Non-microcystin extracellular metabolites of Microcystis aeruginosa impair viability and reproductive gene expression in rainbow trout cell lines.

Microcystis aeruginosa is a ubiquitous freshwater cyanobacterium best known for producing hepatotoxic microcystins; however, this common bloom-forming species also produces myriad biologically active and potentially deleterious other metabolites. Our understanding of the effects of these non-microcystin metabolites on fish is limited. In this study, we evaluated cytotoxicity of extracellular metabolites harvested from both microcystin-producing (MC+) and non-producing (MC-) strains of M. aeruginosa on rainbow trout (Oncorhynchus mykiss) cell lines derived from tissues of the brain, pituitary, heart, gonads, gills, skin, liver, and milt. We also examined the influence of M. aeruginosa exudates (MaE) on the expression of critical reproduction-related genes using the same cell lines. We found that exudates of the MC- M. aeruginosa strain significantly reduced viability in RTBrain, RTgill-W1, and RT-milt5 cell lines and induced significant cellular stress and/or injury in six of the eight cell lines-highlighting potential target tissues of cyanobacterial cytotoxic effects. Observed sublethal consequences of Microcystis bloom exposure occurred with both MC+ and MC- strains' exudates and significantly altered expression of developmental and sex steroidogenic genes. Collectively, our results emphasize the contributions of non-MC metabolites to toxicity of Microcystis-dominated algal blooms and the need to integrate the full diversity of M. aeruginosa compounds-beyond microcystins-into ecotoxicological risk assessments.

Examining animal testing for risk assessment: A WC-12 workshop report.

In toxicology and regulatory testing, the use of animal methods has been both a cornerstone and a subject of intense debate. To continue this discourse a panel and audience representing scientists from various sectors and countries convened at a workshop held during the 12th World Congress on Alternatives and Animal Use in the Life Sciences (WC-12). The ensuing discussion focused on the scientific and ethical considerations surrounding the necessity and responsibility of defending the creation of new animal data in regulatory testing. The primary aim was to foster an open dialogue between the panel members and the audience while encouraging diverse perspectives on the responsibilities and obligations of various stakeholders (including industry, regulatory bodies, technology developers, research scientists, and animal welfare NGOs) in defending the development and subsequent utilization of new animal data. This workshop summary report captures the key elements from this critical dialogue and collective introspection. It describes the intersection of scientific progress and ethical responsibility as all sectors seek to accelerate the pace of 21st century predictive toxicology and new approach methodologies (NAMs) for the protection of human health and the environment.

Using in vitro data to derive acceptable exposure levels: A case study on PBDE developmental neurotoxicity.

BACKGROUND: Current acceptable chemical exposure levels (e.g., tolerable daily intake) are mainly based on animal experiments, which are costly, time-consuming, considered non-ethical by many, and may poorly predict adverse outcomes in humans. OBJECTIVE: To evaluate a method using human in vitro data and biological modeling to calculate an acceptable exposure level through a case study on 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) developmental neurotoxicity (DNT). METHODS: We reviewed the literature on in vitro assays studying BDE-47-induced DNT. Using the most sensitive endpoint, we derived a point of departure using a mass-balance in vitro disposition model and benchmark dose modeling for a 5% response (BMC05) in cells. We subsequently used a pharmacokinetic model of gestation and lactation to estimate administered equivalent doses leading to four different metrics of child brain concentration (i.e., average prenatal, average postnatal, average overall, and maximum concentration) equal to the point of departure. The administered equivalent doses were translated into tolerable daily intakes using uncertainty factors. Finally, we calculated biomonitoring equivalents for maternal serum and compared them to published epidemiological studies of DNT. RESULTS: We calculated a BMC05 of 164 µg/kg of cells for BDE-47 induced alteration of differentiation in neural progenitor cells. We estimated administered equivalent doses of 0.925-3.767 µg/kg/day in mothers, and tolerable daily intakes of 0.009-0.038 µg/kg/day (composite uncertainty factor: 100). The lowest derived biomonitoring equivalent was 19.75 ng/g lipids, which was consistent with reported median (0.9-23 ng/g lipids) and geometric mean (7.02-26.9 ng/g lipids) maternal serum concentrations from epidemiological studies. CONCLUSION: This case study supports using in vitro data and biological modeling as a viable alternative to animal testing to derive acceptable exposure levels.

Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology.

Read-across (RAx) translates available information from well-characterized chemicals to a substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g., EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimization to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular, the RAx guidance, being worked out by the European Commission's H2020 project EU-ToxRisk together with many external partners with regulatory experience, is given.

Advancing nonclinical innovation and safety in pharmaceutical testing.

Nonclinical tests are considered crucial for understanding the safety of investigational medicines. However, the effective translation from nonclinical to human application is limited and must be improved. Drug development stakeholders are working to advance human-based in vitro and in silico methods that may be more predictive of human efficacy and safety in vivo because they enable scientists to model the direct interaction of drugs with human cells, tissues, and biological processes. Here, we recommend test-neutral regulations; increased funding for development and integration of human-based approaches; support for existing initiatives that advance human-based approaches; evaluation of new approaches using human data; establishment of guidelines for procuring human cells and tissues for research; and additional training and educational opportunities in human-based approaches.

Animal research for type 2 diabetes mellitus, its limited translation for clinical benefit, and the way forward.

Obesity and type 2 diabetes mellitus (T2DM) have reached pandemic proportions worldwide, and considerable research efforts have been dedicated to investigating disease pathology and therapeutic options. The two hallmark features of T2DM, insulin resistance and pancreatic dysfunction, have been studied extensively by using various animal models. Despite the knowledge acquired from such models, particularly mechanistic discoveries that sometimes mimic human T2DM mechanisms or pathways, many details of human T2DM pathogenesis remain unknown, therapeutic options remain limited, and a cure has eluded research. Emerging human data have raised concern regarding inter-species differences at many levels (e.g. in gene regulation, pancreatic cytoarchitecture, glucose transport, and insulin secretion regulation), and the subsequent impact of these differences on the clinical translation of animal research findings. Therefore, it is important to recognise and address the translational gap between basic animal-based research and the clinical advances needed to prevent and treat T2DM. The purpose of this report is to identify some limitations of T2DM animal research, and to propose how greater human relevance and applicability of hypothesis-driven basic T2DM research could be achieved through the use of human-based data acquisition at various biological levels. This report addresses how in vitro, in vivo and in silico technologies could be used to investigate particular aspects of human glucose regulation. We do not propose that T2DM animal research has been without value in the identification of mechanisms, pathways, or potential targets for therapies, nor do we claim that human-based methods can provide all the answers. We recognise that the ultimate goal of T2DM animal research is to identify ways to advance the prevention, recognition and treatment of T2DM in humans, but postulate that this is where the use of animal models falls short, despite decades of effort. The best way to achieve this goal is by prioritising human-centred research.

Alzheimer disease research in the 21st century: past and current failures, new perspectives and funding priorities.

Much of Alzheimer disease (AD) research has been traditionally based on the use of animals, which have been extensively applied in an effort to both improve our understanding of the pathophysiological mechanisms of the disease and to test novel therapeutic approaches. However, decades of such research have not effectively translated into substantial therapeutic success for human patients. Here we critically discuss these issues in order to determine how existing human-based methods can be applied to study AD pathology and develop novel therapeutics. These methods, which include patient-derived cells, computational analysis and models, together with large-scale epidemiological studies represent novel and exciting tools to enhance and forward AD research. In particular, these methods are helping advance AD research by contributing multifactorial and multidimensional perspectives, especially considering the crucial role played by lifestyle risk factors in the determination of AD risk. In addition to research techniques, we also consider related pitfalls and flaws in the current research funding system. Conversely, we identify encouraging new trends in research and government policy. In light of these new research directions, we provide recommendations regarding prioritization of research funding. The goal of this document is to stimulate scientific and public discussion on the need to explore new avenues in AD research, considering outcome and ethics as core principles to reliably judge traditional research efforts and eventually undertake new research strategies.

The human subject: an integrative animal model for 21(st) century heart failure research.

Heart failure remains a leading cause of death and it is a major cause of morbidity and mortality affecting tens of millions of people worldwide. Despite decades of extensive research conducted at enormous expense, only a handful of interventions have significantly impacted survival in heart failure. Even the most widely prescribed treatments act primarily to slow disease progression, do not provide sustained survival advantage, and have adverse side effects. Since mortality remains about 50% within five years of diagnosis, the need to increase our understanding of heart failure disease mechanisms and development of preventive and reparative therapies remains critical. Currently, the vast majority of basic science heart failure research is conducted using animal models ranging from fruit flies to primates; however, insights gleaned from decades of animal-based research efforts have not been proportional to research success in terms of deciphering human heart failure and developing effective therapeutics for human patients. Here we discuss the reasons for this translational discrepancy which can be equally attributed to the use of erroneous animal models and the lack of widespread use of human-based research methodologies and address why and how we must position our own species at center stage as the quintessential animal model for 21(st) century heart failure research. If the ultimate goal of the scientific community is to tackle the epidemic status of heart failure, the best way to achieve that goal is through prioritizing human-based, human-relevant research.

A Human-Based Integrated Framework for Alzheimer's Disease Research.

Animal models of Alzheimer's disease (AD) have been extensively utilized for decades in an effort to elucidate the pathophysiological mechanisms of this disease and to test novel therapeutic approaches. However, research success has not effectively translated into therapeutic success for human patients. This translational failure is partially due to the overuse of animal models that cannot accurately recapitulate human AD etiopathogenesis or drug responses and the inadequate use of human-relevant research methods. Here, we propose how to mitigate this translational barrier by employing human-based methods to elucidate disease processes occurring at multiple levels of complexity, accounting for gene and protein expression and the impact of disease at the cellular, tissue/organ, individual, and population levels. In particular, novel human-based cellular and computational models, together with epidemiological and clinical studies, represent the ideal tools to facilitate human-relevant data acquisition, in the effort to better elucidate AD pathogenesis in a human-based setting and design more effective treatments and preventive strategies. Our analysis indicates that a paradigm shift toward human-based, rather than animal-based research is required in the face of the ever-increasing prevalence of AD in the 21st century.

Leptin- and leptin receptor-deficient rodent models: relevance for human type 2 diabetes.

Among the most widely used animal models in obesity-induced type 2 diabetes mellitus (T2DM) research are the congenital leptin- and leptin receptor-deficient rodent models. These include the leptin-deficient ob/ob mice and the leptin receptor-deficient db/db mice, Zucker fatty rats, Zucker diabetic fatty rats, SHR/N-cp rats, and JCR:LA-cp rats. After decades of mechanistic and therapeutic research schemes with these animal models, many species differences have been uncovered, but researchers continue to overlook these differences, leading to untranslatable research. The purpose of this review is to analyze and comprehensively recapitulate the most common leptin/leptin receptor-based animal models with respect to their relevance and translatability to human T2DM. Our analysis revealed that, although these rodents develop obesity due to hyperphagia caused by abnormal leptin/leptin receptor signaling with the subsequent appearance of T2DM-like manifestations, these are in fact secondary to genetic mutations that do not reflect disease etiology in humans, for whom leptin or leptin receptor deficiency is not an important contributor to T2DM. A detailed comparison of the roles of genetic susceptibility, obesity, hyperglycemia, hyperinsulinemia, insulin resistance, and diabetic complications as well as leptin expression, signaling, and other factors that confound translation are presented here. There are substantial differences between these animal models and human T2DM that limit reliable, reproducible, and translatable insight into human T2DM. Therefore, it is imperative that researchers recognize and acknowledge the limitations of the leptin/leptin receptor- based rodent models and invest in research methods that would be directly and reliably applicable to humans in order to advance T2DM management.

Knockout mouse models of insulin signaling: Relevance past and future.

Insulin resistance is a hallmark of type 2 diabetes. In an effort to understand and treat this condition, researchers have used genetic manipulation of mice to uncover insulin signaling pathways and determine the effects of their perturbation. After decades of research, much has been learned, but the pathophysiology of insulin resistance in human diabetes remains controversial, and treating insulin resistance remains a challenge. This review will discuss limitations of mouse models lacking select insulin signaling molecule genes. In the most influential mouse models, glucose metabolism differs from that of humans at the cellular, organ, and whole-organism levels, and these differences limit the relevance and benefit of the mouse models both in terms of mechanistic investigations and therapeutic development. These differences are due partly to immutable differences in mouse and human biology, and partly to the failure of genetic modifications to produce an accurate model of human diabetes. Several factors often limit the mechanistic insights gained from experimental mice to the particular species and strain, including: developmental effects, unexpected metabolic adjustments, genetic background effects, and technical issues. We conclude that the limitations and weaknesses of genetically modified mouse models of insulin resistance underscore the need for redirection of research efforts toward methods that are more directly relevant to human physiology.

Of rodents and men: species-specific glucose regulation and type 2 diabetes research.

Type 2 diabetes mellitus (T2DM) has reached epidemic proportions worldwide and animal models mimicking human T2DM are widely used to study mechanisms of disease and to develop pharmacotherapeutics. Over the last three decades, rodent models of T2DM have yielded more than 50 publications per month; however, many details of human T2DM pathogenesis remain unclear, and means of preventing disease progression remain elusive. This review investigates the reasons for this translational discrepancy by analyzing the experimental evidence from rodent models of T2DM. The analysis revealed significant species-specific differences at every level of glucose regulation, from gene/protein expression, cellular signaling, tissue and organ to whole organism level, when compared with data acquired using human cells, tissues, organs, and populations. Given the extensive species-specific barrier that creates an immutable translational gap, there is an urgent need to further employ and develop human-based research strategies to make significant strides against the current T2DM epidemic.

Adenosine A1 receptors heterodimerize with ß1- and ß2-adrenergic receptors creating novel receptor complexes with altered G protein coupling and signaling.

G protein coupled receptors play crucial roles in mediating cellular responses to external stimuli, and increasing evidence suggests that they function as multiple units comprising homo/heterodimers and hetero-oligomers. Adenosine and ß-adrenergic receptors are co-expressed in numerous tissues and mediate important cellular responses to the autocoid adenosine and sympathetic stimulation, respectively. The present study was undertaken to examine whether adenosine A1ARs heterodimerize with ß1- and/or ß2-adrenergic receptors (ß1R and ß2R), and whether such interactions lead to functional consequences. Co-immunoprecipitation and co-localization studies with differentially epitope-tagged A1, ß1, and ß2 receptors transiently co-expressed in HEK-293 cells indicate that A1AR forms constitutive heterodimers with both ß1R and ß2R. This heterodimerization significantly influenced orthosteric ligand binding affinity of both ß1R and ß2R without altering ligand binding properties of A1AR. Receptor-mediated ERK1/2 phosphorylation significantly increased in cells expressing A1AR/ß1R and A1AR/ß2R heteromers. ß-Receptor-mediated cAMP production was not altered in A1AR/ß1R expressing cells, but was significantly reduced in the A1AR/ß2R cells. The inhibitory effect of the A1AR on cAMP production was abrogated in both A1AR/ß1R and A1AR/ß2R expressing cells in response to the A1AR agonist CCPA. Co-immunoprecipitation studies conducted with human heart tissue lysates indicate that endogenous A1AR, ß1R, and ß2R also form heterodimers. Taken together, our data suggest that heterodimerization between A1 and ß receptors leads to altered receptor pharmacology, functional coupling, and intracellular signaling pathways. Unique and differential receptor cross-talk between these two important receptor families may offer the opportunity to fine-tune crucial signaling responses and development of more specific therapeutic interventions.

Sex differences and the effects of ovariectomy on the ß-adrenergic contractile response.

The presence of sex differences in myocardial ß-adrenergic responsiveness is controversial, and limited studies have addressed the mechanism underlying these differences. Studies were performed using isolated perfused hearts from male, intact female and ovariectomized female mice to investigate sex differences and the effects of ovarian hormone withdrawal on ß-adrenergic receptor function. Female hearts exhibited blunted contractile responses to the ß-adrenergic receptor agonist isoproterenol (ISO) compared with males but not ovariectomized females. There were no sex differences in ß(1)-adrenergic receptor gene or protein expression. To investigate the role of adenylyl cyclase, phosphodiesterase, and the cAMP-signaling cascade in generating sex differences in the ß-adrenergic contractile response, dose-response studies were performed in isolated perfused male and female hearts using forskolin, 3-isobutyl-1-methylxanthine (IBMX), and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP). Males showed a modestly enhanced contractile response to forskolin at 300 nM and 5 µM compared with females, but there were no sex differences in the response to IBMX or CPT-cAMP. The role of the A(1) adenosine receptor (A(1)AR) in antagonizing the ß-adrenergic contractile response was investigated using both the A(1)AR agonist 2-chloro-N(6)-cyclopentyl-adenosine and A(1)AR knockout (KO) mice. Intact females showed an enhanced A(1)AR anti-adrenergic effect compared with males and ovariectomized females. The ß-adrenergic contractile response was potentiated in both male and female A(1)ARKO hearts, with sex differences no longer present above 1 nM ISO. The ß-adrenergic contractile response is greater in male hearts than females, and minor differences in the action of adenylyl cyclase or the A(1)AR may contribute to these sex differences.

Differential effects of adenosine A2a and A2b receptors on cardiac contractility.

The mammalian myocardium expresses four adenosine receptor (AR) subtypes: A(1)AR, A(2a)AR, A(2b)AR, and A(3)AR. The A(1)AR is well known for its profound antiadrenergic effects, but the roles of other AR subtypes in modulating contractility remain inconclusive. Thus, the objective of this study was to determine the direct and indirect effects of A(2a)AR and A(2b)AR on cardiac contractility. Experiments were conducted in paced, constant pressure-perfused isolated hearts from wild-type (WT), A(2a)AR knockout (KO), and A(2b)AR KO mice. The A(2a)AR agonist CGS-21680 did not alter basal contractility or ß-adrenergic receptor agonist isoproterenol (Iso)-mediated positive inotropic responses, and Iso-induced effects were unaltered in A(2a)AR KO hearts. However, A(2a)AR gene ablation resulted in a potentiation of the antiadrenergic effects mediated by the A(1)AR agonist 2-chloro-N-cyclopentyladenosine. The nonselective AR agonist 5'-N-ethylcarboxamido adenosine and the selective A(2b)AR agonist BAY 60-6583 induced coronary flow-independent increases in contractility, but BAY 60-6583 did not alter Iso-induced contractile responses. The A(1)AR antiadrenergic effect was not potentiated in A(2b)AR KO hearts. The expression of all four AR subtypes in the heart and ventricular myocytes was confirmed using real-time quantitative PCR. Taken together, these results indicate that A(2a)AR does not increase cardiac contractility directly but indirectly alters contractility by modulating the A(1)AR antiadrenergic effect, whereas A(2b)AR exerts direct contractile effects but does not alter ß-adrenergic or A(1)AR antiadrenergic effects. These results indicate that multiple ARs differentially modulate cardiac function.

Determination of apparent calcium affinity for endogenously expressed human sarco(endo)plasmic reticulum calcium-ATPase isoform SERCA3.

The sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs) play a crucial role in regulating free cytosolic Ca(2+) concentration in diverse cell types. It has been shown that recombinant SERCA3, when measured in heterologous systems, exhibits low apparent affinity for Ca(2+); however, Ca(2+) affinity of native SERCA3 in an endogenous setting has not been examined. Such a measurement is complicated, because SERCA3 is always coexpressed with the housekeeping isoform SERCA2b. We used a fluorescence-based assay for monitoring continuous Ca(2+) uptake into microsomes to examine the properties of endogenous human SERCA3 and SERCA2b. The kinetic parameters were derived using a cooperative two-component uptake model for Ca(2+) activation, and the values assigned to SERCA3 were confirmed using the highly specific human SERCA3 inhibitory antibody PL/IM430. First, using recombinant human SERCA3 and SERCA2b proteins transiently expressed in HEK-293 cells, we confirmed the previously observed low apparent Ca(2+) affinity for SERCA3 compared with SERCA2b (1.10 +/- 0.04 vs. 0.26 +/- 0.01 microM), and using mixtures of recombinant protein isoforms, we validated the two-component uptake model. Then we determined apparent Ca(2+) affinity for SERCA proteins present endogenously in cultured Jurkat T lymphocytes and freshly isolated human tonsil lymphocytes. The apparent Ca(2+) affinity in these two preparations was 1.04 +/- 0.07 and 1.1 +/- 0.2 muM for SERCA3 and 0.27 +/- 0.02 and 0.26 +/- 0.01 microM for SERCA2b, respectively. Our data demonstrate, for the first time, that affinity for Ca(2+) is inherently lower for SERCA3 expressed in situ than for other SERCA isoforms.