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BACKGROUND: Humans are exposed to tens of thousands of chemical substances that need to be assessed for their potential toxicity. Acute systemic toxicity testing serves as the basis for regulatory hazard classification, labeling, and risk management. However, it is cost- and time-prohibitive to evaluate all new and existing chemicals using traditional rodent acute toxicity tests. In silico models built using existing data facilitate rapid acute toxicity predictions without using animals. OBJECTIVES: The U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Acute Toxicity Workgroup organized an international collaboration to develop in silico models for predicting acute oral toxicity based on five different end points: Lethal Dose 50 (LD50 value, U.S. Environmental Protection Agency hazard (four) categories, Globally Harmonized System for Classification and Labeling hazard (five) categories, very toxic chemicals [LD50 (LD50≤50mg/kg)], and nontoxic chemicals (LD50>2,000mg/kg). METHODS: An acute oral toxicity data inventory for 11,992 chemicals was compiled, split into training and evaluation sets, and made available to 35 participating international research groups that submitted a total of 139 predictive models. Predictions that fell within the applicability domains of the submitted models were evaluated using external validation sets. These were then combined into consensus models to leverage strengths of individual approaches. RESULTS: The resulting consensus predictions, which leverage the collective strengths of each individual model, form the Collaborative Acute Toxicity Modeling Suite (CATMoS). CATMoS demonstrated high performance in terms of accuracy and robustness when compared with in vivo results. DISCUSSION: CATMoS is being evaluated by regulatory agencies for its utility and applicability as a potential replacement for in vivo rat acute oral toxicity studies. CATMoS predictions for more than 800,000 chemicals have been made available via the National Toxicology Program's Integrated Chemical Environment tools and data sets (ice.ntp.niehs.nih.gov). The models are also implemented in a free, standalone, open-source tool, OPERA, which allows predictions of new and untested chemicals to be made. https://doi.org/10.1289/EHP8495.

An adverse outcome pathway (AOP) helps to organize existing knowledge on chemical mode of action, starting with a molecular initiating event such as receptor binding, continuing through key events, and ending with an adverse outcome such as reproductive impairment. AOPs can help identify knowledge gaps where more research is needed to understand the underlying mechanisms, aid in chemical hazard characterization, and guide the development of new testing approaches that use fewer or no animals. A September 2014 workshop in Bethesda, Maryland considered how the AOP concept could improve regulatory assessments of chemical toxicity. Scientists from 21 countries, representing industry, academia, regulatory agencies, and special interest groups, attended the workshop, titled Adverse Outcome Pathways: From Research to Regulation. Workshop plenary presentations were followed by breakout sessions that considered regulatory acceptance of AOPs and AOP-based tools, criteria for building confidence in an AOP for regulatory use, and requirements to build quantitative AOPs and AOP networks. Discussions during the closing session emphasized a need to increase transparent and inclusive collaboration, especially with disciplines outside of toxicology. Additionally, to increase impact, working groups should be established to systematically prioritize and develop AOPs. Multiple collaborative projects and follow-up activities resulted from the workshop.

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.

Earlier we created a chemical hazard database via natural language processing of dossiers submitted to the European Chemical Agency with approximately 10 000 chemicals. We identified repeat OECD guideline tests to establish reproducibility of acute oral and dermal toxicity, eye and skin irritation, mutagenicity and skin sensitization. Based on 350–700+ chemicals each, the probability that an OECD guideline animal test would output the same result in a repeat test was 78%–96% (sensitivity 50%–87%). An expanded database with more than 866 000 chemical properties/hazards was used as training data and to model health hazards and chemical properties. The constructed models automate and extend the read-across method of chemical classification. The novel models called RASARs (read-across structure activity relationship) use binary fingerprints and Jaccard distance to define chemical similarity. A large chemical similarity adjacency matrix is constructed from this similarity metric and is used to derive feature vectors for supervised learning. We show results on 9 health hazards from 2 kinds of RASARs—“Simple” and “Data Fusion”. The “Simple” RASAR seeks to duplicate the traditional read-across method, predicting hazard from chemical analogs with known hazard data. The “Data Fusion” RASAR extends this concept by creating large feature vectors from all available property data rather than only the modeled hazard. Simple RASAR models tested in cross-validation achieve 70%–80% balanced accuracies with constraints on tested compounds. Cross validation of data fusion RASARs show balanced accuracies in the 80%–95% range across 9 health hazards with no constraints on tested compounds. published

Rodent cancer bioassays indicate that the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD), causes increases in both hepatocytic and cholangiocytic tumors. Effects of AHR activation have been evaluated on rodent hepatic stem cells (rHpSCs) versus their descendants, hepatoblasts (rHBs), two lineage stages of multipotent, hepatic precursors with overlapping but also distinct phenotypic traits. This was made possible by defining the first successful culture conditions for ex vivo maintenance of rHpScs consisting of a substratum of hyaluronans and Kubota's medium (KM), a serum-free medium designed for endodermal stem/progenitor cells. Supplementation of KM with leukemia inhibitory factor elicited lineage restriction to rHBs. Cultures were treated with various AHR agonists including TCDD, 6-formylindolo-[3,2-b]carbazole (FICZ), and 3-3'-diindolylmethane (DIM) and then analyzed with a combination of immunocytochemistry, gene expression, and high-content image analysis. The AHR agonists increased proliferation of rHpSCs at concentrations producing a persistent AHR activation as indicated by induction of Cyp1a1. By contrast, treatment with TCDD resulted in a rapid loss of viability of rHBs, even though the culture conditions, in the absence of the agonists, were permissive for survival and expansion of rHBs. The effects were not observed with FICZ and at lower concentrations of DIM. Conclusion: Our findings are consistent with a lineage-dependent mode of action for AHR agonists in rodent liver tumorigenesis through selective expansion of rHpSCs in combination with a toxicity-induced loss of viability of rHBs. These lineage-dependent effects correlate with increased frequency of liver tumors. (Hepatology 2015;61:548-560)

High throughput (HTS) and high content (HCS) screening methods show great promise in changing how hazard and risk assessments are undertaken, but scientific confidence in such methods and associated prediction models needs to be established prior to regulatory use. Using a case study of HTS-derived models for predicting in vivo androgen (A), estrogen (E), thyroid (T) and steroidogenesis (S) endpoints in endocrine screening assays, we compare classification (fitting) models to cross validation (prediction) models. The more robust cross validation models (based on a set of endocrine ToxCast™ assays and guideline in vivo endocrine screening studies) have balanced accuracies from 79% to 85% for A and E, but only 23% to 50% for T and S. Thus, for E and A, HTS results appear promising for initial use in setting priorities for endocrine screening. However, continued research is needed to expand the domain of applicability and to develop more robust HTS/HCS-based prediction models prior to their use in other regulatory applications. Based on the lessons learned, we propose a framework for documenting scientific confidence in HTS assays and the prediction models derived therefrom. The documentation, transparency and the scientific rigor involved in addressing the elements in the proposed Scientific Confidence Framework could aid in discussions and decisions about the prediction accuracy needed for different applications.

The mechanisms regulating the induction of hepatic DNA synthesis by PPARα agonists are currently incompletely understood, and we set out to determine whether there are different mechanisms of induction for PPARα agonists and other hepatic growth agents. High levels of hepatic DNA synthesis (3–7%) were induced by a PPARα agonist, ciprofibrate, and by a PXRα agonist, cyproterone acetate, and liver samples were taken for transcriptomic analysis in a contemporaneous experiment. Microarray analysis of tissue RNAs detected gene induction at 24 hours after dosing, but failed to detect any biologically plausible response at 1–5 hours after dosing. RNA sequencing of control and ciprofibrate samples at 3 hours after dosing revealed 527 perturbed genes, including known PPARα target genes. Seven candidate genes of interest in regulating cell growth and apoptosis were examined by RT-PCR, and were confirmed to be induced by ciprofibrate treatment. Cyproterone acetate, TCPOBOP and partial hepatectomy induced a distinct spectrum of gene induction for ciprofibrate, demonstrating that ciprofibrate induces DNA synthesis through a unique mechanism. These data show that RNA sequencing is a powerful tool for analysis of differentially induced genes in rat liver, and for identification of candidate genes that mediate the induction of DNA synthesis by PPARα agonists.

Toxic equivalency factors (TEFs) for dioxin-like compounds are largely based on relative potency (REP) values derived from biochemical endpoints such as enzyme activity. As of yet, REPs based on gene expression changes have not been accounted for in the TEF values. In this study, primary rat hepatocytes were treated for 24h with 11 concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) ranging from 0.00001 to 100 nM. Differential changes in gene expression were analyzed using analysis of variance to assess the relative contributions of concentration, congener, and the interaction between concentration and congener for each gene. A total of 3283 genes showed significant changes with concentration (false discovery rate < .05 and fold-change ± 1.5 in at least 1 concentration for 1 congener). Among these genes, 399 were significant for both concentration and congener effects indicating parallel concentration-response curves with significant differences in potency. Only 8 genes showed a significant concentration and congener interaction term indicating a minority of genes show nonparallel dose-response curves among the 3 congeners. Benchmark dose (BMD) modeling was used to derive BMD values for induced individual genes and signaling pathways. The REP values for 4-PeCDF and TCDF were generally 3- to 5-fold lower than the World Health Organization (WHO) TEF values on both a gene and pathway basis. These findings suggest that the WHO TEF values may possibly overpredict the potency of these polychlorinated dibenzofuran congeners and demonstrate the importance of identifying functional pathways relevant to the toxicological modes of action for establishing pertinent REPs.

The aryl hydrocarbon receptor (AHR) has been extensively characterized for the essential role it plays in mediating the toxic responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Despite similarities across animal species, species-specific differences exist in the profile of toxicity and sensitivity to TCDD owing, in part, to differences in the AHR. Newer reports have implicated the importance of AHR in the development and regulation of the immune system. Our present studies seek to further explore the essential role of AHR in lymphoid tissue composition, B cell function and the immunological responses after TCDD administration using the recently established AHR KO rats. Comprehensive immune cell phenotyping showed a decrease in the CD8+ T cell, CD11c+ populations and an increase in NKT cells in 3-week-old AHR KO rats compared to the WT controls. The lipopolysaccharide-induced IgM response and proliferation was markedly suppressed in the WT but not in the AHR KO B cells in the presence of TCDD. However, the percentage of LPS-activated IgM+ B cells was significantly higher in the AHR KO B cells as compared to that of WT suggesting the role of AHR in regulating the IgM response. The use of an AHR antagonist further alluded to the endogenous role of AHR in regulating B cell responses in the rat. Overall, the studies report for the first time, comprehensive immune cell phenotyping of the AHR KO rat and the endogenous role of AHR in the regulation of B cell function in the rat.

The Aryl hydrocarbon receptor (AhR) mediates the induction of a variety of xenobiotic metabolism genes. Activation of the AhR occurs through binding to a group of structurally diverse compounds, most notably dioxins, which are exogenous ligands. Isoflavones are part of a family which include some well characterised endogenous AhR ligands. This paper analysed a novel family of these compounds, based on the structure of 2-amino-isoflavone. Initially two luciferase-based cell models, mouse H1L6.1c2 and human HG2L6.1c3, were used to identify whether the compounds had AhR agonistic and/or antagonistic properties. This analysis showed that some of the compounds were weak agonists in mouse and antagonists in human. Further analysis of two of the compounds, Chr-13 and Chr-19, was conducted using quantitative real-time PCR in rat H4IIE and human MCF-7 cells. The results indicated that Chr-13 was an agonist in rat but an antagonist in human cells. Chr-19 was shown to be an agonist in rat but more interestingly, a partial agonist in human. Luciferase induction results not only revealed that subtle differences in the structure of the compound could produce species-specific differences in response but also dictated the ability of the compound to be an AhR agonist or antagonist. Substituted 2-amino-isoflavones represent a novel group of AhR ligands that must differentially interact with the AhR ligand binding domain to produce their species-specific agonist or antagonist activity and future ligand binding analysis and docking studies with these compounds may provide insights into the differential mechanisms of action of structurally similar compounds.

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.

Toxicogenomics was used to examine mRNA expression profiles obtained from primary rat hepatocytes treated for 24 h with 0.01 or 1.0 nM 2,3,7,8-tetrachlorodibenzo- p -dioxin (2,3,7,8-TCDD), 0.02 or 2.0 nM 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF) and 0.1 or 10 nM 2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF). The concentrations of 2,3,4,7,8-PeCDF and 2,3,7,8-TCDF were chosen to be equivalent to 2,3,7,8-TCDD’s concentration based on the toxic equivalency factor/toxic equivalent (TEF/TEQ) method for estimating biological potency. 2,3,7,8-TCDD at 1.0 nM altered the expression of 533 genes; 2,3,4,7,8-PeCDF at 2.0 nM altered 182 genes, and 2,3,7,8-TCDF at 10 nM altered 154 genes. Of these, 57 genes were affected by all three congeners. Agglomerative hierarchical clustering revealed distinct congener-dependent gene subclusters. Principal components analyses of the microarray data revealed that these congeners cluster independently of one another. Data presented here demonstrate that equivalent TEQ concentrations of 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF and 2,3,7,8-TCDF, while altering the expression of a small battery of genes in common, also produce substantial congener specific alterations in gene expression.

While risk assessments are extensively used for guiding critical and resource intensive decisions, assessments that rigorously integrate key exposure and toxicity terms are less often published. This article derives residential soil clean-up levels accounting for ingestion and dermal contact (direct contact criteria [DCC]) for a chlorinated dibenzo-p-dioxin and furan (PCDD/F as toxicity equivalence TEQD/F) impacted site using site-specific information and deterministic and probabilistic methods. In addition, TEQD/F risk assessment has been the subject of extensive scientific and regulatory debate including in-depth comments from two USEPA Science Advisory Boards and the National Academy of Sciences (NAS) on the proposed USEPA Draft Dioxin Risk Assessment. This article presents and applies toxicity values seeking to address the NAS recommendations regarding cancer risk assessment. Deterministic DCC estimates ranged from 19 to 250 ppb through application of linear and nonlinear cancer toxicity valu...

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.

Background The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related dioxin-like chemicals are mediated through binding-dependent activation of the cytosolic aryl hydrocarbon receptor (AHR). The human AHR is a low-affinity receptor relative to most rodents, but some reports suggest that there may be individuals with polymorphic high-affinity receptors, thereby possibly increasing the sensitivity to dioxins in such people. Methods Although no polymorphisms have been reported in the ligand binding region of the AHR in the over 100 reported sequences, we sequenced 108 additional human AHR genes in an effort to further identify single single nucleotide polymorphisms (SNPs) within the open reading frames of the AHR locus. The DNA was sequenced from six ethnic populations that included Japanese, Chinese, European/Caucasian, African–American, South East Asian, and Hispanic. Results Six exonic SNPs were identified; four had been described as previously reported and two seem to be novel. Four of the SNPs identified lead to amino acid changes in the AHR protein and two of the SNPs lead to synonymous substitutions. An additional four SNPs have been reported elsewhere that were not identified in the current analysis. With these new sequences, more than 200 human AHR gene sequences have been analyzed for SNPs. Conclusion The results indicate a very limited presence of polymorphisms in the core ligand binding region of the human AHR. Other regions, such as the transactivation domain, seem to be slightly more polymorphic in the human population and the impact on functionality should be further examined.

Cancer potency estimates were derived for 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) using data collected from the recently published National Toxicology Program bioassay in female Sprague-Dawley rats. By using a toxicokinetic model for 4-PeCDF, the dose-response relationship for combined liver tumors (hepatocellular adenomas and cholangiocarcinomas) in rats was assessed in terms of lifetime average liver concentration and lifetime average adipose concentration with data from both the lifetime and the stop-exposure components of the bioassay. Benchmark dose modeling was performed to estimate tissue concentrations at two points of departure (EC(10) and EC(01) and their 95% upper and lower confidence limits). The same toxicokinetic model with human input values was then used to back-extrapolate human equivalent doses that corresponded to the internal tissue concentration measures at the points of departure. Information regarding the cancer mode of action was used to support the development of several toxicity criterion values based on a nonlinear method, e.g., reference dose or tolerable daily intake. Nonlinear estimates of toxicity criteria based on observed noncancer toxic events as possible precursors to tumor formation were also derived and were similar in value to those based on combined liver tumors. For comparison purposes, linear estimates of cancer potency were also derived.

Temporal analyses were performed on hepatic tissue from immature female C57BL/6 mice in order to compare the gene expression profiles for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibzofuran (TCDF). Time course studies conducted with a single oral dose of 300 microg/kg TCDF or 30 microg/kg TCDD were used to compare differential gene expression on complementary DNA microarrays containing 13,361 features, representing 8194 genes at 2, 4, 8, 12, 24, 72, 120, and 168 h. One hundred and ninety-five genes were identified as differentially regulated by TCDF, of which 116 genes were in common with TCDD, with 109 exhibiting comparable expression profiles (correlation coefficients0.3). In general, TCDF was less effective in eliciting hepatic vacuolization, and differential gene expression compared with TCDD when given at an equipotent dose based on a toxic equivalence factor (TEF) of 0.1 for TCDF, especially 72-h postadministration. For example, the induction of Cyp1a1 messenger RNA by TCDF was less when compared TCDD. Moreover, TCDF induced less severe hepatocyte cytoplasmic vacuolization consistent with lower lipid accumulations which significantly subsided by 120 and 168 h when compared with TCDD. TCDF-elicited responses correlated with their hepatic tissue levels which gradually decreased between 18 and 168 h. Although both compounds elicited comparable gene expression profiles, especially at early time points, the TCDF responses were generally weaker. Collectively, these results suggest that the weaker TCDF responses could be attributed to differences in pharmacokinetics. However, more comprehensive dose-response studies are required at optimal times for each end point of interest in order to investigate the effect of pharmacokinetic differences on relative potencies that are important in establishing TEFs.

This study examines serum levels of 2,3,7,8-substituted chlorinated dioxins and furans, and PCBs for 375 Michigan workers with potential chlorophenol exposure, 37 Worker Referents, and 71 Community Referents. The chlorophenol workers were last exposed to trichlorophenol and/or pentachlorophenol 26-62 years ago. Employees working only in the trichlorophenol units had mean lipid-adjusted 2378-tetrachlorodibenzo-p-dioxin (TCDD) levels of 15.9 ppt compared with 6.5 ppt in the Worker Referents. Employees working only in the pentachlorophenol units had mean lipid-adjusted levels for 123478-H6CDD of 16.1 ppt, 123678-H6CDD of 150.6 ppt, 123789-H6CDD of 20.2 ppt, 1234678-H7CDD of 192.6 ppt, and OCDD of 2,594.0 ppt compared with the Worker Referent levels for the same congeners of 7.5, 74.7, 8.6, 68.7, and 509.1 ppt, respectively. All furan and PCB levels among workers in the trichlorophenol and/or pentachlorophenol departments were similar to the Worker Referents. The Tradesmen who worked throughout the plant had dioxin congener profiles consistent with both trichlorophenol and pentachlorophenol exposures. PCB levels and levels of 23478-P5CDF, 123478-H6CDF, and 123678-H6CDF were also greater in these Tradesmen than in the Worker Referents. The Worker Referent group had higher levels of dioxins and furans than the Community Referents indicating the potential for exposure outside the chlorophenol departments at the site. Distinct patterns of dioxin congeners were found many years after exposure among workers with different chlorophenol exposures. Furthermore, past trichlorophenol exposures were readily distinguishable from past pentachlorophenol exposures based on serum dioxin evaluations among workers. These data can be used to better assess dioxin exposures in future health studies.

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 WileySons, Ltd.

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.

The U.S. Food and Drug Administration's regulatory authority over health claims was clarified in 1990 legislation known as the Nutrition Labeling and Education Act (NLEA). This law established mandatory nutrition labeling for most foods and placed restrictions on the use of food label claims characterizing the levels or health benefits of nutrients in foods. NLEA set a high threshold for the scientific standard under which the U.S. Food and Drug Administration (FDA) may authorize health claims, this standard is known as the significant scientific agreement (SSA) standard. Subsequent legislation known as the Food and Drug Administration Modernization Act (FDAMA) provided an alternative to FDA review of the health claim where an U.S. government scientific body other than FDA concluded that there is SSA for a substance/disease relationship. Courts have since extended the scope of health claims to include qualified health claims (QHC) that are health claims not substantiated on evidence that meets the level of SSA standard, but include a qualifying statement intended to convey to the consumer the level of evidence for the claim. FDA has responded by developing an evidence-based ranking system for scientific data to determine the level of evidence substantiating a health claim. The following is an overview of FDA's regulations and evidence-based method for evaluating health claims.

Non-genotoxic carcinogens act by promoting the clonal expansion of preneoplastic cells by directly or indirectly stimulating cell division or inhibiting cell loss in the target organ. The specific mode-of-action (MoA) by which some non-genotoxic carcinogens ultimately cause cancer is not completely understood. To date, there are several proposed MoAs for non-genotoxic carcinogens, and some of these propose inhibition of apoptosis as one of the key events. In general, inhibition of apoptosis is considered a necessary step for cell survival and in theory can occur in combination or in association with other key promotional events, such as cell proliferation, oxidative stress and inhibition of intercellular communication to promote carcinogenesis. However, the evidence supporting the role of inhibition of apoptosis as a necessary step in promoting specific chemically induced tumors is often debated. To address this evidence, we reviewed studies that utilized prototypical nuclear receptor-mediated hepatocarcinogens. Based on this review, it is proposed that the ability to determine the importance of inhibition of apoptosis as a key event in the MoA for tumor promotion is hampered by the limitations of the methods utilized for its detection. This review provides an assessment of the strengths and limitations of the current methodology used for detection of apoptosis and provides suggestions for improving its detection, thereby strengthening the weight of evidence supporting inhibition of apoptosis as a key event in a MoA for tumor promotion.

There are suspected links between an animal's diet, differentiation status of a target tissue, and sensitivity to chemically induced cancer. We have demonstrated that rats fed AIN93G diets made with soy protein isolate (SPI) or whey protein hydrolysate (WPH) had a lower incidence of 7,12-dimethylbenz(a)anthracene (DMBA)-induced adenocarcinoma than rats fed the same diet made with casein (CAS). The current study was conducted to determine the differentiation status of the mammary glands during development. Offspring of rats (n = 5-10/group) were fed diets made with SPI, WPH, or CAS throughout life (beginning on gestation day 4) and were sacrificed on postnatal day (PND) 21, PND 33, PND 50 or on metaestrous between PND 48 and PND 51. There were no significant differences between the numbers of mammary terminal end buds (TEBs) or lobuloalveoli (LOB) between any of the diets groups at PND 21 or PND 33, but at PND 50 there was an 75% decrease in the mean numbers of TEBs/mm(2) in the SPI- or WPH-fed rats, compared with the CAS-fed rats (p = 0.09 and p = 0.06, respectively). In rats sacrificed in metaestrous, there were no significant differences in the proliferation index (PI) in the TEBs or LOB between any of the diet groups. In metaestrous rats, there were twice as many cells expressing estrogen receptor beta (ERbeta; approximately 60%) compared with estrogen receptor alpha (ERalpha; approximately 30%) in the LOB and 1.5 times more ERbeta (approximately 60%) compared with estrogen receptor alpha (ERalpha, approximately 40%) in the TEBs. There were no diet-dependent differences in expression of ERalpha and ERbeta. Similarly, there were no differences between the diet groups in progesterone receptor (PR) expressing LOB cells. However, in the TEBs there was a diet-dependent difference in PR positive cells with a 34% increase (p < 0.05) in the SPI-fed rats and a 38% increase (p < 0.05) in the WPH-fed rats compared with the CAS-fed rats. These results show that the type of dietary protein alters the phenotype of mammary epithelia in the TEBs. The SPI- and WPH-dependent changes in mammary differentiation may contribute to the reduced sensitivity to DMBA-induced mammary cancer in rats fed these proteins.

Rapid high throughput in vitro screening (HTS) assays are now available for characterizing dose–responses in assays that have been selected for their sensitivity in detecting estrogen-related endpoints. For example, EPA’s ToxCast™ program recently released endocrine assay results for more than 1800 substances and the interagency Tox21 consortium is in the process of releasing data for approximately 10,000 chemicals. But such activity measurements alone fall short for the purposes of priority setting or screening because the relevant exposure context is not considered. Here, we extend the method of exposure:activity profiling by calculating the exposure:activity ratios (EARs) using human exposure estimates and AC50 values for a range of chemicals tested in a suite of seven estrogenic assays in ToxCast™ and Tox21. To provide additional context, relative estrogenic exposure:activity quotients (REEAQ) were derived by comparing chemical-specific EARs to the EAR of the ubiquitous dietary phytoestrogen, genistein (GEN). Although the activity of a substance in HTS-endocrine assays is not a measure of health hazard or risk, understanding how such a dose compares to human exposures provides a valuable additional metric that can be used in decision-making; substances with small EARs and REEAQs would indicate low priority for further endocrine screening or testing.

The HESI RISK21 project formed the Dose-Response/Mode-of-Action Subteam to develop strategies for using all available data (in vitro, in vivo, and in silico) to advance the next-generation of chemical risk assessments. A goal of the Subteam is to enhance the existing Mode of Action/Human Relevance Framework and Key Events/Dose Response Framework (KEDRF) to make the best use of quantitative dose-response and timing information for Key Events (KEs). The resulting Quantitative Key Events/Dose-Response Framework (Q-KEDRF) provides a structured quantitative approach for systematic examination of the dose-response and timing of KEs resulting from a dose of a bioactive agent that causes a potential adverse outcome. Two concepts are described as aids to increasing the understanding of mode of action-Associative Events and Modulating Factors. These concepts are illustrated in two case studies; 1) cholinesterase inhibition by the pesticide chlorpyrifos, which illustrates the necessity of considering quantitative dose-response information when assessing the effect of a Modulating Factor, that is, enzyme polymorphisms in humans, and 2) estrogen-induced uterotrophic responses in rodents, which demonstrate how quantitative dose-response modeling for KE, the understanding of temporal relationships between KEs and a counterfactual examination of hypothesized KEs can determine whether they are Associative Events or true KEs.

The Health and Environmental Sciences Institute (HESI)-coordinated Risk Assessment in the 21st Century (RISK21) project was initiated to develop a scientific, transparent, and efficient approach to the evolving world of human health risk assessment, and involved over 120 participants from 12 countries, 15 government institutions, 20 universities, 2 non-governmental organizations, and 12 corporations. This paper provides a brief overview of the tiered RISK21 framework called the roadmap and risk visualization matrix, and articulates the core principles derived by RISK21 participants that guided its development. Subsequent papers describe the roadmap and matrix in greater detail. RISK21 principles include focusing on problem formulation, utilizing existing information, starting with exposure assessment (rather than toxicity), and using a tiered process for data development. Bringing estimates of exposure and toxicity together on a two-dimensional matrix provides a clear rendition of human safety and risk. The value of the roadmap is its capacity to chronicle the stepwise acquisition of scientific information and display it in a clear and concise fashion. Furthermore, the tiered approach and transparent display of information will contribute to greater efficiencies by calling for data only as needed (enough precision to make a decision), thus conserving animals and other resources.

The RISK21 integrated evaluation strategy is a problem formulation-based exposure-driven risk assessment roadmap that takes advantage of existing information to graphically represent the intersection of exposure and toxicity data on a highly visual matrix. This paper describes in detail the process for using the roadmap and matrix. The purpose of this methodology is to optimize the use of prior information and testing resources (animals, time, facilities, and personnel) to efficiently and transparently reach a risk and/or safety determination. Based on the particular problem, exposure and toxicity data should have sufficient precision to make such a decision. Estimates of exposure and toxicity, bounded by variability and/or uncertainty, are plotted on the X- and Y-axes of the RISK21 matrix, respectively. The resulting intersection is a highly visual representation of estimated risk. Decisions can then be made to increase precision in the exposure or toxicity estimates or declare that the available information is sufficient. RISK21 represents a step forward in the goal to introduce new methodologies into 21st century risk assessment. Indeed, because of its transparent and visual process, RISK21 has the potential to widen the scope of risk communication beyond those with technical expertise.

Under the US laws that FDA enforces, food label claims generally fall into three categories: nutrient content claims, health claims and structure/function claims. Nutrient content claims are statements that characterize the level of a nutrient in a food. Nutrient content claims on food labels must be used in accordance with FDA authorizing regulations. Health claims describe a relationship of a food, food component or dietary supplement in reducing the risk of a disease or health-related condition. Health claims must be evaluated and authorized prior to their use in food labeling. There are three ways by which FDA exercises its oversight in determining which health claims may be used in food labeling. One way is the issuance of authorizing regulations based on FDA’s conclusion that there is significant scientific agreement that the available scientific evidence supports the relationship. A second way is through a notification to FDA of a health claim based on authoritative statements from a scientific body of the US government or the National Academy of Sciences that ensues from that scientific body’s deliberative review of scientific evidence on the substance/disease relationship. The third way provides for qualified health claims where the quality and strength of the scientific evidence falls below that required for FDA to issue an authorizing regulation. Structure/function claims are food label statements that describe the role of a nutrient or dietary supplement ingredient to affect normal structure or function in humans. Structure/function claims have historically appeared on the labels of conventional foods and dietary supplements. However, the Dietary Supplement Health and Education Act of 1994 established special regulatory procedures for the use of such statements on dietary supplement labels.

This article reports on the outcome of FutureTox, a Society of Toxicology (SOT) Contemporary Concepts in Toxicology (CCT) workshop, whose goal was to address the challenges and opportunities associated with implementing 21st century technologies for toxicity testing, hazard identification, and risk assessment. One goal of the workshop was to facilitate an interactive multisector and discipline dialog. To this end, workshop invitees and participants included stakeholders from governmental and regulatory agencies, research institutes, academia, and the chemical and pharmaceutical industry in Europe and the United States. The workshop agenda was constructed to collectively review and discuss the state-of-the-science in these fields, better define the problems and challenges, outline their collective goals for the future, and identify areas of common agreement key to advancing these technologies into practice.

Based on existing data and previous work, a series of studies is proposed as a basis toward a pragmatic early step in transforming toxicity testing. These studies were assembled into a data-driven framework that invokes successive tiers of testing with margin of exposure (MOE) as the primary metric. The first tier of the framework integrates data from high-throughput in vitro assays, in vitro-to-in vivo extrapolation (IVIVE) pharmacokinetic modeling, and exposure modeling. The in vitro assays are used to separate chemicals based on their relative selectivity in interacting with biological targets and identify the concentration at which these interactions occur. The IVIVE modeling converts in vitro concentrations into external dose for calculation of the point of departure (POD) and comparisons to human exposure estimates to yield a MOE. The second tier involves short-term in vivo studies, expanded pharmacokinetic evaluations, and refined human exposure estimates. The results from the second tier studies provide more accurate estimates of the POD and the MOE. The third tier contains the traditional animal studies currently used to assess chemical safety. In each tier, the POD for selective chemicals is based primarily on endpoints associated with a proposed mode of action, whereas the POD for nonselective chemicals is based on potential biological perturbation. Based on the MOE, a significant percentage of chemicals evaluated in the first 2 tiers could be eliminated from further testing. The framework provides a risk-based and animal-sparing approach to evaluate chemical safety, drawing broadly from previous experience but incorporating technological advances to increase efficiency.

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.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

Advances in high throughput and high content (HT/HC) methods such as those used in the fields of toxicogenomics, bioinformatics, and computational toxicology have the potential to improve both the efficiency and effectiveness of toxicity evaluations and risk assessments. However, prior to use, scientific confidence in these methods should be formally established. Traditional validation approaches that define relevance, reliability, sensitivity and specificity may not be readily applicable. HT/HC methods are not exact replacements for in vivo testing, and although run individually, these assays are likely to be used as a group or battery for decision making and use robotics, which may be unique in each laboratory setting. Building on the frameworks developed in the 2010 Institute of Medicine Report on Biomarkers and the OECD 2007 Report on (Q)SAR Validation, we present constructs that can be adapted to address the validation challenges of HT/HC methods. These are flexible, transparent, and require explicit specification of context and purpose of use such that scientific confidence (validation) can be defined to meet different regulatory applications. Using these constructs, we discuss how anchoring the assays and their prediction models to Adverse Outcome Pathways (AOPs) could facilitate the interpretation of results and support scientifically defensible fit-for-purpose applications.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent ligands of the aryl hydrocarbon receptor (AhR). Here, we show that a novel fused mesoionic heterocyclic compound (AZ1) is ∼5-fold more potent than TCDD in both rat and human cell lines at inducing cytochrome P4501A1 RNA. In rat H4IIE cells, AZ1 gave an EC50 = 5.05 pM (95% CI = 2.81–9.09 pM) whereas TCDD had an EC50 = 25.5 pM (95% CI = 18.2–36.0 pM). AZ1 was also more potent than TCDD (5–10-fold) at inducing the AhR-related CYP1A2 and CYP1B1 genes, showing that AZ1 is more potent at inducing multiple genes. In human MCF-7 cells AZ1 gave an EC50 = 65.4 pM (95% CI = 45.6–93.7 pM) and TCDD an EC50 = 241 pM (95% CI = 161–362 pM), showing that AZ1 was more potent than TCDD at inducing CYP1A1 RNA in multiple species. Finally, the compound bound to rat cytosolic AhR with 6-fold higher affinity than TCDD, showing that the highly potent agonism of this substance is mediated via a high affinity for the receptor. This data shows that this novel compound, which shares structural similarities with various naphthoflavones, is a potent ligand of the AhR.

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.

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.

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.

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.

OBJECTIVE: We sought to determine if workers exposed to dioxins in pentachlorophenol (PCP) manufacturing were at increased risk of death from specific causes. METHODS: We examined death rates among 773 workers exposed to chlorinated dioxins during PCP manufacturing from 1937 to 1980 using serum dioxin evaluations to estimate exposures to five dioxins. RESULTS: Deaths from all causes combined, all cancers combined, lung cancer, diabetes, and ischemic heart disease were near expected levels. There were eight deaths from non-Hodgkin lymphoma (standardized mortality ratios = 2.4, 95% CI = 1.0 to 4.8). We observed no trend of increasing risk for any cause of death with increasing dioxin exposure. However, the highest rates of non-Hodgkin lymphoma were found in the highest exposure group (standardized mortality ratios = 4.5, 95% CI = 1.2 to 11.5). CONCLUSIONS: Other than possibly an increased risk of non-Hodgkin lymphoma, we find no other cause of death related to the mixture of the dioxin contaminants found in PCP.

Toxic equivalency factors (TEFs) are assigned to dioxin-like chemicals based on relative potency (REP) values of individual adaptive and toxic responses compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Agilent 4x44K oligonucleotide microarrays were used to examine the hepatic gene expression potency of 2,3,7,8-tetrachlorodibenzofuran (TCDF), relative to TCDD with complementary histopathology, TCDD and TCDF tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay data. Immature ovariectomized C57BL/6 mice were gavaged with 0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 microg/kg TCDD, the World Health Organization TEF-adjusted doses (10 x TCDD dose) of TCDF (0.3, 1, 3, 10, 30, 100, or 300 microg/kg), or sesame oil vehicle and killed at 72 h. Two thousand two hundred eighty-eight and 1347 genes were differentially expressed (P1(t) > 0.90) at one or more doses by TCDD and TCDF, respectively. Automated dose-response modeling (ToxResponse Modeler) identified a total of 1027 and 837 genes with either a sigmoidal, exponential, linear, Gaussian, or quadratic dose-response relationship 72 h after treatment in TCDD and TCDF, respectively. Two hundred seventy genes exhibited a sigmoidal TCDD-induced dose-response (ED(50s) from 0.08 to 42.2 microg/kg) compared to only 179 sigmoidal responsive genes (ED(50s) from 0.74 to 299.9 microg/kg) elicited by TCDF. Of the 1027 TCDD dose-responsive genes, 654 were not examined further due to the lack of a dose response elicited by TCDF. Of the 373 genes that exhibited a TCDD and TCDF dose response, REPs were calculated for the 83 genes that exhibited comparable sigmoidal curve shapes and slopes. The median REP for these 83 genes was 0.10, with a maximum REP of 0.56 and a minimum of 0.01. REPs of 0.04 were also calculated for EROD and increase in relative liver weight (RLW) at 72 h. Collectively, the lower number of TCDF-induced genes compared to TCDD and the 0.04 REPs for EROD activity and increased RLW are not consistent with the TEF of 0.10 for the hepatotoxicity of TCDF in C57BL/6 mice at 72 h.