gene homologues can result in differences among species in their quantitative and qualitative susceptibility and responsiveness to environmental contaminants. In the case of dioxin-like compounds (DLCs), it has been hypothesized that single nucleotide polymorphisms (SNPs) in genes associated with aryl hydrocarbon receptor (AHR)-regulated pathways may result in greater susceptibility to DLC toxicity. A key step in the activation of AHR involves heterodimerization with the AHR nuclear translocator (ARNT) protein before binding to its DNA response element. The objective of this study was to identify SNPs in the human ARNT gene that could potentially affect the sensitivity of AHR-dependent gene transcription. Results from DNA sequencing of 101 human samples demonstrated the presence of five unique SNPs at the ARNT locus, including three non-synonymous SNPs, of which two were novel: V304M and T462A. The genetic frequencies of the non-synonymous SNPs were very low (≤0.02), and the novel SNPs occurred in the Per-ARNT-Sim (PAS) functional domain. In silico analysis indicated that V304M was the only SNP identified in the current population with the potential to significantly alter ARNT protein function. Our findings indicated a very limited occurrence of SNPs with predicted functional consequence in key domains of human ARNT.

An evaluation of single nucleotide polymorphisms in the human aryl hydrocarbon receptor-interacting protein (AIP) gene.

The human aryl hydrocarbon receptor (AHR) is a protein for which there is little evidence of polymorphic variability of functional consequence. It has been hypothesized that potential variability in dioxin sensitivity may be due to polymorphisms in AHR-associated proteins, such as the human AHR-interacting protein (AIP). There are limited data on AIP single nucleotide polymorphisms (SNPs) with potential functional consequences. We sequenced 103 human DNA samples within the open reading frames of the AIP locus using samples from six ethnic populations to further characterize AIP SNPs. Eight exonic SNPs were identified at the AIP locus, including three novel SNPs: T48T, L212L, and V302V. Combined with prior reports, there are now a total of 14 exonic SNPs that have been identified within AIP. Of these, six are non-synonymous and are therefore of potential functional importance, though only two of these (Q228K and A276V) were detected in the current study. The functional consequences of Q228K and A276V are unknown, although functional evidence from AIP SNPs associated with congenital pituitary tumors suggests that such amino acid changes are likely to have no effect or to decrease, rather than increase, sensitivity to dioxins. To date, no non-synonymous SNPs have been detected in the AHR-binding region of AIP.

Automated dose-response analysis and comparative toxicogenomic evaluation of the hepatic effects elicited by TCDD, TCDF, and PCB126 in C57BL/6 mice.

The toxic equivalency factor (TEF) approach recommended by the World Health Organization is used to quantify dioxin-like exposure concentrations for mixtures of polychlorinated dibenzo-dioxins, -furans, and polychlorinated biphenyls (PCBs), including 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Whole-genome microarrays were used to evaluate the hepatic gene expression potency of TCDF and PCB126 relative to TCDD with complementary histopathology, tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay results. Immature ovariectomized C57BL/6 mice were gavaged with 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 µg/kg TCDD and TEF-adjusted doses (TEF for TCDF and PCB126 is 0.1) of TCDF or PCB126 (1, 3, 10, 30, 100, 300, 1000, and 3000 µg/kg of TCDF or PCB126) or sesame oil vehicle and sacrificed 24 h post dose. In general, TCDD, TCDF, and PCB126 tissue levels, as well as histopathological effects, were comparable when comparing TEF-adjusted doses. Automated dose-response modeling (ToxResponse Modeler) of the microarray data identified 210 TCDF and 40 PCB126 genes that exhibited sigmoidal dose-response curves with comparable slopes when compared with TCDD. These similar responses were used to calculate a median TCDF gene expression relative potency (REP) of 0.06 and a median PCB126 gene expression REP of 0.02. REPs of 0.02 were also calculated for EROD induction for both compounds. Collectively, these data suggest that differences in the ability of the liganded aryl hydrocarbon receptor:AhR nuclear translocator complex to elicit differential hepatic gene expression, in addition to pharmacokinetic differences between ligands, influence their potency in immature ovariectomized C57BL/6 mice.

Human and rat primary hepatocyte CYP1A1 and 1A2 induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and 2,3,4,7,8-pentachlorodibenzofuran.

The concentration dose response for aryl hydrocarbon receptor (AHR)-mediated CYP1A1 and CYP1A2 messenger RNA (mRNA) induction and enzyme activity was determined in primary cultures of rat and human hepatocytes for 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 2,3,7,8-tetrachlorodibenzofuran. Eleven different congener concentrations from 0.00001 to 100 nM were used, thus spanning seven orders of magnitude. The Hill model was used to obtain values of EC(x) and maximal response from the individual data sets. No-observed effect concentration values were derived using several statistical methods including Dunnett's test, the Welch-Aspin test, and step-down bilinear regression. Thresholds were estimated using baseline projection methods and a "hockey stick" fitting method. Human hepatocytes were less responsive and less sensitive with respect to CYP1A1 activity and mRNA induction than rats. On the other hand, the human CYP1A2 response was more robust than the response in rats but generally less sensitive. These data allow an evaluation of relative species sensitivities for developing interspecies toxicodynamic adjustment factors, for assessing AHR activation thresholds, and for evaluating relative congener potencies. Overall, these data support the position that humans are less sensitive than rats to these AHR-dependent end points and support the use of a data-derived adjustment factor of 1.0 or less for extrapolating between rats and humans.

Estimates of cancer potency of 2,3,7,8-tetrachlorodibenzo(p)dioxin using linear and nonlinear dose-response modeling and toxicokinetics.

Linear and nonlinear toxicity criteria were derived for 2,3,7,8-tetrachlorodibenzo(p)dioxin (TCDD) using the recent National Toxicology Program rat cancer bioassay. Dose-response relationships were assessed for combined liver tumors based on lifetime average liver concentrations (LALCs) estimated with a toxicokinetic model. Rat LALC estimates at the 1% point of departure (POD) were obtained with benchmark dose (BMD) modeling to yield the BMD(01) in terms of LALC. The same toxicokinetic model was used to back-extrapolate the human-equivalent external dose (HED). A linear cancer slope factor (CSF) with a value of 1 x 10(-4) per pg/kg/day was calculated as the ratio between the benchmark response rate and the HED at the lower confidence limit of the benchmark dose (BMDL)(01). A nonlinear reference dose (RfD) with a value of 100 pg/kg/day was developed from the BMD(01) value by applying uncertainty factors to rat internal and human external doses. The RfD was 100 times higher than the 10(-4) risk-specific dose (RSD) based on the linear CSF. For comparison, BMD(01) and BMDL(01) values were developed for key events in the tumor promotion mode of action (MOA) of TCDD. This MOA involves dysregulation of the normal function of the aryl hydrocarbon receptor and its associated biological processes and results in pathologies that drive tumor promotion and progression. The BMD(01) values for key events were consistent with the timing of the key events within the MOA and provided support for the choices of the 1% tumor rate as a POD and dichotomous Hill model for representing receptor-mediated carcinogenicity. Because a threshold toxicity criterion most accurately reflects the MOA, the RfD for TCDD with a value of 100 pg/kg/day is considered appropriate for regulatory purposes, consistent with a 2006 NRC panel's recommendation to develop a threshold-based cancer potency factor for TCDD and with the methodology in U.S. Environmental Protection Agency's Cancer Guidelines.