Cellular quality control during gametogenesis.

A hallmark of aging is the progressive accumulation of cellular damage. Age-induced damage arises due to a decrease in organelle function along with a decline in protein quality control. Although somatic tissues deteriorate with age, the germline must maintain cellular homeostasis in order to ensure the production of healthy progeny. While germline quality control has been primarily studied in multicellular organisms, recent evidence suggests the existence of gametogenesis-specific quality control mechanisms in unicellular eukaryotes, highlighting the evolutionary conservation of meiotic events beyond chromosome morphogenesis. Notably, budding yeast eliminates age-induced damage during meiotic differentiation, employing novel organelle and protein quality control mechanisms to produce young and healthy gametes. Similarly, organelle and protein quality control is present in metazoan gametogenesis; however, whether and how these mechanisms contribute to cellular rejuvenation requires further investigation. Here, we summarize recent findings that describe organelle and protein quality control in budding yeast gametogenesis, examine similar quality control mechanisms in metazoan development, and identify research directions that will improve our understanding of meiotic cellular rejuvenation.

Categorizing the characteristics of human carcinogens: a need for specificity.

The International Agency for Research on Cancer (IARC) has recently proposed employing "ten key characteristics of human carcinogens" (TKCs) to determine the potential of agents for harmful effects. The TKCs seem likely to confuse the unsatisfactory correlation from testing regimes that have ignored the differences evident when cellular changes are compared in short and long-lived species, with their very different stem cell and somatic cell phylogenies. The proposed characteristics are so broad that their use will lead to an increase in the current unacceptably high rate of false positives. It could be an informative experiment to take well-established approved therapeutics with well-known human safety profiles and test them against this new TKC paradigm. Cancers are initiated and driven by heritable and transient changes in gene expression, expand clonally, and progress via additional associated acquired mutations and epigenetic alterations that provide cells with an evolutionary advantage. The genotoxicity testing protocols currently employed and required by regulation, emphasize testing for the mutational potential of the test agent. Two-year, chronic rodent cancer bioassays are intended to test for the entire spectrum of carcinogenic transformation. The use of cytotoxic doses causing increased, sustained cell proliferation that facilitates accumulated genetic damage leads to a high false-positive rate of tumor induction. Current cancer hazard assessment protocols and weight-of-the-evidence analysis of agent-specific cancer risk align poorly with the pathogenesis of human carcinoma and so need modernization and improvement in ways suggested here.

sNASP and ASF1A function through both competitive and compatible modes of histone binding.

Histone chaperones are proteins that interact with histones to regulate the thermodynamic process of nucleosome assembly. sNASP and ASF1 are conserved histone chaperones that interact with histones H3 and H4 and are found in a multi-chaperoning complex in vivo Previously we identified a short peptide motif within H3 that binds to the TPR domain of sNASP with nanomolar affinity. Interestingly, this peptide motif is sequestered within the known ASF1-H3-H4 interface, raising the question of how these two proteins are found in complex together with histones when they share the same binding site. Here, we show that sNASP contains at least two additional histone interaction sites that, unlike the TPR-H3 peptide interaction, are compatible with ASF1A binding. These surfaces allow ASF1A to form a quaternary complex with both sNASP and H3-H4. Furthermore, we demonstrate that sNASP makes a specific complex with H3 on its own in vitro, but not with H4, suggesting that it could work upstream of ASF1A. Further, we show that sNASP and ASF1A are capable of folding an H3-H4 dimer in vitro under native conditions. These findings reveal a network of binding events that may promote the entry of histones H3 and H4 into the nucleosome assembly pathway.

Human relevance of rodent liver tumors: Key insights from a Toxicology Forum workshop on nongenotoxic modes of action.

The Toxicology Forum sponsored a workshop in October 2016, on the human relevance of rodent liver tumors occurring via nongenotoxic modes of action (MOAs). The workshop focused on two nuclear receptor-mediated MOAs (Constitutive Androstane Receptor (CAR) and Peroxisome Proliferator Activated Receptor-alpha (PPARα), and on cytotoxicity. The goal of the meeting was to review the state of the science to (1) identify areas of consensus and differences, data gaps and research needs; (2) identify reasons for inconsistencies in current regulatory positions; and (3) consider what data are needed to demonstrate a specific MOA, and when additional research is needed to rule out alternative possibilities. Implications for quantitative risk assessment approaches were discussed, as were implications of not considering MOA and dose in hazard characterization and labeling schemes. Most, but not all, participants considered the CAR and PPARα MOAs as not relevant to humans based on quantitative and qualitative differences. In contrast, cytotoxicity is clearly relevant to humans, but a threshold applies. Questions remain for all three MOAs concerning what data are necessary to determine the MOA and to what extent it is necessary to exclude other MOAs.

Obfuscating transparency?

Several recent and prominent articles in Science and Nature deliberately mischaracterized the nature of genuine scientific evidence. Those articles take issue with the United States Environmental Protection Agency's recent proposal to structure its policies and rules only from studies with transparently published raw data. The articles claim it is an effort to obfuscate with transparency, by eliminating a host of studies not offering raw data. A remarkable declaration by a Science editorial is that properly trained experts can verify the scientific evidence of studies without access to raw data, We assert the Agency's proposal must be sustained. Transparency in reporting is a fundamental ethical imperative of objective scientific research justifying massive official regulations and policies. Putative hazards bereft of independent scientific evidence will continue to stoke public anxieties, calling for precautionary regulations and policies. These should rely not on spurious science but on transparent tradeoffs between the smallest exposures compatible with utility and with social perceptions of affordable precaution.

Origin of the TTC values for compounds that are genotoxic and/or carcinogenic and an approach for their re-evaluation.

The threshold of toxicological concern (TTC) approach is a resource-effective de minimis method for the safety assessment of chemicals, based on distributional analysis of the results of a large number of toxicological studies. It is being increasingly used to screen and prioritize substances with low exposure for which there is little or no toxicological information. The first step in the approach is the identification of substances that may be DNA-reactive mutagens, to which the lowest TTC value is applied. This TTC value was based on the analysis of the cancer potency database and involved a number of assumptions that no longer reflect the state-of-the-science and some of which were not as transparent as they could have been. Hence, review and updating of the database is proposed, using inclusion and exclusion criteria reflecting current knowledge. A strategy for the selection of appropriate substances for TTC determination, based on consideration of weight of evidence for genotoxicity and carcinogenicity is outlined. Identification of substances that are carcinogenic by a DNA-reactive mutagenic mode of action and those that clearly act by a non-genotoxic mode of action will enable the protectiveness to be determined of both the TTC for DNA-reactive mutagenicity and that applied by default to substances that may be carcinogenic but are unlikely to be DNA-reactive mutagens (i.e. for Cramer class I-III compounds). Critical to the application of the TTC approach to substances that are likely to be DNA-reactive mutagens is the reliability of the software tools used to identify such compounds. Current methods for this task are reviewed and recommendations made for their application.

A High Through-put Platform for Recombinant Antibodies to Folded Proteins.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.

Illustrative case using the RISK21 roadmap and matrix: prioritization for evaluation of chemicals found in drinking water.

The HESI-led RISK21 effort has developed a framework supporting the use of twenty-first century technology in obtaining and using information for chemical risk assessment. This framework represents a problem formulation-based, exposure-driven, tiered data acquisition approach that leads to an informed decision on human health safety to be made when sufficient evidence is available. It provides a transparent and consistent approach to evaluate information in order to maximize the ability of assessments to inform decisions and to optimize the use of resources. To demonstrate the application of the framework's roadmap and matrix, this case study evaluates a large number of chemicals that could be present in drinking water. The focus is to prioritize which of these should be considered for human health risk as individual contaminants. The example evaluates 20 potential drinking water contaminants, using the tiered RISK21 approach in combination with graphical representation of information at each step, using the RISK21 matrix. Utilizing the framework, 11 of the 20 chemicals were assigned low priority based on available exposure data alone, which demonstrated that exposure was extremely low. The remaining nine chemicals were further evaluated, using refined estimates of toxicity based on readily available data, with three deemed high priority for further evaluation. In the present case study, it was determined that the greatest value of additional information would be from improved exposure models and not from additional hazard characterization.

Computational modeling identifies key gene regulatory interactions underlying phenobarbital-mediated tumor promotion.

Gene regulatory interactions underlying the early stages of non-genotoxic carcinogenesis are poorly understood. Here, we have identified key candidate regulators of phenobarbital (PB)-mediated mouse liver tumorigenesis, a well-characterized model of non-genotoxic carcinogenesis, by applying a new computational modeling approach to a comprehensive collection of in vivo gene expression studies. We have combined our previously developed motif activity response analysis (MARA), which models gene expression patterns in terms of computationally predicted transcription factor binding sites with singular value decomposition (SVD) of the inferred motif activities, to disentangle the roles that different transcriptional regulators play in specific biological pathways of tumor promotion. Furthermore, transgenic mouse models enabled us to identify which of these regulatory activities was downstream of constitutive androstane receptor and ß-catenin signaling, both crucial components of PB-mediated liver tumorigenesis. We propose novel roles for E2F and ZFP161 in PB-mediated hepatocyte proliferation and suggest that PB-mediated suppression of ESR1 activity contributes to the development of a tumor-prone environment. Our study shows that combining MARA with SVD allows for automated identification of independent transcription regulatory programs within a complex in vivo tissue environment and provides novel mechanistic insights into PB-mediated hepatocarcinogenesis.

Mode of action and human relevance analysis for nuclear receptor-mediated liver toxicity: A case study with phenobarbital as a model constitutive androstane receptor (CAR) activator.

The constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are important nuclear receptors involved in the regulation of cellular responses from exposure to many xenobiotics and various physiological processes. Phenobarbital (PB) is a non-genotoxic indirect CAR activator, which induces cytochrome P450 (CYP) and other xenobiotic metabolizing enzymes and is known to produce liver foci/tumors in mice and rats. From literature data, a mode of action (MOA) for PB-induced rodent liver tumor formation was developed. A MOA for PXR activators was not established owing to a lack of suitable data. The key events in the PB-induced liver tumor MOA comprise activation of CAR followed by altered gene expression specific to CAR activation, increased cell proliferation, formation of altered hepatic foci and ultimately the development of liver tumors. Associative events in the MOA include altered epigenetic changes, induction of hepatic CYP2B enzymes, liver hypertrophy and decreased apoptosis; with inhibition of gap junctional intercellular communication being an associative event or modulating factor. The MOA was evaluated using the modified Bradford Hill criteria for causality and other possible MOAs were excluded. While PB produces liver tumors in rodents, important species differences were identified including a lack of cell proliferation in cultured human hepatocytes. The MOA for PB-induced rodent liver tumor formation was considered to be qualitatively not plausible for humans. This conclusion is supported by data from a number of epidemiological studies conducted in human populations chronically exposed to PB in which there is no clear evidence for increased liver tumor risk.

A proposed framework for assessing risk from less-than-lifetime exposures to carcinogens.

Quantitative methods for estimation of cancer risk have been developed for daily, lifetime human exposures. There are a variety of studies or methodologies available to address less-than-lifetime exposures. However, a common framework for evaluating risk from less-than-lifetime exposures (including short-term and/or intermittent exposures) does not exist, which could result in inconsistencies in risk assessment practice. To address this risk assessment need, a committee of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute conducted a multisector workshop in late 2009 to discuss available literature, different methodologies, and a proposed framework. The proposed framework provides a decision tree and guidance for cancer risk assessments for less-than-lifetime exposures based on current knowledge of mode of action and dose-response. Available data from rodent studies and epidemiological studies involving less-than-lifetime exposures are considered, in addition to statistical approaches described in the literature for evaluating the impact of changing the dose rate and exposure duration for exposure to carcinogens. The decision tree also provides for scenarios in which an assumption of potential carcinogenicity is appropriate (e.g., based on structural alerts or genotoxicity data), but bioassay or other data are lacking from which a chemical-specific cancer potency can be determined. This paper presents an overview of the rationale for the workshop, reviews historical background, describes the proposed framework for assessing less-than-lifetime exposures to potential human carcinogens, and suggests next steps.

Arteriovenous malformation identification after conservative management of placenta percreta with uterine artery embolization and adjunctive therapy.

Placenta percreta is a complication of pregnancy with significant morbidity and mortality rates. Conservative management may be considered when fertility preservation is desired or to possibly reduce morbidity when there is invasion of pelvic structures. We present 3 cases of antenatally diagnosed placenta percreta that were managed conservatively. A finding after the operation included the identification of arteriovenous malformations.

Meiotic cellular rejuvenation is coupled to nuclear remodeling in budding yeast.

Production of healthy gametes in meiosis relies on the quality control and proper distribution of both nuclear and cytoplasmic contents. Meiotic differentiation naturally eliminates age-induced cellular damage by an unknown mechanism. Using time-lapse fluorescence microscopy in budding yeast, we found that nuclear senescence factors - including protein aggregates, extrachromosomal ribosomal DNA circles, and abnormal nucleolar material - are sequestered away from chromosomes during meiosis II and subsequently eliminated. A similar sequestration and elimination process occurs for the core subunits of the nuclear pore complex in both young and aged cells. Nuclear envelope remodeling drives the formation of a membranous compartment containing the sequestered material. Importantly, de novo generation of plasma membrane is required for the sequestration event, preventing the inheritance of long-lived nucleoporins and senescence factors into the newly formed gametes. Our study uncovers a new mechanism of nuclear quality control and provides insight into its function in meiotic cellular rejuvenation.

Risk assessment of toxaphene and its breakdown products: time for a change?

Technical toxaphene (TT) was last used in commerce in about 1982. Any environmental exposure to toxaphene in this century is to environmentally degraded forms of toxaphene, termed weathered toxaphene. Several hundred chlorinated bornane congeners have been identified in technical toxaphene. The degradation of technical toxaphene to weathered toxaphene can result in various congener mixtures, but the primary mode of degradation is dechlorination. The U.S. Environmental Protection Agency (EPA) presently estimates the risk of exposure to toxaphene by relying upon rat and mouse toxicology studies performed on technical toxaphene. No adjustment is made for the dechlorination of toxaphene in the environment. The European Union (EU), however, has modeled toxaphene risks from eating fish with chlorinated bornane residues through a series of studies on toxaphene degraded by either ultraviolet light, or biodegradation in fish. The EU risk assessment relies upon rat liver studies in vivo and mouse in vitro studies on the inhibition of gap junction intercellular communication (GJIC). This article reviews the current state of knowledge of technical and weathered toxaphene toxicology. We discuss the various current methods and opportunities to advance the risk assessment of weathered toxaphene beyond the existing U.S. EPA assessment of technical toxaphene.

Predicting future human and environmental health challenges: the Health and Environmental Sciences Institute's scientific mapping exercise.

To predict important strategic issues in product safety during the next 10 years, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute initiated a mapping exercise to evaluate which issues are likely to be of societal, scientific, and regulatory importance to regulatory authorities, the HESI membership, and the scientific community at large. Scientists representing government, academia, and industry participated in the exercise. Societal issues identified include sensitive populations, alternative therapies, public education on the precautionary principle, obesity, and aging world populations. Scientific issues identified include cancer testing, children's health, mixtures and co-exposures, sensitive populations, idiosyncratic reactions, "omics" or bioinformatics, and environmental toxicology. Regulatory issues identified include national and regional legislation on chemical safety, exposure inputs, new technologies, transitioning new science into regulations and guidelines, conservative default factors, data quality, and sensitive populations. Because some issues were identified as important in all three areas (e.g. sensitive populations), a comprehensive approach to assessment and management is needed to ensure consideration of societal, scientific, and regulatory implications. The resulting HESI Combined Challenges Map is not intended to offer a universal description of challenges in safety assessment, nor is it intended to address, advocate, or manage the prioritized issues. Rather, the map focuses on and predicts issues likely to be central to the strategic agendas of individual companies and regulatory authorities in the developed world. Many of these issues will become increasingly important in the future in rapidly developing economies, such as India and China. The scientific mapping exercise has particular value to the toxicology community because it represents the contributions of key scientists from around the world from government, academia, and industry.

Correction: Goodbye to the bioassay.

[This corrects the article DOI: 10.1039/C8TX00004B.].