Applying new approach methodologies to assess next-generation tobacco and nicotine products.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and "roadmaps" to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

An Effective Program to Reduce Malpractice Claims and Payments in a Large Orthopaedic Practice.

BACKGROUND: High reliability in health care requires a balance between intentionally designed systems and individual professional accountability. One element of accountability includes a process for addressing clinicians whose practices are associated with a disproportionate share of patient complaints. This study aimed to evaluate the impact of the Patient Advocacy Reporting System (PARS), a tiered intervention model to reduce patient complaints about clinicians. METHODS: A retrospective cohort study was conducted involving a southeastern U.S. orthopaedic group practice. The study assessed the implementation of the PARS program and subsequent malpractice claims from 2004 to 2020. RESULTS: The implementation of PARS was associated with an 83% reduction in malpractice claims cost per high-risk clinician after intervention (p = 0.05; Wilcoxon rank sum test). The overall practice group experienced an 87% reduction in mean annual claims cost per clinician (p = 0.007; segmented regression). The successful adoption required essential elements such as PARS champions, peer messengers, an Office of Patient Affairs, and a clear statement of practice values and professionalism expectations at the time of onboarding. CONCLUSIONS: The PARS program was successfully adopted within a surgical specialty group as a part of ongoing risk prevention and management efforts. The period following PARS was associated with a retrospectively measured reduction in malpractice claim costs. The PARS program can be effectively implemented in a large, single-specialty orthopaedic practice setting and, although not necessarily causal, was, in our case, associated with a period of reduced malpractice claim costs. CLINICAL RELEVANCE: We have learned in previous research that there are clear links between professionalism and patient outcomes (e.g., surgical complications), but agree that the focus here on medical malpractice is not directly clinical.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro.

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157-0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

An electrophysiological characterization of naturally occurring tobacco alkaloids and their action on human α4ß2 and α7 nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptor (nAChR) subtype-selective pharmacological profiles of tobacco alkaloids are essential for understanding the physiological effects of tobacco products. In this study, automated electrophysiology was used to functionally characterize the effects of distinct groups of tobacco alkaloids on human α4ß2 and α7 nAChRs. We found that, in tobacco alkaloids, pyridine as a hydrogen bond acceptor and a basic nitrogen atom at a distance of 4-7 Šare pharmacophoric elements necessary for molecular recognition by α4ß2 and α7 nAChRs with various degrees of selectivity, potency, and efficacy. While four alkaloids-nicotine, nornicotine, anabasine and R-anatabine-potently activated α4ß2, they were also weak agonists of α7 nAChRs. Nicotine was the most potent agonist of α4ß2, while anabasine elicited the highest activation of α7. None of the tobacco alkaloids enhanced nAChR activity elicited by the endogenous ligand acetylcholine; therefore, none was considered to be a positive allosteric modulator (PAM) of either α4ß2 or α7 nAChRs. In contrast, we identified tobacco alkaloids, such as the tryptophan metabolite 6-hydroxykynurenic acid, that decreased the activity of both α4ß2 and α7 nAChRs. Our study identified a class of alkaloids with positive and negative effects against human α4ß2 and α7 nAChRs. It also revealed human α4ß2 to be the principal receptor for sensing the most abundant alkaloids in tobacco leaves.

Lysosomotropic-related limitations of the BALB/c 3T3 cell-based neutral red uptake assay and an alternative testing approach for assessing e-liquid cytotoxicity.

Cytotoxicity assays are used to quantify the cytotoxic potential of chemicals. The neutral red uptake (NRU) assay is one of these assays and is routinely used in the pharmaceutical, cosmetic, and tobacco industries. In the context of e-cigarette development, an NRU assay-based screen was implemented to evaluate the cytotoxic potential of e-liquids. E-liquids induced a biphasic response in the BALB/c 3T3-based assay. The NRU initially increased in a concentration-dependent manner before decreasing following treatment with higher concentrations until NRU was abolished. Experiments were performed to characterize the mechanism underlying this biphasic signal. Nicotine alone was found to induce the same biphasic effects, while inducing concentration-dependent decreases in relative cell counts (RCC). Imaging and flow cytometry data revealed that the increases in NRU likely resulted from nicotine-induced vacuolization via a lysosomotropic mechanism. In support of this, two lysosomotropic agents, chloroquine and lapatinib, induced similar profiles. Nicotine's effects were also translatable, as brain-, lung-, bone marrow-, and smooth muscle-derived mammalian cells responded with the biphasic NRU signal. However, like RCC, three other cytotoxicity endpoints, resazurin, adenosine triphosphate, and water soluble tetrazolium salt (WST)-8, were not subject to these effects. The WST-8 assay is proposed as an alternative to screen the cytotoxic potential of e-liquids.

Mode-of-action analysis of the effects induced by nicotine in the in vitro micronucleus assay.

Nicotine's genotoxic potential has been extensively studied in vitro. While the results of mammalian cell-based studies have inferred that it can potentially damage chromosomes, in general and with few exceptions, adverse DNA effects have been observed primarily at supraphysiological concentrations in nonregulatory assays that provide little information on its mode-of-action (MoA). In this study, a modern-day regulatory genotoxicity assessment was conducted using a flow cytometry-based in vitro micronucleus (MN) assay, Good Laboratory Practice study conditions, Chinese hamster ovary cells of known provenance, and acceptance/evaluation criteria from the current OECD Test Guideline 487. Nicotine concentrations up to 3.95 mM had no effect on background levels of DNA damage; however, concentrations above the point-of-departure range of 3.94-4.54 mM induced increases in MN and hypodiploid nuclei, indicating a possible aneugenicity hazard. Follow-up experiments designed to elucidate nicotine's MoA revealed cellular vacuolization, accompanying distortions in microtubules, inhibition of tubulin polymerization, centromere-positive DNA, and multinucleate cells at MN-inducing concentrations. Vacuoles likely originated from acidic cellular compartments (e.g., lysosomes). Remarkably, genotoxicity was suppressed by chemicals that raised the luminal pH of these organelles. Other endpoints (e.g., changes in phosphorylated histones) measured in the study cast doubt on the biological relevance of this apparent genotoxicity. In addition, three major nicotine metabolites, including cotinine, had no MN effects but nornicotine induced a nicotine-like profile. It is possible that nicotine's lysosomotropic properties drive the genotoxicity observed in vitro; however, the potency and mechanistic insights revealed here indicate that it is likely of minimal physiological relevance for nicotine consumers. Environ. Mol. Mutagen. 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Evaluation of the Tobacco Heating System 2.2. Part 2: Chemical composition, genotoxicity, cytotoxicity, and physical properties of the aerosol.

The chemical composition, in vitro genotoxicity, and cytotoxicity of the mainstream aerosol from the Tobacco Heating System 2.2 (THS2.2) were compared with those of the mainstream smoke from the 3R4F reference cigarette. In contrast to the 3R4F, the tobacco plug in the THS2.2 is not burnt. The low operating temperature of THS2.2 caused distinct shifts in the aerosol composition compared with 3R4F. This resulted in a reduction of more than 90% for the majority of the analyzed harmful and potentially harmful constituents (HPHCs), while the mass median aerodynamic diameter of the aerosol remained similar. A reduction of about 90% was also observed when comparing the cytotoxicity determined by the neutral red uptake assay and the mutagenic potency in the mouse lymphoma assay. The THS2.2 aerosol was not mutagenic in the Ames assay. The chemical composition of the THS2.2 aerosol was also evaluated under extreme climatic and puffing conditions. When generating the THS2.2 aerosol under "desert" or "tropical" conditions, the generation of HPHCs was not significantly modified. When using puffing regimens that were more intense than the standard Health Canada Intense (HCI) machine-smoking conditions, the HPHC yields remained lower than when smoking the 3R4F reference cigarette with the HCI regimen.

Identification of endocrine cells of the stomach that express acid-sensitive background potassium (K(2P)9.1/TASK3) channels.

The family of two-pore domain potassium (K(2P)) channels is important in setting and controlling the background potassium current of excitable cells. This study examines the localisation of the acid-sensitive channel, K(2P)9.1 (TASK3), in cells of the gastric mucosa. We observed K(2P)9.1 immunoreactivity in endocrine cells of the mucosal glands of the guinea-pig, rat and mouse but the channels were not detected in parietal, chief, or mucous cells. K(2P)9.1 channel immunoreactivity was consistently co-localised with histidine decarboxylase immunopositive enterochromaffin-like (ECL) cells, and with the majority of ghrelin immunoreactive X/A cells. Localisation in somatostatin immunoreactive D cells was rare in the guinea-pig, and did not occur in the stomach of rat, but, in the mouse, K(2P)9.1 channels were observed in the majority of somatostatin-immunoreactive D cells. Conversely, sections taken from the guinea-pig and mouse stomachs, but not rat stomach, revealed K(2P)9.1 in gastrin-containing G cells. These results demonstrate the presence of K(2P)9.1 channels in the entero-endocrine ECL, G and D cell populations of the stomach that regulate acid secretion through the release of histamine, gastrin and somatostatin. K(2P)9.1 channels were located in the ghrelin X/A cells that regulate food intake.

A sphingosine-1-phosphate-activated calcium channel controlling vascular smooth muscle cell motility.

In a screen of potential lipid regulators of transient receptor potential (TRP) channels, we identified sphingosine-1-phosphate (S1P) as an activator of TRPC5. We explored the relevance to vascular biology because S1P is a key cardiovascular signaling molecule. TRPC5 is expressed in smooth muscle cells of human vein along with TRPC1, which forms a complex with TRPC5. Importantly, S1P also activates the TRPC5-TRPC1 heteromultimeric channel. Because TRPC channels are linked to neuronal growth cone extension, we considered a related concept for smooth muscle. We find S1P stimulates smooth muscle cell motility, and that this is inhibited by E3-targeted anti-TRPC5 antibody. Ion permeation involving TRPC5 is crucial because S1P-evoked motility is also suppressed by the channel blocker 2-aminoethoxydiphenyl borate or a TRPC5 ion-pore mutant. S1P acts on TRPC5 via two mechanisms, one extracellular and one intracellular, consistent with its bipolar signaling functions. The extracellular effect appears to have a primary role in S1P-evoked cell motility. The data suggest S1P sensing by TRPC5 calcium channel is a mechanism contributing to vascular smooth muscle adaptation.

Calcium-sensing mechanism in TRPC5 channels contributing to retardation of neurite outgrowth.

The calcium- and sodium-permeable transient receptor potential channel TRPC5 has an inhibitory role in neuronal outgrowth but the mechanisms governing its activity are poorly understood. Here we propose a mechanism involving the neuronal calcium sensor-1 (NCS-1) protein. Inhibitory mutants of TRPC5 and NCS-1 enhance neurite outgrowth similarly. Mutant NCS-1 does not inhibit surface-expression of TRPC5 but generally suppresses channel activity, irrespective of whether it is evoked by carbachol, store depletion, lanthanides or elevated intracellular calcium. NCS-1 and TRPC5 are in the same protein complex in rat brain and NCS-1 directly binds to the TRPC5 C-terminus. The data suggest protein-protein interaction between NCS-1 and TRPC5, and involvement of this protein complex in retardation of neurite outgrowth.

Human TRPC5 channel activated by a multiplicity of signals in a single cell.

Here we explore the activation mechanisms of human TRPC5, a putative cationic channel that was cloned from a region of the X chromosome associated with mental retardation. No basal activity was evident but activity was induced by carbachol stimulation of muscarinic receptors independently of Ca2+ release. This is 'receptor activation', as described for mouse TRPC5. In addition, and in the absence of receptor stimulation, extracellular gadolinium (0.1 mm) activated TRPC5, an effect that was mimicked by 5-20 mm extracellular Ca2+ with intracellular Ca2+ buffered. We refer to this as 'external ionic activation'. TRPC5 was also activated by modest elevation of [Ca2+]i in the absence of GTP--'calcium activation'. A putative fourth activation mechanism is a signal from depleted intracellular Ca2+ stores. Consistent with this idea, human TRPC5 was activated by a standard store-depletion/Ca2+ re-entry protocol, an effect that was difficult to explain by calcium activation. Multiplicity of TRPC5 activation was demonstrated in single cells and thus not dependent on heterogeneity of expression levels or cellular context. Therefore, human TRPC5 is activated by a range of stimuli, avoiding dependence on a single critical activator as in many other ion channels. One of these stimuli would seem to be a change in Ca2+ handling by the endoplasmic reticulum.

Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation.

TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.