Lineage-dependent effects of aryl hydrocarbon receptor agonists contribute to liver tumorigenesis.

UNLABELLED: 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.

Adverse outcome pathways: From research to regulation scientific workshop report.

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.

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.

An exposure:activity profiling method for interpreting high-throughput screening data for estrogenic activity--proof of concept.

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.

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.

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 critical assessment of the methodologies to investigate the role of inhibition of apoptosis in rodent hepatocarcinogenesis.

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.

The use of mode of action information in risk assessment: quantitative key events/dose-response framework for modeling the dose-response for key events.

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.

A 21st century roadmap for human health risk assessment.

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.

Risk assessment in the 21st century: roadmap and matrix.

Abstract 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.

Developing scientific confidence in HTS-derived prediction models: lessons learned from an endocrine case study.

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.

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.

Use and validation of HT/HC assays to support 21st century toxicity evaluations.

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.

The human AHR: identification of single nucleotide polymorphisms from six ethnic populations.

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.

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole is a ligand and shows species-specific partial agonism of the aryl hydrocarbon receptor.

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203) and related compounds are a series of anti-cancer candidate pharmaceuticals, that have been shown to activate the AhR. We show that these compounds are high-affinity ligands for the rat AhR, but a quantitative assay for their ability to induce CYP1A1 RNA in H4IIEC3 cells, a measure of activation of the AhR, showed a poor relationship between affinity for the AhR and ability to induce CYP1A1 RNA. 5F 203, an agonist with low potency, was able to antagonise the induction of CYP1A1 RNA by TCDD, while IH 445, a potent agonist, did not antagonise the induction of CYP1A1 RNA by TCDD, and Schild analysis confirmed 5F 203 to be a potent antagonist of the induction of CYP1A1 RNA by TCDD in H4IIEC3 cells. In contrast, several benzothiazoles show potent induction of CYP1A1 RNA in human MCF-7 cells, and 5F 203 is unable to detectably antagonise the induction of CYP1A1 RNA in MCF-7 cells, showing a species difference in antagonism. Evaluation of the anti-proliferative activity of benzothiazoles showed that the ability to agonise the AhR correlated with growth inhibition both in H4IIEC3 cells for a variety of benzothiazoles, and between H4IIEC3 and MCF-7 cells for 5F 203, suggesting an important role of agonism of the AhR in the anti-proliferative activity of benzothiazoles.

FDA perspectives on health claims for food labels.

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.

Immunological characterization of the aryl hydrocarbon receptor (AHR) knockout rat in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

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.

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.

Altered mammary gland differentiation and progesterone receptor expression in rats fed soy and whey proteins.

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.

FutureTox: building the road for 21st century toxicology and risk assessment practices.

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.