Search

Engineered carbon nanotubes (CNT) are currently used in many industrial products. Multi-walled carbon nanotubes (MWCNT) exhibit unique physicochemical properties and due to their low density and small size are easily aerosolized, making workplace exposure a potential risk to human health. MWCNT are similar to Asbestos in terms of their high aspect-ratio and thus may pose an asbestos-like inhalation hazard, including sporadic pleural Mesothelioma. Mitsui-7 MWCNT (MWCNT-7) have been classified by IARC as a Group 2B carcinogen. Importantly, physicochemically-altered MWCNT synthesized with nitrogen (MWCNT-ND) have been found to elicit attenuated toxicity; however, their pathogenicity in the pleura is unknown. Using a mouse model of direct injection into the pleural cavity, we compared the molecular changes that occur at the mesothelium over 20-months following injection with an occupationally-relevant dose of MWCNT-7, MWCNT-ND, Asbestos or carbon black. When compared to Asbestos or MWCNT-7, mice injected with MWCNT-ND did not develop respiratory symptoms. In contrast, in cell lines established from pleural effusions from MWCNT-7 or Asbestos-exposed mice with pre-neoplastic lesions or tumours, Whole-Exome-Sequencing and RNASeq revealed common molecular changes in both lesions and tumours. This identified genes involved in driving progression of pleural inflammatory lesions to Mesothelioma, whereas pre-neoplastic lesion-derived cell lines allow dissection of the hazard mechanism of pathogenic fibres at the molecular level early in disease development and thus may help predict genotoxic potency in exposed human populations. Our data demonstrate that physicochemical alteration of MWCNT (e.g. synthesis with nitrogen) attenuates pathogenicity and may offer an alternative for use in industrial applications.

Analysis of wind turbine blades using beam or shell models presents difficulties in accurately capturing the torsional stiffness and local 3D stress fields. Instead, modeling torsional effects accurately often necessitates three-dimensional analysis as achieved with solid elements in finite element analysis. The use of solid elements and complex local mesh refinement algorithms are often required to capture the three-dimensional stress fields in critical regions, which results in systems with a large number of degrees of freedom. The present work proposes using variable kinematics finite elements to analyze wind turbine blades. Variable kinematic elements use a higher-order shape function to represent the displacement field in an element, enabling a more refined kinematic description of displacements. Previous works have shown that higher-order elements with variable kinematics can obtain accurate 3D stress fields with fewer degrees of freedom than conventional solid models. Using p-refinement furthermore allows for local refinement without requiring remeshing. By allowing the kinematics to be directional, the accuracy and degrees of freedom can be tailored to be closely related to the structure.

Exposure to asbestos causes pathological changes in the pleural cavity, including malignant mesothelioma (MPM). Length-dependent retention of asbestos fibres in the pleural cavity is crucial for disease development. Chronic inflammation induced by biopersistent asbestos fibres plays a key role in carcinogenesis. Engineered CarbonNanoTubes (CNT) are similar to long-fibre asbestos (LFA) in terms of their high aspect-ratio and thus may pose an asbestos-like inhalation hazard, with evidence from our laboratory and others showing the carcinogenic potential of Long-CNT. Asbestos-induced MPM is diagnosed most commonly at late stage; as a result, little is known about the genetic or epigenetic aberrations occurring in pre-malignant pleural lesions and whether they align with deletions/mutations commonly found in end-stage asbestos-exposed patient tumours. Using a mouse model of direct injection into the pleural cavity, we compared the molecular changes that occur at the mesothelium following injection with an occupationally-relevant dose of Long-CNT or LFA over 20-months. We show a common molecular signature induced by Long-CNT and LFA throughout disease progression eventually leading to the development of sporadic MPM. DNA methylation, gene expression profiles, as well as specific genes are similarly altered in the presence of Long-CNT and LFA, at matched longitudinal exposure times. For the first time, these data uncover the Adverse Outcome Pathway (AOP) from initial Long-CNT exposure through to induction of pre-neoplastic lesions and the development of sporadic pleural mesothelioma, replicating the pathogenesis of end-stage asbestos-induced human disease. This reinforces concerns that high aspect-ratio CNT may pose an asbestos-like hazard, including sporadic malignant mesothelioma.

The accessibility of cell surface proteins makes them tractable for targeting by cancer immunotherapy, but identifying suitable targets remains challenging. Here we describe plasma membrane profiling of primary human myeloma cells to identify an unprecedented number of cell surface proteins of a primary cancer. We used a novel approach to prioritize immunotherapy targets and identified a cell surface protein not previously implicated in myeloma, semaphorin-4A (SEMA4A). Using knock-down by short-hairpin RNA and CRISPR/nuclease-dead Cas9 (dCas9), we show that expression of SEMA4A is essential for normal myeloma cell growth in vitro, indicating that myeloma cells cannot downregulate the protein to avoid detection. We further show that SEMA4A would not be identified as a myeloma therapeutic target by standard CRISPR/Cas9 knockout screens because of exon skipping. Finally, we potently and selectively targeted SEMA4A with a novel antibody–drug conjugate in vitro and in vivo.

Background The development and adoption of health care common data models (CDMs) has addressed some of the logistical challenges of performing research on data generated from disparate health care systems by standardizing data representations and leveraging standardized terminology to express clinical information consistently. However, transforming a data system into a CDM is not a trivial task, and maintaining an operational, enterprise capable CDM that is incrementally updated within a data warehouse is challenging. Objectives To develop a quality assurance (QA) process and code base to accompany our incremental transformation of the Department of Veterans Affairs Corporate Data Warehouse health care database into the Observational Medical Outcomes Partnership (OMOP) CDM to prevent incremental load errors. Methods We designed and implemented a multistage QA) approach centered on completeness, value conformance, and relational conformance data-quality elements. For each element we describe key incremental load challenges, our extract, transform, and load (ETL) solution of data to overcome those challenges, and potential impacts of incremental load failure. Results Completeness and value conformance data-quality elements are most affected by incremental changes to the CDW, while updates to source identifiers impact relational conformance. ETL failures surrounding these elements lead to incomplete and inaccurate capture of clinical concepts as well as data fragmentation across patients, providers, and locations. Conclusion Development of robust QA processes supporting accurate transformation of OMOP and other CDMs from source data is still in evolution, and opportunities exist to extend the existing QA framework and tools used for incremental ETL QA processes.

The increasing reliance of aerospace structures on numerical analyses encourages the development of accurate, yet computationally efficient, models. Finite element (FE) beam models have, in particular, become widely used approximations during preliminary design stages and to investigate novel concepts, for example, aeroelastic tailoring. Over the last 50 years, developments in hp-FE methods based on elements of variable size (h) and polynomial degree (p) have helped reduce the computational cost of numerical analyses. Concurrently, many structures, including aircraft wings and wind turbine blades, have gradually increased in length, slenderness, and complexity. As a result, modern blades and wings regularly operate beyond the range of linear deformation, requiring nonlinear analyses for which hp-FE methods are often not readily applicable. The aim of this paper is, therefore, to derive a corotational FE formulation for enriched three-, four-, and five-noded beam elements, suitable for nonlinear hp-FE refinement. To this end, the mathematical formulation is derived to incorporate enriched elements within the corotational FE beam framework. The proposed formulation is then validated against multiple nonlinear benchmark problems and an experimental case study.

The carcinogenic potential of a non-genotoxic pyrethroid imiprothrin was examined in rats and mice. There was no carcinogenicity in rats up to a maximum dose of 5000 ppm of the diet. There was a higher (p = 0.03) incidence of lung adenocarcinomas at 7000 ppm in males, and females showed an increasing (p 0.01) trend in the incidence of lung adenomas and combined lung adenoma/adenocarcinomas. Additional step sections of lung demonstrated no significant increases in any tumor at p 0.05, although an increasing trend with dose was observed among females. We argue that, the 7000 ppm dose exceeded the Maximum Tolerated Dose (MTD) for both sexes, based on systemic toxicity as evidenced by body weight gain reduction (both sexes) and high mortality (females). If the 7000 ppm dose is therefore removed from consideration, there are not significant (p 0.05) increases in tumor formation. Moreover, a consideration of multiple comparisons reveals that the lung tumor increases observed are totally consistent with what would be expected by chance alone. Based on high susceptibility of this mouse strain for the appearance of lung tumors and the lack of a statistically significant increase in tumors by appropriate analysis, the mouse study does not indicate a carcinogenic effect of imiprothrin, and thus no classification for carcinogenicity is warranted.

Recommendations (best practices) are provided by the Society of Toxicologic Pathology’s Adversity Working Group for making consistent interpretations of test article–related effects as “adverse” and assigning a “no observed adverse effect level” (NOAEL) in nonclinical toxicity studies. Adverse is a term indicating “harm” to the test animal, while nonadverse indicates lack of harm. Adverse findings in the study reports should be defined in relation to effects on the test species used and within the context of the given study. Test article–related effects should be described on their own merits, and decisions to consider them as adverse or nonadverse should be justified. Related effects may be discussed together; in particular, markers of toxicity that are not in and of themselves adverse ideally should be discussed in conjunction with the causal toxicity to determine adversity. Adverse findings should be identified in subreports (clinical data, pathology data, etc.) if sufficient information is available, and/or in the final study report as individual or grouped findings, but study NOAELs should be established at the level of the overall study report. Interpretations such as “not biologically relevant” or “not toxicologically important” should be avoided unless defined and supported by scientific rationale. Decisions defining adverse findings and the NOAEL in final study reports should combine the expertise of all contributing scientific disciplines. Where possible, use of NOAELs in data tables should be linked to explanatory text that places them in context. Ideally, in nonclinical summary documents, NOAELs from multiple studies are considered together in defining the most important adverse responses in the most sensitive species. These responses are then considered along with an understanding of their likely mechanisms, as well as other information such as variability in species sensitivity, comparative pathology, reversibility and progression, kinetics, and metabolism of the test substance to help assess human risk.

This study examines the impact of diet on health in different districts of mid-19th century London. Surveys of London diets and living condition were compared with mortality data between 1851 and 1880. Despite an abundance of fresh foods reaching London, the very poor labouring population living in the inner boroughs between 1850 and 1861 had great difficulty obtaining sufficient nourishment because of its cost. This population showed high death rates from infectious diseases, notably pulmonary tuberculosis, which was endemic and is typically associated with poor nutrition. This high death rate was exacerbated by more deaths from gastrointestinal infections associated with a polluted water supply from the river Thames. By contrast, the poor in the outer suburbs enjoyed both more nutritious diets and cleaner water which was associated with lower death rates comparable to those in rural Britain. Outer suburbs retained a relatively rural life-style associated with cleaner water and an abundance of locally grown food. In the following two decades, there was a significant reduction in the death rates from gastrointestinal infections in the inner boroughs which correlated with the major improvements in London’s water supply. The decline in death rates from tuberculosis and other infectious disease was inconsistent and increased in some boroughs, suggesting patchy economic improvement and a persisting limited ability of many of London’s poor to afford a nutritious diet.

Background S100 proteins have been implicated in various aspects of cancer, including epithelial-mesenchymal transitions (EMT), invasion and metastasis, and also in inflammatory disorders. Here we examined the impact of individual members of this family on the invasion of pancreatic ductal adenocarcinoma (PDAC) cells, and their regulation by EMT and inflammation. Methods Invasion of PDAC cells was analysed in zebrafish embryo xenografts and in transwell invasion assays. Expression and regulation of S100 proteins was studied in vitro by immunoblotting, quantitative PCR and immunofluorescence, and in pancreatic lesions by immunohistochemistry. Results Whereas the expression of most S100 proteins is characteristic for epithelial PDAC cell lines, S100A4 and S100A6 are strongly expressed in mesenchymal cells and upregulated by ZEB1. S100A4/A6 and epithelial protein S100A14 respectively promote and represses cell invasion. IL-6/11-STAT3 pathway stimulates expression of most S100 proteins. ZEB1 synergises with IL-6/11-STAT3 to upregulate S100A4/A6, but nullifies the effect of inflammation on S100A14 expression. Conclusion EMT/ZEB1 and IL-6/11-STAT3 signalling act independently and congregate to establish the expression pattern of S100 proteins, which drives invasion. Although ZEB1 regulates expression of S100 family members, these effects are masked by IL-6/11-STAT3 signalling, and S100 proteins cannot be considered as bona fide EMT markers in PDAC.

In this paper, the sensitivity of the structural integrity of wind turbine blades to debonding of the shear web from the spar cap was investigated. In this regard, modal analysis, static and fatigue testing were performed on a 45.7 m blade for three states of the blade: (i) as received blade (ii) when a crack of 200 mm was introduced between the web and the spar cap and (iii) when the crack was extended to 1000 mm. Calibration pull-tests for all three states of the blade were performed to obtain the strain-bending moment relationship of the blade according to the estimated target bending moment (BM) which the blade is expected to experience in its service life. The resultant data was used to apply appropriate load in the fatigue tests. The blade natural frequencies in flapwise and edgewise directions over a range of frequency domain were found by modal testing for all three states of the blade. The blade first natural frequency for each state was used for the flapwise fatigue tests. These were performed in accordance with technical specification IEC TS 61400-23. The fatigue results showed that, for a 200 mm crack between the web and spar cap at 9 m from the blade root, the crack did not propagate at 50% of the target BM up to 62,110 cycles. However, when the load was increased to 70% of target BM, some damages were detected on the pressure side of the blade. When the 200 mm crack was extended to 1000 mm, the crack began to propagate when the applied load exceeded 100% of target BM and the blade experienced delaminations, adhesive joint failure, compression failure and sandwich core failure.

This article addresses a number of concepts related to the selection and modelling of carcinogenicity data for the calculation of a Margin of Exposure. It follows up on the recommendations put forward by the International Life Sciences Institute - European branch in 2010 on the application of the Margin of Exposure (MoE) approach to substances in food that are genotoxic and carcinogenic. The aims are to provide practical guidance on the relevance of animal tumour data for human carcinogenic hazard assessment, appropriate selection of tumour data for Benchmark Dose Modelling, and approaches for dealing with the uncertainty associated with the selection of data for modelling and, consequently, the derived Point of Departure (PoD) used to calculate the MoE. Although the concepts outlined in this article are interrelated, the background expertise needed to address each topic varies. For instance, the expertise needed to make a judgement on biological relevance of a specific tumour type is clearly different to that needed to determine the statistical uncertainty around the data used for modelling a benchmark dose. As such, each topic is dealt with separately to allow those with specialised knowledge to target key areas of guidance and provide a more in-depth discussion on each subject for those new to the concept of the Margin of Exposure approach.

Upscaling wind turbines has resulted in levelised cost of energy (LCoE) reductions. However, larger turbine diameters pose significant design challenges, often with conflicting requirements. For example, non-linear dynamics of aeroelastic tailored blades must be accurately predicted whilst, for the sake of efficient gradient-based design, it is also desirable to simplify the numerical definition of such blades—keeping design variables (DVs) to a minimum. This work presents and validates two features of the ATOM code (Aeroelastic Turbine Optimisation Methods), developed at the University of Bristol, that enable accurate and efficient modelling of large-scale wind turbine blades. Both an efficient parameterisation method and high-order beam elements illustrate the capacity for increasing the speed of gradient evaluations whilst accurately predicting blade dynamics—either by reducing DVs or simulation time. As a preliminary validation, aero-servo-elastic simulations from ATOM and an industry-standard software—DNV GL Bladed—are compared against field measurements gathered from an existing 7 MW turbine.

The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying lesions observed in the soft tissues including skeletal muscle as well as the mesothelium of rats and mice. The standardized nomenclature of lesions presented in this document is also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous developmental and aging lesions as well as those induced by exposure to test materials. A widely accepted and utilized international harmonization of nomenclature for lesions in soft tissues, skeletal muscle and mesothelium in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists. (DOI: 10.1293/tox.26.1S; J Toxicol Pathol 2013; 26: 1S–26S)

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) project is a joint initiative of the Societies of Toxicologic Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP), and North America (STP) to develop an internationally accepted nomenclature and diagnostic criteria for nonproliferative and proliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature and diagnostic criteria for classifying lesions in the digestive system including the salivary glands and the exocrine pancreas of laboratory rats and mice. Most lesions are illustrated by color photomicrographs. The standardized nomenclature, the diagnostic criteria, and the photomicrographs are also available electronically on the Internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and age related lesions as well as lesions induced by exposure to test items. Relevant infectious and parasitic lesions are included as well. A widely accepted and utilized international harmonization of nomenclature and diagnostic criteria for the digestive system will decrease misunderstandings among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Some naturally occurring flavonols, exemplified by quercetin, seem to possess experimental cancer chemopreventive efficacy. Modulation of p53 is a mechanism thought to contribute to their activity. The hypothesis was tested that a synthetic flavonol, 3′,4′,5′-trimethoxyflavonol (TMFol), can interfere with tumor development and p53 expression in two models of colorectal carcinogenesis, ApcMin mice and human-derived HCT116 adenocarcinoma–bearing nude mice. Mice received TMFol with their diet (0.2%) from weaning to week 16 in the case of ApcMin or from either day 7 before (“TMFol early”) or day 7 after (“TMFol late”) tumor inoculation in HCT116 mice. The ability of TMFol to affect tumor proliferation or apoptosis, as reflected by staining for Ki-67 or cleaved caspase-3, respectively, was studied in HCT116 tumors. TMFol tumor levels were measured by high-performance liquid chromatography. Consumption of TMFol reduced small intestinal adenoma burden in ApcMin mice by 47%, compared with control mice (P < 0.002). The TMFol early regimen approximately halved HCT116 tumor size (P < 0.05), decreased tumor proliferation, and increased apoptosis, whereas the TMFol late regimen had no significant effect when compared with controls. In tumor tissues from mice, in which TMFol reduced tumor development, p53 expression was increased 3-fold in ApcMin and 1.5-fold in HCT116 tumor–bearing mice (P = 0.02). TMFol increased p53 also in cells derived from these tumors. TMFol was detected in HCT116 tumors, but levels did not correlate with tumor burden. TMFol was not mutagenic in the Ames test. The results suggest that chemical modification of the flavonol structure may generate safe and efficacious cancer chemopreventive agents. Cancer Prev Res; 3(8); 929–39. ©2010 AACR.

Summary The aim of this study was to examine the impact of regional diets on the health of the poor in mid-Victorian Britain. Contemporary surveys of regional diets and living condition were reviewed. This information was compared with mortality data from Britain over the same period. Although there was an overall improvement in life expectancy during the latter part of the 19th century, there were large regional differences in lifestyle, diet and mortality rates. Dietary surveys showed that the poor labouring population in isolated rural areas of England, in the mainland and islands of Scotland and in the west of Ireland enjoyed the most nutritious diets. These regions also showed the lowest mortality rates in Britain. This was not simply the result of better sanitation and less mortality from food and waterborne infections but also fewer deaths from pulmonary tuberculosis, which is typically associated with better nutrition. These more isolated regions where a peasant-style culture provided abundant locally produced cheap foodstuffs such as potatoes, vegetables, whole grains, and milk and fish, were in the process of disappearing in the face of increasing urbanisation. This was to the detriment of many rural poor during the latter half of the century. Conversely, increasing urbanisation, with its improved transport links, brought greater availability and diversity of foods to many others. It was this that that led to an improved nutrition and life expectancy for the majority in urbanising Britain, despite the detrimental effects of increasing food refinement.

Summary Mesothelioma is a fatal tumor of the pleura and is strongly associated with asbestos exposure. The molecular mechanisms underlying the long latency period of mesothelioma and driving carcinogenesis are unknown. Moreover, late diagnosis means that mesothelioma research is commonly focused on end-stage disease. Although disruption of the CDKN2A (INK4A/ARF) locus has been reported in end-stage disease, information is lacking on the status of this key tumor suppressor gene in pleural lesions preceding mesothelioma. Manufactured carbon nanotubes (CNTs) are similar to asbestos in terms of their fibrous shape and biopersistent properties and thus may pose an asbestos-like inhalation hazard. Here we show that instillation of either long CNTs or long asbestos fibers into the pleural cavity of mice induces mesothelioma that exhibits common key pro-oncogenic molecular events throughout the latency period of disease progression. Sustained activation of pro-oncogenic signaling pathways, increased proliferation, and oxidative DNA damage form a common molecular signature of long-CNT- and long-asbestos-fiber-induced pathology. We show that hypermethylation of p16/Ink4a and p19/Arf in CNT- and asbestos-induced inflammatory lesions precedes mesothelioma; this results in silencing of Cdkn2a (Ink4a/Arf) and loss of p16 and p19 protein, consistent with epigenetic alterations playing a gatekeeper role in cancer. In end-stage mesothelioma, silencing of p16/Ink4a is sustained and deletion of p19/Arf is detected, recapitulating human disease. This study addresses the long-standing question of which early molecular changes drive carcinogenesis during the long latency period of mesothelioma development and shows that CNT and asbestos pose a similar health hazard., Highlights • Long-fiber carbon nanotubes induce malignant mesothelioma with loss of Cdkn2a • Long-fiber carbon nanotube tumors replicate asbestos-induced mesothelioma in humans • Cdkn2a hypermethylation during disease latency precedes fiber-induced mesothelioma • Long-fiber carbon nanotubes pose an asbestos-like hazard, Manufactured carbon nanotubes (CNTs) show structural similarities to asbestos, strongly associated with the fatal tumor mesothelioma. Chernova et al. show that CNTs and asbestos indeed pose similar health hazards and address the long-standing question of which early molecular changes drive carcinogenesis during mesothelioma’s long latency period.

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) represents a non-invasive biomarker for oxidative stress and may be useful for monitoring chemotherapeutic and chemopreventive interventions associated with cancer-related alterations in oxidative stress. We describe the development and validation of two separate liquid chromatography/tandem mass spectrometry (LC/MS/MS) selected reaction monitoring (SRM) methods for the determination of 8-oxodG and creatinine in both murine and human urine using stable isotope labelled internal standards. Levels of 8-oxodG were normalised to creatinine. The LC/MS/MS methods were applied to two chemoprevention studies utilising tea polyphenols in humans and TRAMP (TRansgenic Adenocarcinoma of the Mouse Prostate) mice. Patients with benign prostatic hyperplasia received 1 g/day of green tea polyphenols (GTP), 1 g/day of black tea theaflavins (BTT) or no treatment for 4 weeks. TRAMP mice received GTP (0.05% in drinking water) for 4 or 25 weeks. Prostate pathology in TRAMP mice was not affected by GTP. Levels of 8-oxodG were not altered by tea polyphenols in either mice or humans. In TRAMP mice, urinary 8-oxodG levels were elevated with increasing age (p < 0.0001) but not changed by the presence of prostate tumours. In conclusion, the LC/MS/MS SRM methods described here are ideally suited for the accurate determination of 8-oxodG and creatinine in urine samples from both clinical and pre-clinical studies.

Silibinin, a flavonolignan from milk thistle seeds, possesses cancer chemopreventive properties in rodent models of carcinogenesis. We tested the hypotheses that silibinin or silipide, silibinin formulated with phospholipids, delays tumour development in TRAMP or Apc(Min) mice, genetic models of prostate or intestinal malignancies, respectively. Mice received silibinin or silipide with their diet (0.2% silibinin equivalents) from weaning. Intervention with silipide reduced the size of well differentiated TRAMP adenocarcinomas by 31%. Silipide and silibinin decreased the incidence of poorly differentiated carcinomas by 61% compared to mice on control diet. Silipide decreased plasma levels of insulin-like growth factor (IGF)-1 by 36%. Levels of circulating IGF binding protein (IGFBP)-3 in mice on silipide or silibinin were 3.9- or 5.9-fold, respectively, elevated over those in control TRAMP mice. In Apc(Min) mice silibinin, but not silipide, had only a marginal adenoma number-reducing effect. The results cautiously support the advancement of silipide to the stage of clinical investigation in prostate cancer.

Intracellular reactive oxygen species (ROS) may cause oxidative DNA damage, resulting in the formation of adducts such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N(2) malondialdehyde-2'-deoxyguanosine (M(1)dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. We tested two hypotheses in human prostate cancer cells grown in vitro and in a xenograft model: (1) treatment of androgen-sensitive cells with DHT increases levels of oxidative DNA adduct levels; (2) flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. Levels of M(1)dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry. M(1)dG and 8-oxo-dG levels were significantly higher than control levels in LNCaP cells exposed to supra-physiological concentrations (25-100 nM) of DHT (both P

Peroxisome proliferator-activated receptors (PPAR) are involved in the pathogenesis of insulin resistance, diabetes, and related complications. Consequently, the identification of PPAR subtypes and the potential for their activation provides promising therapeutic targets for the management of type 2 diabetes mellitus. Available data from rodent carcinogenicity studies, however, demonstrate that PPAR agonists can be tumorigenic in one or more species of rodents at multiple sites. In 2005, the Health and Environmental Sciences Institute (HESI) PPAR Agonist Project Committee was established by a group of pharmaceutical companies to advance research on and to understand the modes of action and human relevance of this emerging rodent tumor data for PPAR agonists. Since the most commonly observed tumor types reported in rodents are hemangiosarcomas, fibrosarcomas and liposarcomas, the PPAR Agonist Project Committee approved a Pathology Working Group (PWG) to develop consensus of morphologic criteria for tumor diagnoses and consistency of diagnoses across multiple studies for hemangiosarcomas in mice and hamsters and liposarcomas/fibrosarcomas in rats. Therefore, the focus of the PWG review was to establish consistent tumor diagnostic criteria, to assess evidence of potentially preneoplastic changes and to identify distinguishing morphologic differences which may exist between spontaneous changes present in control animals with similar changes from treated animals. Specific diagnostic criteria and nomenclature are recommended for the classification of proliferative vascular lesions which may be present in mice or hamsters and for proliferative mesenchymal changes in rats in studies that are conducted with PPAR agonists.

Brown rice is a staple dietary constituent in Asia, whereas rice consumed in the Western world is generally white, obtained from brown rice by removal of the bran. We tested the hypothesis that rice bran interferes with development of tumours in TAg, TRansgenic Adenocarcinoma of the Mouse Prostate (TRAMP) or Apc(Min) mice, genetic models of mammary, prostate and intestinal carcinogenesis, respectively. Mice received rice bran (30%) in AIN-93G diet throughout their post-weaning lifespan. In TAg and TRAMP mice, rice bran did not affect carcinoma development. In TRAMP or wild-type C57Bl6/J mice, dietary rice bran increased kidney weight by 18 and 20%, respectively. Consumption of rice bran reduced numbers of intestinal adenomas in Apc(Min) mice by 51% (P0.01), compared to mice on control diet. In parallel, dietary rice bran decreased intestinal haemorrhage in these mice, as reflected by increased haematocrit. At 10% in the diet, rice bran did not significantly retard Apc(Min) adenoma development. Likewise, low-fibre rice bran (30% in the diet) did not affect intestinal carcinogenesis, suggesting that the fibrous constituents of the bran mediate chemopreventive efficacy. The results suggest that rice bran might be beneficially evaluated as a putative chemopreventive intervention in humans with intestinal polyps.

Resveratrol is widely promoted as a potential cancer chemopreventive agent, but a lack of information on the optimal dose prohibits rationally designed trials to assess efficacy. To challenge the assumption that "more is better," we compared the pharmacokinetics and activity of a dietary dose with an intake 200 times higher. The dose-response relationship for concentrations generated and the metabolite profile of [(14)C]-resveratrol in colorectal tissue of cancer patients helped us to define clinically achievable levels. In Apc(Min) mice (a model of colorectal carcinogenesis) that received a high-fat diet, the low resveratrol dose suppressed intestinal adenoma development more potently than did the higher dose. Efficacy correlated with activation of adenosine monophosphate-activated protein kinase (AMPK) and increased expression of the senescence marker p21. Nonlinear dose responses were observed for AMPK and mechanistic target of rapamycin (mTOR) signaling in mouse adenoma cells, culminating in autophagy and senescence. In human colorectal tissues exposed to low dietary concentrations of resveratrol ex vivo, we measured enhanced AMPK phosphorylation and autophagy. The expression of the cytoprotective NAD(P)H dehydrogenase, quinone 1 (NQO1) enzyme was also increased in tissues from cancer patients participating in our [(14)C]-resveratrol trial. These findings warrant a revision of developmental strategies for diet-derived agents designed to achieve cancer chemoprevention.

Highlights • Curcumin + FOLFOX inhibits growth of primary cancer stem cell (CSC) spheroid models. • Curcumin + FOLFOX decreases expression of CSC markers in primary CSC spheroid models. • Curcumin enhances proapoptotic effects of chemotherapy in explant culture. • Curcumin is safe and tolerable in combination with FOLFOX chemotherapy. • Curcumin is perceived by patients as an acceptable daily adjunct to chemotherapy., In vitro and pre-clinical studies have suggested that addition of the diet-derived agent curcumin may provide a suitable adjunct to enhance efficacy of chemotherapy in models of colorectal cancer. However, the majority of evidence for this currently derives from established cell lines. Here, we utilised patient-derived colorectal liver metastases (CRLM) to assess whether curcumin may provide added benefit over 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) in cancer stem cell (CSC) models. Combination of curcumin with FOLFOX chemotherapy was then assessed clinically in a phase I dose escalation study. Curcumin alone and in combination significantly reduced spheroid number in CRLM CSC models, and decreased the number of cells with high aldehyde dehydrogenase activity (ALDHhigh/CD133−). Addition of curcumin to oxaliplatin/5-FU enhanced anti-proliferative and pro-apoptotic effects in a proportion of patient-derived explants, whilst reducing expression of stem cell-associated markers ALDH and CD133. The phase I dose escalation study revealed curcumin to be a safe and tolerable adjunct to FOLFOX chemotherapy in patients with CRLM (n = 12) at doses up to 2 grams daily. Curcumin may provide added benefit in subsets of patients when administered with FOLFOX, and is a well-tolerated chemotherapy adjunct.

While brown rice is a staple dietary constituent in Asia, rice consumed in the Western world is generally white, obtained from brown rice by removal of the bran. Rice bran contains the flavone tricin, which has been shown to inhibit colon cancer cell growth. We tested the hypothesis that tricin interferes with adenoma formation in the ApcMin mouse. Mice received tricin (0.2%) in their American Institute of Nutrition 93G diet throughout their postweaning life span (4–18 weeks). Consumption of tricin reduced numbers of intestinal adenomas by 33% (P < 0.05) compared with mice on control diet. We explored whether tricin may exert its effect via inhibition of cyclooxygenase (COX) enzymes. Its effect on COX activity was assessed in purified enzyme preparations in vitro and its ability to reduce prostaglandin E2 (PGE2) levels in human colon–derived human colon epithelial cell (HCEC) and HCA-7 cells in vitro and in ApcMin mice in vivo. Tricin inhibited activity of purified COX-1 and COX-2 enzyme preparations with IC50 values of ∼1 μmol/L. At 5 μmol/L, it reduced PGE2 production in HCEC or HCA-7 cells by 36% (P < 0.01) and 35% (P < 0.05), respectively. COX-2 expression was reduced by tricin weakly in HCEC and unaffected in HCA-7 cells. PGE2 levels in the small intestinal mucosa and blood of ApcMin mice that had received tricin were reduced by 34% (P < 0.01) and 40% (P < 0.05), respectively, compared with control mice. The results suggest that tricin should be further evaluated as a putative colorectal cancer chemopreventive agent.

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.

Tricin, a flavone found in rice bran, inhibits the growth of human-derived malignant MDA-MB-468 breast tumour cells at submicromolar concentrations. As part of the exploration of tricin as a potential cancer chemopreventive agent, we investigated the duration and cell cycle specificity of growth inhibition elicited by tricin in vitro and the effect of tricin on the development of MDA-MB-468 tumours grown in immune-compromised MF-1 mice in vivo. Preincubation of MDA-MB-468 cells with tricin (1-40 microM) for 72 h compromised cell growth after tricin removal, and such irreversibility was not observed in human breast-derived nonmalignant HBL-100 cells. Tricin (>/=5 microM) arrested MDA-MB-468 cells in the G2/M phase of the cell cycle without inducing apoptosis as adjudged by annexin V staining. In nude mice consumption of tricin with the diet (0.2%, w w(-1)) from 1 week prior to MDA-MB-468 cell implantation failed to impede tumour development. Steady-state levels of tricin in plasma, breast tumour tissue and intestinal mucosa, as measured by HPLC, were 0.13 microM and 0.11 and 63 nmol g(-1), respectively. Cells were exposed to tricin (0.11, 1.1 or 11 microM) in vitro for 72 h and then implanted into mice. The volume of tumours in animals bearing cells pre-exposed to 11 microM tricin was less than a third of that in mice with control cells, while tumours from cells incubated with 0.1 or 1.1 microM tricin were indistinguishable from controls. These results suggest that the potent breast tumour cell growth-inhibitory activity of tricin in vitro does not directly translate into activity in the nude mouse bearing the MDA MB-468 tumour. While the results do not support the notion that tricin is a promising candidate for breast cancer chemoprevention, its high levels in the gastrointestinal tract after dietary intake render exploration of its ability to prevent colorectal carcinogenesis propitious.

The need for careful testing of new drugs in animal models before study in humans has been recognised by physicians since the First World War. Now, first human studies on new drugs are subject to detailed government guidelines, which in the European Union are presently being reinforced through the wide-ranging Clinical Trials Directive. However, despite their long history and widespread application, these guidelines are empirical and have been formulated with a paucity of critical scientific evidence. Here, we review the principles and the available, albeit limited, evidence that support the design and conduct of preclinical studies in a way that permits effective and safe first-dose studies of potential new medicines in humans.

ET-743, an experimental antitumor drug with promising activity in sarcoma, breast and ovarian carcinoma, is currently under phase 2 clinical evaluation. It is hepatotoxic in animals and patients. We tested the hypothesis that indole-3-carbinol (I3C), the hydrolysis product of glucosinolates occurring in cruciferous vegetables, may protect against ET-743-induced hepatotoxicity in the female Wistar rat, the animal species with the highest sensitivity toward the adverse hepatic effect of this drug. Hepatotoxicity was adjudged by measurement of plasma levels of bilirubin, alkaline phosphatase (ALP) and aspartate aminotransferase (AST) and by liver histopathology. The effect of I3C on the kinetics of ET-743 in rat plasma and liver was investigated by high-pressure liquid chromatography. The effect of I3C on the antitumor efficacy of ET-743 was explored in rats bearing the 13762 mammary carcinoma. ET-743 (40 microg/kg i.v.) alone caused an elevation of plasma bilirubin, ALP and AST levels and degeneration and patchy focal necrosis of bile duct epithelial cells. Addition of I3C to the diet (0.5%) for 6 days prior to ET-743 administration almost completely abolished manifestations of hepatotoxicity. In contrast, a dietary concentration of 0.1% I3C did not protect, nor did dietary diindolylmethane (0.2%), an acid-catalyzed condensation product of I3C. Ingestion by rats of I3C for 6 days prior to ET-743 (40 microg/kg i.v.) decreased plasma but not hepatic concentrations of ET-743 compared to animals that received ET-743 alone. I3C did not interfere with the antitumor efficacy of ET-743. The results suggest that ingestion of I3C may counteract the unwanted effect of ET-743 in the liver. I3C should be investigated as a hepatoprotectant in patients who receive ET-743 therapy.

Two endothelin antagonists, ZD1611 (3- {4-[3-(3-methoxy-5-methylpyrazin-2-ylsulfamoyl)-2-pyridyl]phenyl }-2,2-dimethylpropanoic acid) and ZD2574 (2-(4-isobutylphenyl)- N-(3-methoxy-5-methylpyrazin-2-yl)pyridine-3-sulfonamide), selective for the ETA receptor and intended for use in pulmonary hypertension, were tested in Beagle dogs at various doses for periods of up to 4 weeks. These studies included in vivo telemetric hemodynamic assessment, full histopathological and ultrastructural pathological evaluation of coronary arteries. Both drugs produced arteritis in small- and medium-sized coronary arteries after single or multiple doses, some of which were at or below the ED50. The distribution of lesions was predominantly in extramural arteries over the atria and atrioventricular groove of the right side of the heart and consisted of epicardial hemorrhage and arteritis. Systemic arteritis was also present at a lower incidence than the coronary arteritis, was located at different sites and appeared inconsistently. Ultrastructural changes in coronary arteries suggested that damage was the result of mechanical factors. Although these patterns of vascular injury possessed features in common with those induced in dogs by high doses of vasodilating antihypertensive drugs and inotropic agents, they were atypical, as there was no left ventricular myocardial necrosis, papillary muscle damage, or subendocardial hemorrhage suggestive of ischaemia or excessive inotropism. Moreover, physiological monitoring showed no evidence of exaggerated systemic hypotension or reflex tachycardia at doses associated with vascular damage. Consequently, the changes might be the result of a localized pharmacological process such as intense, prolonged vasodilatation in unsupported arteries that are well endowed with endothelin receptors and particularly sensitive to endothelin antagonism.

In toxicity studies, the examination of tissue sections for pathological changes is the principle method for the identification of organ toxicity and characterisation of the hazard of novel drugs for humans. Study of the patterns of pathological alterations also represents an important means of developing an understanding of the mechanism of toxicity. However as pathological change frequently represents a final common expression of diverse processes, additional functional information is often required for a clear understanding of the mechanisms of toxicity. This is exemplified in the evaluation of the effects of drugs on the beagle dog cardiovascular system where an understanding of mechanisms is crucial in the assessment of human risk. Particular patterns of drug-induced structural change in the myocardium or blood vessels are frequently linked to specific mechanisms of toxicity. However, assessment based on the interpretation of patterns of cardiovascular pathology alone may be misleading. Quite different changes in cardiac and vascular function or direct cellular toxicity may also be manifest by pathological features in common. Therefore, a clear understanding of mechanism frequently requires additional in vivo or in vitro physiological, pharmacological, biochemical or other mechanistic information. The beagle dog remains an important model for the study of cardiovascular toxicity because in this species, haemodynamic changes and pathological alterations can be related in a way that provides the basis for the safe study in humans of novel drugs with cardiovascular activity.

Summary Over 100 marketed drugs induce neoplasia when administered at high doses to rats and mice for periods of up to two years. Despite their diverse chemical structures and biological activities, these compounds produce a relatively limited range of tumour types in rodents, most commonly in the liver. Tumours usually develop only after long periods of time following high exposure to drug. The main exceptions are DNA-reactive anticancer drugs such as alkylating agents which produce tumours rapidly in rodents in several organs. In this laboratory, mouse carcinogenicity studies are performed using the C57BL/10J strain. This strain infrequently develops hepatic tumours spontaneously but it is sensitive to the effects of DNA-reactive carcinogens. Moreover, hepatic neoplasms regularly develop in male but not female C57BL/10J mice following long-term treatment with non-genotoxic drugs that produce hepatic enlargement associated with diverse hepatocellular effects. Studies in this strain with the tumorigenic liver enlarger, phenobarbitone, have shown that although such liver enlargement is characterised by a brief burst of hepatocyte replication, this is associated with persistent regional modulation of hepatic growth stimulatory and inhibitory factors and their associated receptors. These findings indicate that there is a sustained alteration to the internal hepatic environment characterised by regional alterations to the balance of hepatocyte mitogens and inhibitors of replication and their respective receptors. Thus, the development of hepatocellular tumours in C57BL/10J mice following two-year treatment with non-genotoxic drugs appears to be a regular response of an organ to an exaggerated and long-term disruption of its homeostasis. Agents that produce tumours in rodents in this way seem likely to pose little or no risk to humans if administered under appropriate clinical circumstances at doses which show no significant disruption of organ homeostasis. However, drugs that produce this type of response need to be distinguished from those that induce unusual and rapid patterns of tumour development because these agents may have high tumorigenic potency of potential hazard to humans.

Publisher Summary During the course of drug development, the effects of novel compounds on the reproductive system are examined in specialized reproductive tests. However, these studies are infrequently conducted before the first dose of a novel pharmaceutical to humans. Hence, the only assessment of the male reproductive organs performed prior to the first administration of a novel therapeutic agent to man is histological examination of the male sex organs in conventional toxicity studies in rodent and nonrodent species. For this reason, this assessment should be performed using meticulous techniques. This approach has been agreed in the consensus guidelines from the International Conference on Harmonisation following the studies that showed histopathological examination of the male reproductive organs in the rodent 4-week toxicity study was more sensitive in detecting effects than fertility studies. In mammals the male sex organs comprise the paired testes and the accessory reproductive organs: prostate, seminal vesicles, coagulating glands, ampullary glands, and epididymis. Cytochemical or immunocytochemical demonstration of acid phosphatase has been shown to be a good indicator of the functional integrity of prostatic secretory cells, particularly as acid phosphatase is a major secretory component, which is androgen-dependent in man and laboratory animals. It has been shown that there is a close immunological cross-reactivity of antibody against acid phosphatase from the rat ventral prostate, the canine, and human prostate.

Chapter 4 describes the normal features and pathologic changes that can occur in the blood, bone marrow, lymph nodes, spleen and thymus in laboratory animals both spontaneously and as a result of drug treatment. It provides comparison between changes to these tissues observed in preclinical toxicity studies with those reported in the hemopoietic and immune systems in patients treated with drugs. It discusses the distinct mechanisms involved. It summarizes technical considerations in the assessment of the hemopoietic and immune systems as well as immunohistochemical analysis of lymphoid cells in different species. It also deals with the proliferative and neoplastic changes that can affect the cells of the hemopoietic and lymphoid series particularly those linked to immunosuppression and anticancer therapies. It discusses the lack of relevance of rodent histiocytic sarcomas and large granular cell leukemias for humans.

Fos-related antigen 1 (Fra-1) is a Fos family member overexpressed in several types of human cancers. Here, we report that Fra-1 is highly expressed in the muscle-invasive form of the carcinoma of the bladder (80%) and to a lesser extent in superficial bladder cancer (42%). We demonstrate that in this type of cancer Fra-1 is regulated via a C-terminal instability signal and C-terminal phosphorylation. We show that manipulation of Fra-1 expression levels in bladder cancer cell lines affects cell morphology, motility and proliferation. The gene coding for AXL tyrosine kinase is directly upregulated by Fra-1 in bladder cancer and in other cell lines. Importantly, our data demonstrate that AXL mediates the effect of Fra-1 on tumour cell motility but not on cell proliferation. We suggest that AXL may represent an attractive therapeutic target in cancers expressing high Fra-1 levels.

The purpose of this chapter is to discuss the importance of the pathologic alterations that can be induced by drugs in the liver of laboratory animals and their implication for hepatic toxicity in patients. It describes anatomy and physiology of the liver of the laboratory species in some detail and discusses how these may differ from humans. Drug-induced hepatic alterations described include changes to hepatic weight, hypertrophy, hyperplasia, storage disorders, pathology of the bile duct and gallbladder as well as the frequently occurring neoplasms in the rodent liver and their implications for human therapy. The chapter also describes a similar range of changes that can be seen in the exocrine pancreas and the endocrine alterations in the islets of Langerhans.

Publisher Summary The potential importance of the effect of xenobiotics on the female reproductive system and the foetus dictates strict regulatory requirements for specific preclinical reproductive safety studies. Reproductive studies are directed primarily toward examination of functional perturbations of reproductive performance and morphological effects on the foetus. Consequently, pathological assessment of the female reproductive organs in repeated dose toxicity studies remains an important part of the preclinical assessment of novel drugs. Ovarian and reproductive functions are intimately linked to hypothalamic and pituitary gland activity. The hypothalamic-pituitary axis serves as the primary control center for reproductive hormones. Although drug-induced effects on the reproductive system are either mediated directly through action on the peripheral sex organs or indirectly through the hypothalamus, these mechanisms are not sharply separable. Histopathological evaluation of the female reproductive system is also complicated by the morphological changes that occur during the oestrous or menstrual cycle. Age-related cessation of ovulatory function in laboratory animals and in humans is preceded by a transitional phase characterized by increasing variability in ovarian cycling time.