Mitochondrial complex I promotes kidney cancer metastasis.

Most kidney cancers are metabolically dysfunctional1-4, but how this dysfunction affects cancer progression in humans is unknown. We infused 13C-labelled nutrients in over 80 patients with kidney cancer during surgical tumour resection. Labelling from [U-13C]glucose varies across subtypes, indicating that the kidney environment alone cannot account for all tumour metabolic reprogramming. Compared with the adjacent kidney, clear cell renal cell carcinomas (ccRCCs) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in ex vivo organotypic cultures, indicating that suppressed labelling is tissue intrinsic. [1,2-13C]acetate and [U-13C]glutamine infusions in patients, coupled with measurements of respiration in isolated human kidney and tumour mitochondria, reveal lower electron transport chain activity in ccRCCs that contributes to decreased oxidative and enhanced reductive TCA cycle labelling. However, ccRCC metastases unexpectedly have enhanced TCA cycle labelling compared with that of primary ccRCCs, indicating a divergent metabolic program during metastasis in patients. In mice, stimulating respiration or NADH recycling in kidney cancer cells is sufficient to promote metastasis, whereas inhibiting electron transport chain complex I decreases metastasis. These findings in humans and mice indicate that metabolic properties and liabilities evolve during kidney cancer progression, and that mitochondrial function is limiting for metastasis but not growth at the original site.

Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation.

Asthma resulting from sensitisation of the respiratory tract to chemicals is an important occupational health issue, presenting many toxicological challenges. Most importantly there are no recognised predictive methods for respiratory allergens. Nevertheless, it has been found that all known chemical respiratory allergens elicit positive responses in assays for skin sensitising chemicals. Thus, chemicals failing to induce a positive response in skin sensitisation assays such as the local lymph node assay (LLNA) lack not only skin sensitising activity, but also the potential to cause respiratory sensitisation. However, it is unclear whether it will be possible to regard chemicals that are negative in in vitro skin sensitisation tests also as lacking respiratory sensitising activity. To address this, the behaviour of chemical respiratory allergens in the LLNA and in recently validated non-animal tests for skin sensitisation have been examined. Most chemical respiratory allergens are positive in one or more newly validated non-animal test methods, although the situation varies between individual assays. The use of an integrated testing strategy could provide a basis for recognition of most respiratory sensitising chemicals. However, a more complete picture of the performance characteristics of such tests is required before specific recommendations can be made.

The challenge of the application of 'omics technologies in chemicals risk assessment: Background and outlook.

This survey by the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) highlights that 'omics technologies are generally not yet applied to meet standard information requirements during regulatory hazard assessment. While they are used within weight-of-evidence approaches to investigate substances' modes-of-action, consistent approaches for the generation, processing and interpretation of 'omics data are not applied. To date, no 'omics technology has been standardised or validated. Best practices for performing 'omics studies for regulatory purposes (e.g., microarrays for transcriptome profiling) remain to be established. Therefore, three frameworks for (i) establishing a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) 'omics data processing; and (iii) quantitative WoE approaches to interpret 'omics data have been developed, that are presented in this journal supplement. Application of the frameworks will enable between-study comparison of results, which will facilitate the regulatory applicability of 'omics data. The frameworks do not constitute prescriptive protocols precluding any other data analysis method, but provide a baseline for analysis that can be applied to all data allowing ready cross-comparison. Data analysis that does not follow the frameworks can be justified and the resulting data can be compared with the Framework-based common analysis output.

Framework for the quantitative weight-of-evidence analysis of 'omics data for regulatory purposes.

A framework for the quantitative weight-of-evidence (QWoE) analysis of 'omics data for regulatory purposes is presented. The QWoE framework encompasses seven steps to evaluate 'omics data (also together with non-'omics data): (1) Hypothesis formulation, identification and weighting of lines of evidence (LoEs). LoEs conjoin different (types of) studies that are used to critically test the hypothesis. As an essential component of the QWoE framework, step 1 includes the development of templates for scoring sheets that predefine scoring criteria with scores of 0-4 to enable a quantitative determination of study quality and data relevance; (2) literature searches and categorisation of studies into the pre-defined LoEs; (3) and (4) quantitative assessment of study quality and data relevance using the respective pre-defined scoring sheets for each study; (5) evaluation of LoE-specific strength of evidence based upon the study quality and study relevance scores of the studies conjoined in the respective LoE; (6) integration of the strength of evidence from the individual LoEs to determine the overall strength of evidence; (7) characterisation of uncertainties and conclusion on the QWoE. To put the QWoE framework in practice, case studies are recommended to confirm the relevance of its different steps, or to adapt them as necessary.

A generic Transcriptomics Reporting Framework (TRF) for 'omics data processing and analysis.

A generic Transcriptomics Reporting Framework (TRF) is presented that lists parameters that should be reported in 'omics studies used in a regulatory context. The TRF encompasses the processes from transcriptome profiling from data generation to a processed list of differentially expressed genes (DEGs) ready for interpretation. Included within the TRF is a reference baseline analysis (RBA) that encompasses raw data selection; data normalisation; recognition of outliers; and statistical analysis. The TRF itself does not dictate the methodology for data processing, but deals with what should be reported. Its principles are also applicable to sequencing data and other 'omics. In contrast, the RBA specifies a simple data processing and analysis methodology that is designed to provide a comparison point for other approaches and is exemplified here by a case study. By providing transparency on the steps applied during 'omics data processing and analysis, the TRF will increase confidence processing of 'omics data, and regulatory use. Applicability of the TRF is ensured by its simplicity and generality. The TRF can be applied to all types of regulatory 'omics studies, and it can be executed using different commonly available software tools.

Framework for the quality assurance of 'omics technologies considering GLP requirements.

'Omics technologies are gaining importance to support regulatory toxicity studies. Prerequisites for performing 'omics studies considering GLP principles were discussed at the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) Workshop Applying 'omics technologies in Chemical Risk Assessment. A GLP environment comprises a standard operating procedure system, proper pre-planning and documentation, and inspections of independent quality assurance staff. To prevent uncontrolled data changes, the raw data obtained in the respective 'omics data recording systems have to be specifically defined. Further requirements include transparent and reproducible data processing steps, and safe data storage and archiving procedures. The software for data recording and processing should be validated, and data changes should be traceable or disabled. GLP-compliant quality assurance of 'omics technologies appears feasible for many GLP requirements. However, challenges include (i) defining, storing, and archiving the raw data; (ii) transparent descriptions of data processing steps; (iii) software validation; and (iv) ensuring complete reproducibility of final results with respect to raw data. Nevertheless, 'omics studies can be supported by quality measures (e.g., GLP principles) to ensure quality control, reproducibility and traceability of experiments. This enables regulators to use 'omics data in a fit-for-purpose context, which enhances their applicability for risk assessment.

Applying 'omics technologies in chemicals risk assessment: Report of an ECETOC workshop.

Prevailing knowledge gaps in linking specific molecular changes to apical outcomes and methodological uncertainties in the generation, storage, processing, and interpretation of 'omics data limit the application of 'omics technologies in regulatory toxicology. Against this background, the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a workshop Applying 'omics technologies in chemicals risk assessment that is reported herein. Ahead of the workshop, multi-expert teams drafted frameworks on best practices for (i) a Good-Laboratory Practice-like context for collecting, storing and curating 'omics data; (ii) the processing of 'omics data; and (iii) weight-of-evidence approaches for integrating 'omics data. The workshop participants confirmed the relevance of these Frameworks to facilitate the regulatory applicability and use of 'omics data, and the workshop discussions provided input for their further elaboration. Additionally, the key objective (iv) to establish approaches to connect 'omics perturbations to phenotypic alterations was addressed. Generally, it was considered promising to strive to link gene expression changes and pathway perturbations to the phenotype by mapping them to specific adverse outcome pathways. While further work is necessary before gene expression changes can be used to establish safe levels of substance exposure, the ECETOC workshop provided important incentives towards achieving this goal.

Guidance on the selection of cohorts for the extended one-generation reproduction toxicity study (OECD test guideline 443).

The extended one-generation reproduction toxicity study (EOGRTS; OECD test guideline 433) is a new and technically complex design to evaluate the putative effects of chemicals on fertility and development, including effects upon the developing nervous and immune systems. In addition to offering a more comprehensive assessment of developmental toxicity, the EOGRTS offers important improvements in animal welfare through reduction and refinement in a modular study design. The challenge to the practitioner is to know how the modular aspects of the study should be triggered on the basis of prior knowledge of a particular chemical, or on earlier findings in the EOGRTS itself, requirements of specific regulatory frameworks notwithstanding. The purpose of this document is to offer guidance on science-based triggers for these extended evaluations.

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop.

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context.

Antithrombin activity and central venous catheter-associated thrombosis in critically ill children at high risk of bleeding.

BACKGROUND: Normalization of antithrombin activity may prevent catheter-associated thrombosis in critically ill children at high risk of bleeding. OBJECTIVES: To characterize the temporal pattern of antithrombin activity, assess its association with catheter-associated thrombosis and clinically relevant bleeding, and evaluate its relationship with thrombin generation in these children. METHODS: In this prospective cohort study, critically ill children <18 years old at high risk of bleeding with central venous catheter were eligible. Antithrombin activity and thrombin generation were measured from platelet-poor plasma and after in vitro antithrombin supplementation. Systematic surveillance ultrasound was performed to diagnose thrombosis. Children were followed for bleeding. RESULTS: We enrolled 8 infants (median age: 0.2 years, IQR: 0.2, 0.3 years) and 72 older children (median age: 14.3 years, IQR: 9.1, 16.1 years). Mean antithrombin on the day of catheter insertion was 64 IU/dL (SD: 32 IU/dL) in infants and 83 IU/dL (SD: 35 IU/dL) in older children. Antithrombin normalized by the day of catheter removal. Thrombosis developed in 27 children, while 31 children bled. Thrombosis (regression coefficient: 0.008, 95% CI: -0.01, 0.03) and bleeding (regression coefficient: -0.0007, 95% CI: -0.02, 0.02) were not associated with antithrombin. Antithrombin was not correlated with in vivo change in endogenous thrombin potential (correlation coefficient: -0.07, 95% CI: -0.21, 0.08). In vitro supplementation reduced endogenous thrombin potential (correlation coefficient: -0.78; 95% CI: -0.95, -0.23). CONCLUSION: These findings may not support normalization of antithrombin activity to prevent catheter-associated thrombosis in critically ill children at high risk of bleeding.

Mitochondrial metabolism in primary and metastatic human kidney cancers.

Most kidney cancers display evidence of metabolic dysfunction1-4 but how this relates to cancer progression in humans is unknown. We used a multidisciplinary approach to infuse 13C-labeled nutrients during surgical tumour resection in over 70 patients with kidney cancer. Labeling from [U-13C]glucose varies across cancer subtypes, indicating that the kidney environment alone cannot account for all metabolic reprogramming in these tumours. Compared to the adjacent kidney, clear cell renal cell carcinomas (ccRCC) display suppressed labelling of tricarboxylic acid (TCA) cycle intermediates in vivo and in organotypic slices cultured ex vivo, indicating that suppressed labeling is tissue intrinsic. Infusions of [1,2-13C]acetate and [U-13C]glutamine in patients, coupled with respiratory flux of mitochondria isolated from kidney and tumour tissue, reveal primary defects in mitochondrial function in human ccRCC. However, ccRCC metastases unexpectedly have enhanced labeling of TCA cycle intermediates compared to primary ccRCCs, indicating a divergent metabolic program during ccRCC metastasis in patients. In mice, stimulating respiration in ccRCC cells is sufficient to promote metastatic colonization. Altogether, these findings indicate that metabolic properties evolve during human kidney cancer progression, and suggest that mitochondrial respiration may be limiting for ccRCC metastasis but not for ccRCC growth at the site of origin.

Diiodomethyl-p-tolylsulfone: evaluation of the mode of action for reproductive toxicity.

The mode of action (MOA) underpinning the reproductive toxicity of diiodomethyl-p-tolylsulfone (DIMPTS) is excess systemic iodine levels, resulting in hypothyroidism. This MOA evaluation also addresses the potential for toxicity and adverse health outcomes during critical windows of development for different tissues. The data indicate that testicular development in the neonate represents the tissue and life-stage that are most sensitive to iodine toxicity. Life-stage specific dosimetry appears to be a major determinant of this sensitivity, with the neonate being exposed to higher levels of iodine than the fetus during the period of testicular development, in particular Sertoli cell maturation and differentiation. While no reports could be found in the literature linking excess iodine exposure in humans to testicular toxicity, there is evidence that neonates born to mothers with excessive iodine intake do exhibit signs of transient hypothyroidism. Although there are major physiological and temporal differences in testicular development and Sertoli cell replication between the rat and human, it is not inconceivable that continuous long term exposures to excess iodine first from maternal milk and then in the diet through to the onset of puberty could affect testicular development. However, exposures to iodinated substances - such as DIMPTS - contribute less than 1% of the required daily iodine intake for normal fetal and neonatal development and, consequently, continuous exposure to excess iodine during the pre-pubertal period is unlikely. As exposures to DIMPTS are both very low and sporadic in nature it is not likely that they represent any risk to health at any life-stage.

Risk assessment for consumer exposure to toluene diisocyanate (TDI) derived from polyurethane flexible foam.

Polyurethanes (PU) are polymers made from diisocyanates and polyols for a variety of consumer products. It has been suggested that PU foam may contain trace amounts of residual toluene diisocyanate (TDI) monomers and present a health risk. To address this concern, the exposure scenario and health risks posed by sleeping on a PU foam mattress were evaluated. Toxicity benchmarks for key non-cancer endpoints (i.e., irritation, sensitization, respiratory tract effects) were determined by dividing points of departure by uncertainty factors. The cancer benchmark was derived using the USEPA Benchmark Dose Software. Results of previous migration and emission data of TDI from PU foam were combined with conservative exposure factors to calculate upper-bound dermal and inhalation exposures to TDI as well as a lifetime average daily dose to TDI from dermal exposure. For each non-cancer endpoint, the toxicity benchmark was divided by the calculated exposure to determine the margin of safety (MOS), which ranged from 200 (respiratory tract) to 3×10(6) (irritation). Although available data indicate TDI is not carcinogenic, a theoretical excess cancer risk (1×10(-7)) was calculated. We conclude from this assessment that sleeping on a PU foam mattress does not pose TDI-related health risks to consumers.

Knowledge-based potentials in protein design.

Knowledge-based potentials are statistical parameters derived from databases of known protein properties that empirically capture aspects of the physical chemistry of protein structure and function. These potentials play a key role in protein design by improving the accuracy of physics-based models of interatomic interactions and enhancing the computational efficiency of the design process by limiting the complexity of searching sequence space. Recently, knowledge-based potentials (in isolation or in combination with physics-based potentials) have been applied to the modification of existing protein function, the redesign of natural protein folds and the complete design of a non-natural protein fold. In addition, knowledge-based potentials appear to be providing important information about the global topology of amino acid interactions in natural proteins. A detailed study of the methods and products of these protein design efforts promises to greatly expand our understanding of proteins and the evolutionary process that created them.

Skin and respiratory chemical allergy: confluence and divergence in a hybrid adverse outcome pathway.

Sensitisation of the respiratory tract to chemicals resulting in respiratory allergy and allergic asthma is an important occupational health problem, and presents toxicologists with no shortage of challenges. A major issue is that there are no validated or, even widely recognised, methods available for the identification and characterisation of chemical respiratory allergens, or for distinguishing respiratory allergens from contact allergens. The first objective here has been review what is known (and what is not known) of the mechanisms through which chemicals induce sensitisation of the respiratory tract, and to use this information to construct a hybrid Adverse Outcome Pathway (AOP) that combines consideration of both skin and respiratory sensitisation. The intention then has been to use the construction of this hybrid AOP to identify areas of commonality/confluence, and areas of departure/divergence, between skin sensitisation and sensitisation of the respiratory tract. The hybrid AOP not only provides a mechanistic understanding of how the processes of skin and respiratory sensitisation differ, buy also a means of identifying areas of uncertainty about chemical respiratory allergy that benefit from a further investment in research.

Building a developmental toxicity ontology.

BACKGROUND: As more information is generated about modes of action for developmental toxicity and more data are generated using high-throughput and high-content technologies, it is becoming necessary to organize that information. This report discussed the need for a systematic representation of knowledge about developmental toxicity (i.e., an ontology) and proposes a method to build one based on knowledge of developmental biology and mode of action/ adverse outcome pathways in developmental toxicity. METHODS: This report is the result of a consensus working group developing a plan to create an ontology for developmental toxicity that spans multiple levels of biological organization. RESULTS: This report provide a description of some of the challenges in building a developmental toxicity ontology and outlines a proposed methodology to meet those challenges. As the ontology is built on currently available web-based resources, a review of these resources is provided. Case studies on one of the most well-understood morphogens and developmental toxicants, retinoic acid, are presented as examples of how such an ontology might be developed. DISCUSSION: This report outlines an approach to construct a developmental toxicity ontology. Such an ontology will facilitate computer-based prediction of substances likely to induce human developmental toxicity.

Thresholds in chemical respiratory sensitisation.

There is a continuing interest in determining whether it is possible to identify thresholds for chemical allergy. Here allergic sensitisation of the respiratory tract by chemicals is considered in this context. This is an important occupational health problem, being associated with rhinitis and asthma, and in addition provides toxicologists and risk assessors with a number of challenges. In common with all forms of allergic disease chemical respiratory allergy develops in two phases. In the first (induction) phase exposure to a chemical allergen (by an appropriate route of exposure) causes immunological priming and sensitisation of the respiratory tract. The second (elicitation) phase is triggered if a sensitised subject is exposed subsequently to the same chemical allergen via inhalation. A secondary immune response will be provoked in the respiratory tract resulting in inflammation and the signs and symptoms of a respiratory hypersensitivity reaction. In this article attention has focused on the identification of threshold values during the acquisition of sensitisation. Current mechanistic understanding of allergy is such that it can be assumed that the development of sensitisation (and also the elicitation of an allergic reaction) is a threshold phenomenon; there will be levels of exposure below which sensitisation will not be acquired. That is, all immune responses, including allergic sensitisation, have threshold requirement for the availability of antigen/allergen, below which a response will fail to develop. The issue addressed here is whether there are methods available or clinical/epidemiological data that permit the identification of such thresholds. This document reviews briefly relevant human studies of occupational asthma, and experimental models that have been developed (or are being developed) for the identification and characterisation of chemical respiratory allergens. The main conclusion drawn is that although there is evidence that the acquisition of sensitisation to chemical respiratory allergens is a dose-related phenomenon, and that thresholds exist, it is frequently difficult to define accurate numerical values for threshold exposure levels. Nevertheless, based on occupational exposure data it may sometimes be possible to derive levels of exposure in the workplace, which are safe. An additional observation is the lack currently of suitable experimental methods for both routine hazard characterisation and the measurement of thresholds, and that such methods are still some way off. Given the current trajectory of toxicology, and the move towards the use of non-animal in vitro and/or in silico) methods, there is a need to consider the development of alternative approaches for the identification and characterisation of respiratory sensitisation hazards, and for risk assessment.

Brood reduction in temperate and sub-tropical ospreys.

In an effort to understand patterns and causes of nestling loss in Ospreys (Pandion haliaetus), I studied brood reduction in three eastern U.S. Osprey colonies during 1978 and 1979. The colonies, located in Florida Bay (1) and on coastal Long Island, N.Y. (2), differed in the average daily amount of food delivered to nestlings; Florida nests received 43% and 11% less fish per day than nests in the two N.Y. colonies, largely because latitude and season restricted day length and thus foraging time for the winter-breeding Florida Ospreys. Increased distance from stable food sources accounted for the lower rate of feeding at one of the N.Y. colonies. Variation in clutch size in the three colonies reflected differences in latitude more than in food availability; average clutch sizes in Long Island were larger than Florida clutches by 0.5 of an egg, but were similar to each other and to those in other northeastern U.S. Osprey populations.Increased nestling loss coincided with reduced food delivery rates and, in food stressed colonies, this loss was 2-3 times greater than any recorded for Ospreys. Starvation was the primary cause of nestling death, with mortality concentrated on third chicks, which hatched on average 3.9 d later and from eggs 5.6% smaller than chicks hatching first. Sibling aggression accounted for the preferential feeding of older nestmates,but only in colonies or nests where food was limited. Aggressive chicks nearly always stopped fighting after being fed. This behavior provided a reversible mechanism for controling brood reduction that was based on nutrition. Growth rates of young measured during the first half of the growth period were more variable between colonies than within nests. This is interpreted as reflecting both the differences in colony food delivery rates as well as the evolutionary pressures of sibling competition to equalize the growth of nestmates.

Fine-needle aspiration cytology in the management of salivary gland tumors: an Australian experience.

We conducted a retrospective study of 104 cases of salivary gland tumors that were initially assessed by fine-needle aspiration biopsy (FNAB). Based on subsequent histopathologic analysis of excised specimens, we found that preoperative FNAB was highly sensitive and specific for both benign and malignant tumors-including the most common, pleomorphic adenomas and squamous cell carcinomas, respectively. Despite its possible drawbacks, we conclude that preoperative FNAB is a useful tool in the management of salivary gland tumors.