Developing a pragmatic consensus procedure supporting the ICH S1B(R1) weight of evidence carcinogenicity assessment.

The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review. First, it is acknowledged that the six WoE factors described in the addendum form an integrated network of evidence within a holistic assessment framework that is used synergistically to analyze and explain safety signals. Second, the proposed standardized procedure builds upon different considerations related to the primary sources of evidence, mechanistic analysis, alternative methodologies and novel investigative approaches, metabolites, and reliability of the data and other acquired information. Each of the six WoE factors is described highlighting how they can contribute evidence for the overall WoE assessment. A suggested reporting format to summarize the cross-integration of evidence from the different WoE factors is also presented. This work also notes that even if a 2-year rat study is ultimately required, creating a WoE assessment is valuable in understanding the specific factors and levels of human carcinogenic risk better than have been identified previously with the 2-year rat bioassay alone.

In silico approaches in carcinogenicity hazard assessment: case study of pregabalin, a nongenotoxic mouse carcinogen.

In silico toxicology protocols are meant to support computationally-based assessments using principles that ensure that results can be generated, recorded, communicated, archived, and then evaluated in a uniform, consistent, and reproducible manner. We investigated the availability of in silico models to predict the carcinogenic potential of pregabalin using the ten key characteristics of carcinogens as a framework for organizing mechanistic studies. Pregabalin is a single-species carcinogen producing only one type of tumor, hemangiosarcomas in mice via a nongenotoxic mechanism. The overall goal of this exercise is to test the ability of in silico models to predict nongenotoxic carcinogenicity with pregabalin as a case study. The established mode of action (MOA) of pregabalin is triggered by tissue hypoxia, leading to oxidative stress (KC5), chronic inflammation (KC6), and increased cell proliferation (KC10) of endothelial cells. Of these KCs, in silico models are available only for selected endpoints in KC5, limiting the usefulness of computational tools in prediction of pregabalin carcinogenicity. KC1 (electrophilicity), KC2 (genotoxicity), and KC8 (receptor-mediated effects), for which predictive in silico models exist, do not play a role in this mode of action. Confidence in the overall assessments is considered to be medium to high for KCs 1, 2, 5, 6, 7 (immune system effects), 8, and 10 (cell proliferation), largely due to the high-quality experimental data. In order to move away from dependence on animal data, development of reliable in silico models for prediction of oxidative stress, chronic inflammation, immunosuppression, and cell proliferation will be critical for the ability to predict nongenotoxic compound carcinogenicity.

Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches.

Neurotoxicology is the study of adverse effects on the structure or function of the developing or mature adult nervous system following exposure to chemical, biological, or physical agents. The development of more informative alternative methods to assess developmental (DNT) and adult (NT) neurotoxicity induced by xenobiotics is critically needed. The use of such alternative methods including in silico approaches that predict DNT or NT from chemical structure (e.g., statistical-based and expert rule-based systems) is ideally based on a comprehensive understanding of the relevant biological mechanisms. This paper discusses known mechanisms alongside the current state of the art in DNT/NT testing. In silico approaches available today that support the assessment of neurotoxicity based on knowledge of chemical structure are reviewed, and a conceptual framework for the integration of in silico methods with experimental information is presented. Establishing this framework is essential for the development of protocols, namely standardized approaches, to ensure that assessments of NT and DNT based on chemical structures are generated in a transparent, consistent, and defendable manner.

Evidence for a semisolid phase state of aerosols and droplets relevant to the airborne and surface survival of pathogens.

The phase state of respiratory aerosols and droplets has been linked to the humidity-dependent survival of pathogens such as SARS-CoV-2. To inform strategies to mitigate the spread of infectious disease, it is thus necessary to understand the humidity-dependent phase changes associated with the particles in which pathogens are suspended. Here, we study phase changes of levitated aerosols and droplets composed of model respiratory compounds (salt and protein) and growth media (organic-inorganic mixtures commonly used in studies of pathogen survival) with decreasing relative humidity (RH). Efflorescence was suppressed in many particle compositions and thus unlikely to fully account for the humidity-dependent survival of viruses. Rather, we identify organic-based, semisolid phase states that form under equilibrium conditions at intermediate RH (45 to 80%). A higher-protein content causes particles to exist in a semisolid state under a wider range of RH conditions. Diffusion and, thus, disinfection kinetics are expected to be inhibited in these semisolid states. These observations suggest that organic-based, semisolid states are an important consideration to account for the recovery of virus viability at low RH observed in previous studies. We propose a mechanism in which the semisolid phase shields pathogens from inactivation by hindering the diffusion of solutes. This suggests that the exogenous lifetime of pathogens will depend, in part, on the organic composition of the carrier respiratory particle and thus its origin in the respiratory tract. Furthermore, this work highlights the importance of accounting for spatial heterogeneities and time-dependent changes in the properties of aerosols and droplets undergoing evaporation in studies of pathogen viability.

Principles and Procedures for Assessment of Acute Toxicity Incorporating In Silico Methods.

Acute toxicity in silico models are being used to support an increasing number of application areas including (1) product research and development, (2) product approval and registration as well as (3) the transport, storage and handling of chemicals. The adoption of such models is being hindered, in part, because of a lack of guidance describing how to perform and document an in silico analysis. To address this issue, a framework for an acute toxicity hazard assessment is proposed. This framework combines results from different sources including in silico methods and in vitro or in vivo experiments. In silico methods that can assist the prediction of in vivo outcomes (i.e., LD50) are analyzed concluding that predictions obtained using in silico approaches are now well-suited for reliably supporting assessment of LD50-based acute toxicity for the purpose of GHS classification. A general overview is provided of the endpoints from in vitro studies commonly evaluated for predicting acute toxicity (e.g., cytotoxicity/cytolethality as well as assays targeting specific mechanisms). The increased understanding of pathways and key triggering mechanisms underlying toxicity and the increased availability of in vitro data allow for a shift away from assessments solely based on endpoints such as LD50, to mechanism-based endpoints that can be accurately assessed in vitro or by using in silico prediction models. This paper also highlights the importance of an expert review of all available information using weight-of-evidence considerations and illustrates, using a series of diverse practical use cases, how in silico approaches support the assessment of acute toxicity.

In silico approaches in organ toxicity hazard assessment: Current status and future needs for predicting heart, kidney and lung toxicities.

The kidneys, heart and lungs are vital organ systems evaluated as part of acute or chronic toxicity assessments. New methodologies are being developed to predict these adverse effects based on in vitro and in silico approaches. This paper reviews the current state of the art in predicting these organ toxicities. It outlines the biological basis, processes and endpoints for kidney toxicity, pulmonary toxicity, respiratory irritation and sensitization as well as functional and structural cardiac toxicities. The review also covers current experimental approaches, including off-target panels from secondary pharmacology batteries. Current in silico approaches for prediction of these effects and mechanisms are described as well as obstacles to the use of in silico methods. Ultimately, a commonly accepted protocol for performing such assessment would be a valuable resource to expand the use of such approaches across different regulatory and industrial applications. However, a number of factors impede their widespread deployment including a lack of a comprehensive mechanistic understanding, limited in vitro testing approaches and limited in vivo databases suitable for modeling, a limited understanding of how to incorporate absorption, distribution, metabolism, and excretion (ADME) considerations into the overall process, a lack of in silico models designed to predict a safe dose and an accepted framework for organizing the key characteristics of these organ toxicants.

In silico approaches in organ toxicity hazard assessment: current status and future needs in predicting liver toxicity.

Hepatotoxicity is one of the most frequently observed adverse effects resulting from exposure to a xenobiotic. For example, in pharmaceutical research and development it is one of the major reasons for drug withdrawals, clinical failures, and discontinuation of drug candidates. The development of faster and cheaper methods to assess hepatotoxicity that are both more sustainable and more informative is critically needed. The biological mechanisms and processes underpinning hepatotoxicity are summarized and experimental approaches to support the prediction of hepatotoxicity are described, including toxicokinetic considerations. The paper describes the increasingly important role of in silico approaches and highlights challenges to the adoption of these methods including the lack of a commonly agreed upon protocol for performing such an assessment and the need for in silico solutions that take dose into consideration. A proposed framework for the integration of in silico and experimental information is provided along with a case study describing how computational methods have been used to successfully respond to a regulatory question concerning non-genotoxic impurities in chemically synthesized pharmaceuticals.

In Silico Approaches In Carcinogenicity Hazard Assessment: Current Status and Future Needs.

Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.

Principles and procedures for handling out-of-domain and indeterminate results as part of ICH M7 recommended (Q)SAR analyses.

The International Council for Harmonization (ICH) M7 guideline describes a hazard assessment process for impurities that have the potential to be present in a drug substance or drug product. In the absence of adequate experimental bacterial mutagenicity data, (Q)SAR analysis may be used as a test to predict impurities' DNA reactive (mutagenic) potential. However, in certain situations, (Q)SAR software is unable to generate a positive or negative prediction either because of conflicting information or because the impurity is outside the applicability domain of the model. Such results present challenges in generating an overall mutagenicity prediction and highlight the importance of performing a thorough expert review. The following paper reviews pharmaceutical and regulatory experiences handling such situations. The paper also presents an analysis of proprietary data to help understand the likelihood of misclassifying a mutagenic impurity as non-mutagenic based on different combinations of (Q)SAR results. This information may be taken into consideration when supporting the (Q)SAR results with an expert review, especially when out-of-domain results are generated during a (Q)SAR evaluation.

Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca(2+)-ATPase by Competitive Inhibition of [γ-(32)P]TNP-8N3-ATP Photolabeling.

The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca(2+)-ATPase, taking advantage of the specific photolabeling of Lys(492) in the Ca(2+)-ATPase by [γ-(32)P]2',3'-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5'-triphosphate ([γ-(32)P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca(2+)-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-(32)P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

Regreening in spathes of Zantedeschia after anthesis: its physiology and control by fructification and hormones.

The mature pigmented spathe of Zantedeschia is characterized by a developmental process, wherein the spathe regreens after anthesis and prior to senescence of the inflorescence. Previous research has shown that spathe regreening involves redifferentiation of chloroplasts and re-accumulation of chlorophyll, but the detailed physiological changes associated with regreening are still largely unknown. Using Zantedeschia aethiopica and the Zantedeschia pentlandii variety 'Best Gold' as models, this study explores the physiological mechanism and possible roles of fructification, 6-benzylaminopurine (BAP) and gibberellin (GA3 ) in induction or progression of spathe regreening. Application of BAP stimulated regreening in spathe tissue of 'Best Gold' by enhancing accumulation of carotenoid and chlorophyll, and also increasing stacking of grana. In contrast, GA3 retarded formation of double-membrane lamella during chloroplast redifferentiation, thus delaying the onset of regreening. We suggest that these actions of BAP and GA3 have a synergistic effect in delaying the onset of regreening in 'Best Gold' so that when applied together retardation of chlorophyll accumulation, chloroplast redifferentiation and accumulation of carotenoids were enhanced. The elimination of fructification did not prevent the occurrence of regreening in either Zantedeschia model plants, indicating that fructification was not a prerequisite for the induction of regreening. It is still unclear how regreening in Zantedeschia is triggered. We propose that the onset of regreening in Zantedeschia is likely to be a genetically programmed event.

Mechanisms of glyphosate resistance in two perennial ryegrass (Lolium perenne) populations.

BACKGROUND: Perennial ryegrass (Lolium perenne) has developed resistance to glyphosate within New Zealand vineyards following many years of herbicide application. The objectives of this work were to confirm resistance within two populations obtained from affected vineyards, and to determine the mechanism of resistance to glyphosate. RESULTS: Population O was confirmed to have a 25-fold resistance to glyphosate, whereas population J had a sevenfold resistance. Results of genotyping assays demonstrated a single nucleotide substitution at codon 106 of 5-enolpyruvylshikimate-3-phosphate synthase in population O but not population J. Glyphosate-resistant and glyphosate-susceptible populations did not differ in glyphosate absorption. However, in both resistant populations, much more of the absorbed (14) C-glyphosate remained in the treated leaf than occurred in the susceptible population. Significantly more glyphosate was found in the pseudostem region of susceptible plants compared with resistant plants. CONCLUSION: Both target-site and non-target-site mechanisms of glyphosate resistance were found in the perennial ryegrass population with 25-fold resistance, whereas only the non-target-site mechanism of resistance was found in the population with sevenfold resistance. This is the first study of the mechanism of glyphosate resistance in perennial ryegrass.

Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase.

The mechanism of ATP modulation of E2P dephosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase wild type and mutant forms was examined in nucleotide binding studies of states analogous to the various intermediates of the dephosphorylation reaction, obtained by binding of metal fluorides, vanadate, or thapsigargin. Wild type Ca(2+)-ATPase displays an ATP affinity of 4 µM for the E2P ground state analog, 1 µM for the E2P transition state and product state analogs, and 11 µM for the E2 dephosphoenzyme. Hence, ATP binding stabilizes the transition and product states relative to the ground state, thereby explaining the accelerating effect of ATP on dephosphorylation. Replacement of Phe(487) (N-domain) with serine, Arg(560) (N-domain) with leucine, or Arg(174) (A-domain) with alanine or glutamate reduces ATP affinity in all E2/E2P intermediate states. Alanine substitution of Ile(188) (A-domain) increases the ATP affinity, although ATP acceleration of dephosphorylation is disrupted, thus indicating that the critical role of Ile(188) in ATP modulation is mechanistically based rather than being associated with the binding of nucleotide. Mutants with alanine replacement of Lys(205) (A-domain) or Glu(439) (N-domain) exhibit an anomalous inhibition by ATP of E2P dephosphorylation, due to ATP binding increasing the stability of the E2P ground state relative to the transition state. The ATP affinity of Ca(2)E2P, stabilized by inserting four glycines in the A-M1 linker, is similar to that of the E2P ground state, but the Ca(2+)-free E1 state of this mutant exhibits 3 orders of magnitude reduction of ATP affinity.

Flagellar oscillation: a commentary on proposed mechanisms.

Eukaryotic flagella and cilia have a remarkably uniform internal 'engine' known as the '9+2' axoneme. With few exceptions, the function of cilia and flagella is to beat rhythmically and set up relative motion between themselves and the liquid that surrounds them. The molecular basis of axonemal movement is understood in considerable detail, with the exception of the mechanism that provides its rhythmical or oscillatory quality. Some kind of repetitive 'switching' event is assumed to occur; there are several proposals regarding the nature of the 'switch' and how it might operate. Herein I first summarise all the factors known to influence the rate of the oscillation (the beating frequency). Many of these factors exert their effect through modulating the mean sliding velocity between the nine doublet microtubules of the axoneme, this velocity being the determinant of bend growth rate and bend propagation rate. Then I explain six proposed mechanisms for flagellar oscillation and review the evidence on which they are based. Finally, I attempt to derive an economical synthesis, drawing for preference on experimental research that has been minimally disruptive of the intricate structure of the axoneme. The 'provisional synthesis' is that flagellar oscillation emerges from an effect of passive sliding direction on the dynein arms. Sliding in one direction facilitates force-generating cycles and dynein-to-dynein synchronisation along a doublet; sliding in the other direction is inhibitory. The direction of the initial passive sliding normally oscillates because it is controlled hydrodynamically through the alternating direction of the propulsive thrust. However, in the absence of such regulation, there can be a perpetual, mechanical self-triggering through a reversal of sliding direction due to the recoil of elastic structures that deform as a response to the prior active sliding. This provisional synthesis may be a useful basis for further examination of the problem.

A multiwell electrochemical biosensor for real-time monitoring of the behavioural changes of cells in vitro.

We report the development of a multiwell biosensor for detecting changes in the electrochemical open circuit potential (OCP) generated by viable human cells in vitro. The instrument features eight culture wells; each containing three gold sensors around a common silver/silver chloride reference electrode, prepared using screen-printed conductive inks. The potential applications of the device were demonstrated by monitoring rheumatoid synovial fibroblasts (RSF) and HepG2 hepatocarcinoma cells in response to chemical and biological treatments. This technology could provide an alternative to conventional end-point assays used in the fields of chemotherapy, toxicology and drug discovery.

A study of synchronisation between the flagella of bull spermatozoa, with related observations.

Flagellar synchronisation has been observed between bull spermatozoa as they swam in a viscous medium, confined to a glass surface. This process is of interest in understanding the regulation of flagellar oscillation in general. Exact and persisting synchrony between bull spermatozoa occurred only when the spermatozoan heads were tightly coupled mechanically. For these cells, viscous coupling between the flagella was not by itself sufficient to establish synchronisation. Immediately on synchronisation, with the spermatozoan heads superposed, the paired spermatozoa showed rises in conjoint beat frequency, wave velocity and swimming velocity, i.e. in nearly all cases, the new conjoint values were greater than those shown by either of the two singleton spermatozoa. In our interpretation of these results, we put forward hydrodynamic arguments for seeing the primary change as a rise in wave velocity, via a decreased viscous resistance to bend propagation. Mechanistically, the rise in beat frequency is mysterious unless, as we suggest, it is consequential to the rise in wave velocity, and mediated by an as-yet-unknown mechanical feedback process. The rise in swimming velocity is not surprising given the rise in wave velocity but there is evidence for an additional influence due to a subtle re-orientation of the conjoint spermatozoan heads, such that they experienced less frictional drag.

Critical interaction of actuator domain residues arginine 174, isoleucine 188, and lysine 205 with modulatory nucleotide in sarcoplasmic reticulum Ca2+-ATPase.

ATP plays dual roles in the reaction cycle of the sarcoplasmic reticulum Ca2+-ATPase by acting as the phosphorylating substrate as well as in nonphosphorylating (modulatory) modes accelerating conformational transitions of the enzyme cycle. Here we have examined the involvement of actuator domain residues Arg174, Ile188, Lys204, and Lys205 by mutagenesis. Alanine mutations to these residues had little effect on the interaction of the Ca2E1 state with nucleotide or on the HnE 2 to Ca2E1 transition of the dephosphoenzyme. The phosphoenzyme processing steps, Ca2E1P to E2P and E2P dephosphorylation, and their stimulation by MgATP/ATP were markedly affected by mutations to Arg174, Ile188, and Lys205. Replacement of Ile188 with alanine abolished nucleotide modulation of dephosphorylation but not the modulation of the Ca2E1P to E2P transition. Mutation to Arg174 interfered with nucleotide modulation of either of the phosphoenzyme processing steps, indicating a significant overlap between the modulatory nucleotide-binding sites involved. Mutation to Lys205 enhanced the rates of the phosphoenzyme processing steps in the absence of nucleotide and disrupted the nucleotide modulation of the Ca2E1P to E2P transition. Remarkably, the mutants with alterations to Lys205 showed an anomalous inhibition by ATP of the dephosphorylation, and in the alanine mutant the affinity for the inhibition by ATP was indistinguishable from that for stimulation by ATP of the wild type. Hence, the actuator domain is an important player in the function of ATP as modulator of phosphoenzyme processing, with Arg174, Ile188, and Lys205 all being critically involved, although in different ways. The data support a variable site model for the modulatory effects with the nucleotide binding somewhat differently in each of the conformational states occurring during the transport cycle.

Cell separation with horizontal cell electrophoresis under near-isopycnic conditions on a "density cushion".

This report describes an improvement made to the horizontal cell electrophoresis methodology. It involves using two liquid layers differing in density to produce an interface described as a "density cushion". The electrophoretic system that employed an anti-convective porous matrix to separate red blood cells (RBC) and charged dyes effectively was found to be unsuitable for some other mammalian cells. The "density cushion" method was found to be more versatile and applicable to studies on the separation of a variety of cell types. The experiments described show the differences between the electrophoretic mobilities of a human eosinophilic leukaemia cell line (Eol-1) and RBC, both with and without the modification of the cell surface properties.

Electrochemical monitoring of rat mammary adenocarcinoma cells: an in vitro assay for anticancer drug selection.

We report the application of an electrochemical biosensor (Oncoprobe; Marks & Clerk, Manchester, UK) to determine whether changes in the open circuit potential (OCP) of rat mammary adenocarcinoma cells (MTLn3) treated in vitro with four cytotoxic anticancer drugs could predict their effects in vivo. MTLn3 cells were seeded onto sensors, then exposed to each anticancer compound (cisplatin, doxorubicin, paclitaxel, or vinblastine), and monitored for 44 hours. Electrochemical monitoring in vitro detected OCP responses to all four drugs, with cisplatin and doxorubicin producing greater changes over a shorter period than vinblastine and paclitaxel. Syngeneic MTLn3 cells were used to generate palpable tumors in 50 female Fischer 344 rats. Animals were divided into five equal groups; on day 12 four of the groups received an anticancer drug, and one received a saline control. Fourteen days later the animals were killed, and primary tumor weights were determined. Tumors from cisplatin- and doxorubicin-treated rats were significantly reduced in weight compared to the control, paclitaxel-, and vinblastine-treated groups. The anticancer drug-induced changes observed through real-time electrochemical monitoring of MTLn3 cells in vitro correlated well with the in vivo animal model, unlike the conventional end-point assays of lactate dehydrogenase release and Alamar Blue.

Neurofascin as a novel target for autoantibody-mediated axonal injury.

Axonal injury is considered the major cause of disability in patients with multiple sclerosis (MS), but the underlying effector mechanisms are poorly understood. Starting with a proteomics-based approach, we identified neurofascin-specific autoantibodies in patients with MS. These autoantibodies recognize the native form of the extracellular domains of both neurofascin 186 (NF186), a neuronal protein concentrated in myelinated fibers at nodes of Ranvier, and NF155, the oligodendrocyte-specific isoform of neurofascin. Our in vitro studies with hippocampal slice cultures indicate that neurofascin antibodies inhibit axonal conduction in a complement-dependent manner. To evaluate whether circulating antineurofascin antibodies mediate a pathogenic effect in vivo, we cotransferred these antibodies with myelin oligodendrocyte glycoprotein-specific encephalitogenic T cells to mimic the inflammatory pathology of MS and breach the blood-brain barrier. In this animal model, antibodies to neurofascin selectively targeted nodes of Ranvier, resulting in deposition of complement, axonal injury, and disease exacerbation. Collectively, these results identify a novel mechanism of immune-mediated axonal injury that can contribute to axonal pathology in MS.