Mode of action human relevance (species concordance) framework: Evolution of the Bradford Hill considerations and comparative analysis of weight of evidence.

The mode of action human relevance (MOA/HR) framework increases transparency in systematically considering data on MOA for end (adverse) effects and their relevance to humans. This framework continues to evolve as experience increases in its application. Though the MOA/HR framework is not designed to address the question of "how much information is enough" to support a hypothesized MOA in animals or its relevance to humans, its organizing construct has potential value in considering relative weight of evidence (WOE) among different cases and hypothesized MOA(s). This context is explored based on MOA analyses in published assessments to illustrate the relative extent of supporting data and their implications for dose-response analysis and involved comparisons for chemical assessments on trichloropropane, and carbon tetrachloride with several hypothesized MOA(s) for cancer. The WOE for each hypothesized MOA was summarized in narrative tables based on comparison and contrast of the extent and nature of the supporting database versus potentially inconsistent or missing information. The comparison was based on evolved Bradford Hill considerations rank ordered to reflect their relative contribution to WOE determinations of MOA taking into account increasing experience in their application internationally. This clarification of considerations for WOE determinations as a basis for comparative analysis is anticipated to contribute to increasing consistency in the application of MOA/HR analysis and potentially, transparency in separating science judgment from public policy considerations in regulatory risk assessment.

Development of a putative adverse outcome pathway network for male rat reproductive tract abnormalities with specific considerations for the androgen sensitive window of development.

Structured approaches like the adverse outcome pathway (AOP) framework offer great potential for depicting complex toxicological processes in a manner that can facilitate informed integration of mechanistic information in regulatory decisions. While this concept provides a structure for organizing evidence and facilitates consistency in evidence integration; the process, inputs, and manner in which AOPs and AOP networks are developed is still evolving. Following the OECD guiding principles of AOP development, we propose three AOPs for male reproductive tract abnormalities and derive a putative AOP network. The AOPs were developed using a fundamental understanding of the developmental biology of the organs of interest, paying close attention to the gestational timing of key events (KEs) to very specifically inform the domain of life stage applicability for the key event relationships (KERs). Chemical stressor data primarily from studies on low molecular weight phthalates (LMWPs) served to 'bound' the pathways of focus in this dynamic period of development and were integrated with the developmental biology data through an iterative process to define KEs and conclude on the extent of evidence in support of the KERs. The AOPs developed describe the linkage between 1) a decrease in Insl3 gene expression and cryptorchidism, 2) the sustained expression of Coup-tfII and hypospadias and 3) the sustained expression of Coup-tfII and altered Wolffian duct development/ epididymal agenesis. A putative AOP network linking AOP2 and AOP3 through decreased steroidogenic biosynthetic protein expression and converging of all AOPS at the population level impaired fertility adverse outcome is proposed. The network depiction specifies and displays the KEs aligned with their occurrence in gestational time. The pathways and network described herein are intended to catalyze collaborative initiatives for expansion into a larger network to enable effective data collection and inform alternative approaches for identifying stressors impacting this sensitive period of male reproductive tract development.

The AF4-mimetic peptide, PFWT, induces necrotic cell death in MV4-11 leukemia cells.

Despite ongoing success in the treatment of childhood acute lymphoblastic leukemia, patients harboring translocations involving the MLL gene at chromosome 11q23 remain resistant to treatment. To improve outcomes, novel therapeutics designed to target the unusual biology of these leukemias need to be developed. Previously, we identified an interaction between the two most common MLL fusion proteins, AF4 and AF9, and designed a synthetic peptide (PFWT) capable of disrupting this interaction. PFWT induced cell death in leukemia cells expressing MLL-AF4 with little effect on the colony forming potential of hematopoietic progenitor cells, suggesting the AF4-AF9 complex is an important pharmacological target for leukemia therapy and PFWT is a promising chemotherapeutic prototype. In these studies, we demonstrate that PFWT induces death by necrosis in MV4-11 cells. Cell death is characterized by rapid loss of plasma membrane integrity with maintenance of nuclear membrane integrity, and is independent of caspase activation, DNA fragmentation, and mitochondrial membrane depolarization. PFWT-mediated necrosis is inhibited by the serine protease inhibitor TLCK, suggesting this death pathway is regulated. Given the resistance of t(4;11) leukemias to conventional chemotherapeutic agents that induce apoptosis, further identification of the molecular events mediating this death process should uncover new avenues for therapeutic intervention.

The bone marrow niche, stem cells, and leukemia: impact of drugs, chemicals, and the environment.

Hematopoietic stem cells (HSCs) are a unique population of somatic stem cells that can both self-renew for long-term reconstitution of HSCs and differentiate into hematopoietic progenitor cells (HPCs), which in turn give rise, in a hierarchical manner, to the entire myeloid and lymphoid lineages. The differentiation and maturation of these lineages occurs in the bone marrow (BM) niche, a microenvironment that regulates self-renewal, survival, differentiation, and proliferation, with interactions among signaling pathways in the HSCs and the niche required to establish and maintain homeostasis. The accumulation of genetic mutations and cytogenetic abnormalities within cells of the partially differentiated myeloid lineage, particularly as a result of exposure to benzene or cytotoxic anticancer drugs, can give rise to malignancies like acute myeloid leukemia and myelodysplastic syndrome. Better understanding of the mechanisms driving these malignancies and susceptibility factors, both within HPCs and cells within the BM niche, may lead to the development of strategies for prevention of occupational and cancer therapy-induced disease.

Epigallocatechin gallate inhibits aryl hydrocarbon receptor gene transcription through an indirect mechanism involving binding to a 90 kDa heat shock protein.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known to mediate the toxic effects of numerous environmental contaminants, including the polycyclic aromatic hydrocarbons (PAHs). Historically, binding of PAHs to the AhR and the events leading to the generation of DNA adducts have been associated with chemical carcinogenesis. Previous investigations have implicated green tea (GT) as affording protection against PAH-induced cancers in animal models. Investigations in our laboratory have demonstrated that the GT polyphenol epigallocatechin gallate (EGCG) is capable of antagonizing AhR-mediated gene transcription, implicating inhibition of AhR signaling as a potential chemopreventive mechanism. This line of investigation was directed at elucidating the molecular mechanism of this antagonism. Competitive binding assays strongly suggest that EGCG does not bind to the AhR ligand binding site, indicating this compound functions through a mechanism unlike that of typical AhR antagonists. Affinity chromatography experiments implicate an indirect mechanism of action involving direct binding of EGCG to the AhR chaperone protein, hsp90. This induces an AhR conformation capable of nuclear localization but incapable of binding DNA. These altered signaling events correlate with the formation of a complex with sedimentation characteristics different from those of the latent or ligand-activated AhR. These data implicate a model in which EGCG inhibits release of hsp90 from the AhR, stabilizing the complex in an intermediary state associated with XAP2. This is the first time EGCG has been demonstrated to directly bind hsp90 and the first indication that GT may exert its chemopreventive effects through an interaction with the common chaperone hsp90.

Species-specific transcriptional activity of synthetic flavonoids in guinea pig and mouse cells as a result of differential activation of the aryl hydrocarbon receptor to interact with dioxin-responsive elements.

To investigate possible species-specificity of aryl hydrocarbon receptor (AhR)-mediated signal transduction pathways, activities of 2,3,7,8-tetrochlorodibenzo-p-dioxin (TCDD) and six synthetic flavonoids were evaluated in mouse hepatoma and guinea pig adenocarcinoma cells transfected with an AhR-responsive luciferase reporter. Rank order potency in these two cell lines was similar for the ability of these flavonoids to antagonize TCDD-induced reporter gene expression. However, in the presence of flavone alone, a species-specific difference in agonist activity was observed. In guinea pig cells, several flavonoids demonstrated agonist activity up to 50% of the maximum TCDD response. In mouse cells, however, no significant agonist activity was observed at the same concentrations based on luciferase enzyme activity, protein expression, and mRNA analysis. Moreover, competitive ligand-binding assays, using [(3)H]TCDD in cytosolic fractions, demonstrated that 3'-methoxy-4'-nitroflavone had a similar IC(50) in both recombinant cell lines, suggesting that the flavone has similar binding affinity to receptors from both species. However, electrophoretic mobility shift assay using the cytosolic fractions demonstrated that this flavone elicited binding to the DRE by guinea pig but not mouse AhR complex. The dependence of the AhR in this differential interaction was further demonstrated using in vitro synthesized guinea pig and mouse Ah receptors and mouse Arnt. Together, these data suggest that the differential agonist/antagonist activity of these flavone derivatives is caused by the efficacy of these flavonoids in eliciting an AhR conformation that recognizes regulatory response elements in a species-specific manner.