In vitro mutagenicity of selected environmental carcinogens and their metabolites in MutaMouse FE1 lung epithelial cells.

Chemicals in commerce or under development must be assessed for genotoxicity; assessment is generally conducted using validated assays (e.g. Tk mouse lymphoma assay) as part of a regulatory process. Currently, the MutaMouse FE1 cell mutagenicity assay is undergoing validation for eventual use as a standard in vitro mammalian mutagenicity assay. FE1 cells have been shown to be metabolically competent with respect to some cytochrome P450 (CYP) isozymes; for instance, they can convert the human carcinogen benzo[a]pyrene into its proximate mutagenic metabolite. However, some contradictory results have been noted for other genotoxic carcinogens that require two-step metabolic activation (e.g. 2-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoxaline). Here, we examined three known or suspected human carcinogens, namely acrylamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (4-ABP), together with their proximate metabolites (i.e. glycidamide, N-OH-PhIP and N-OH-4-ABP), to aid in the validation of the FE1 cell mutagenicity assay. Assessments of the parent compounds were conducted both in the presence and absence of an exogenous metabolic activation mixture S9; assessments of the metabolites were in the absence of S9. The most potent compound was N-OH-PhIP -S9, which elicited a mutant frequency (MF) level 5.3-fold over background at 5 µM. There was a 4.3-fold increase for PhIP +S9 at 5 µM, a 1.7-fold increase for glycidamide -S9 at 3.5 mM and a 1.5-fold increase for acrylamide +S9 at 4 mM. Acrylamide -S9 elicited a marginal 1.4-fold MF increase at 8 mM. Treatment with PhIP -S9, 4-ABP ±S9 and N-OH-4-ABP -S9 failed to elicit significant increases in lacZ MF with any of the treatment conditions tested. Gene expression of key CYP isozymes was quantified by RT-qPCR. Cyp1a1, 1a2 and 1b1 are required to metabolise PhIP and 4-ABP. Results showed that treatment with both compounds induced expression of Cyp1a1 and Cyp1b1 but not Cyp1a2. Cyp2e1, which catalyses the bioactivation of acrylamide to glycidamide, was not induced after acrylamide treatment. Overall, our results confirm that the FE1 cell mutagenicity assay has the potential for use alongside other, more traditional in vitro mutagenicity assays.

The utility of metabolic activation mixtures containing human hepatic post-mitochondrial supernatant (S9) for in vitro genetic toxicity assessment.

In vitro genotoxicity assessment routinely employs an exogenous metabolic activation mixture to simulate mammalian metabolism. Activation mixtures commonly contain post-mitochondrial liver supernatant (i.e. S9) from chemically induced Sprague Dawley rats. Although Organization for Economic Cooperation and Development (OECD) test guidelines permit the use of other S9 preparations, assessments rarely employ human-derived S9. The objective of this study is to review and evaluate the use of human-derived S9 for in vitro genetic toxicity assessment. All available published genotoxicity assessments employing human S9 were compiled for analysis. To facilitate comparative analyses, additional matched Ames data using induced rat liver S9 were obtained for certain highly cited chemicals. Historical human and induced rat S9 quality control reports from Moltox were obtained and mined for enzyme activity and mutagenic potency data. Additional in vitro micronucleus data were experimentally generated using human and induced rat S9. The metabolic activity of induced rat S9 was found to be higher than human S9, and linked to high mutagenic potency results. This study revealed that human S9 often yields significantly lower Salmonella mutagenic potency values, especially for polycyclic aromatic hydrocarbons, aflatoxin B1 and heterocyclic amines (~3- to 350-fold). Conversely, assessment with human S9 activation yields higher potency for aromatic amines (~2- to 50-fold). Outliers with extremely high mutagenic potency results were observed in the human S9 data. Similar trends were observed in experimentally generated mammalian micronucleus cell assays, however human S9 elicited potent cytotoxicity L5178Y, CHO and TK6 cell lines. Due to the potential for reduced sensitivity and the absence of a link between enzyme activity levels and mutagenic potency, human liver S9 is not recommended for use alone in in vitro genotoxicity screening assays; however, human S9 may be extremely useful in follow-up tests, especially in the case of chemicals with species-specific metabolic differences, such as aromatic amines.

Mouthguard Use and Awareness of Junior Rugby League Players in the Gold Coast, Australia: A Need for More Education.

OBJECTIVE: To investigate knowledge, attitude, and awareness of appropriate mouthguard use, as well as the management of traumatic dental injuries (TDIs). DESIGN: Cross-sectional survey. SETTING: Community survey. PARTICIPANTS: Four hundred ninety-four junior rugby league players and their coaches in the Gold Coast, Australia. INTERVENTIONS: Data were collected anonymously during training sessions by means of a survey questionnaire, followed by a presentation on mouthguards and the management of TDIs. MAIN OUTCOME MEASURES: Descriptive statistics on the frequency distribution of the demographic characteristics, knowledge, and use of mouthguards as well as the relationships between the type of mouthguard worn and questions related to mouthguard use. RESULTS: The majority of players surveyed reported wearing a mouthguard (68.2%). Cost (40.1%) and not believing that they work (35.7%) were listed as the main reasons for not doing so. The boil-and-bite type was the most used by players (64.7%). Almost 44% of coaches and 50% of players responded that only a dentist can manage an avulsed tooth, that a tooth had to be reinserted within 15 minutes (40.6% and 21.9%, respectively), and that either water or milk can be used to transport an avulsed tooth to the dentist (100% and 82.2%, respectively). CONCLUSIONS: Education related to the correct use of mouthguards and the management of TDIs should be ongoing for players, their caregivers, and their coaches and should be required as part of the training of coaches to prevent injury in rugby league. CLINICAL RELEVANCE: This study found that although the overwhelming majority of junior rugby league coaches encourage the wearing of mouthguards, only two-thirds of the players do so. Players' compliance to wear mouthguards is largely influenced by their perception of efficacy and its comfort. Educational campaigns with involvement of dental practitioners, related to the correct use of mouthguards and the management of TDIs, should be ongoing for players, their caregivers, and their coaches and should also be required as part of the training of coaches.

The mutagenic activity of select azo compounds in MutaMouse target tissues in vivo and primary hepatocytes in vitro.

The mutagenicity of Direct Black 38, Sudan I, and Para Red were evaluated in the in vivo MutaMouse assay and the in vitro MutaMouse primary hepatocyte (PH) assay. Direct Black 38 is an International Agency for Research on Cancer (IARC) Group 1 carcinogen and a prototypical benzidine-based azo compound that requires azo-reduction to yield a DNA-reactive metabolite. Sudan I and Para Red are structurally related azo compounds that have been detected as illegal contaminants in foods. Sudan I is an in vivo mutagen, and both it and Para Red are known to be mutagenic in vitro. Sudan I is oxidized by hepatic and/or bladder enzymes to yield a mutagenic metabolite, but little is known about Para Red. In the present study, Direct Black 38 elicited a significant mutagenic response in the bone marrow, glandular stomach, small intestine and colon in vivo, and in PHs in vitro. Sudan I elicited a weak positive response in the bone marrow and a marginally significant treatment effect in the bladder (p = 0.059); it did not elicit a significant response in PHs in vitro. Para Red elicited a positive response in the colon, as well as in PHs in vitro, albeit at a cytotoxic concentration. The findings are well aligned with the known mechanisms of action of Direct Black 38 and Sudan I; they suggest that intestinal azo-reduction plays an important role in the activation of Para Red. The MutaMouse pH results illustrate the ability of this assay to detect chemicals requiring azo-reduction; however, they also demonstrate a gap in applicability domain, as MutaMouse PHs elicit a negative response following exposure to Sudan I. Elucidation of the mechanisms underlying this gap will require further study.

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part II: Assay performance for the identification of mutagenic chemicals.

As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their "normal" phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct-acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration-dependent increases in mutant frequency (MF) ranging from 2.6- to 14.4-fold over the control. None of the four nonmutagens, including two misleading, or "false," positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3-nitrobenzanthrone [3-NBA], benzo[a]pyrene [BaP], and aflatoxin B1 [AFB1]) to least (i.e., N-ethyl-N-nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in an in vitro gene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348-360, 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes, Part I: Isolation, structural, genetic, and biochemical characterization.

To develop an improved in vitro mammalian cell gene mutation assay, it is imperative to address the known deficiencies associated with existing assays. Primary hepatocytes isolated from the MutaMouse are ideal for an in vitro gene mutation assay due to their metabolic competence, their "normal" karyotype (i.e., neither transformed nor immortalized), and the presence of the MutaMouse transgene for rapid and reliable mutation scoring. The cells were extensively characterized to confirm their utility. Freshly isolated cells were found to have a hepatocyte-like morphology, predominantly consisting of binucleated cells. These cells maintain hepatocyte-specific markers for up to 3 days in culture. Analyses revealed a normal murine hepatocyte karyotype with a modal ploidy number of 4n. Fluorescence in situ hybridization analysis confirmed the presence of the lambda shuttle vector on chromosome 3. The doubling time was determined to be 22.5 ± 3.3 h. Gene expression and enzymatic activity of key Phase I and Phase II metabolic enzymes were maintained for at least 8 and 24 h in culture, respectively. Exposure to ß-naphthoflavone led to approximately 900- and 9-fold increases in Cyp1a1 and Cyp1a2 gene expression, respectively, and approximately twofold induction in cytochrome P450 (CYP) 1A1/1A2 activity. Exposure to phenobarbital resulted in an approximately twofold increase in CYP 2B6 enzyme activity. Following this characterization, it is evident that MutaMouse primary hepatocytes have considerable promise for in vitro mutagenicity assessment. The performance of these cells in an in vitro gene mutation assay is assessed in Part II. Environ. Mol. Mutagen. 60:331-347, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

In vitro mammalian cell mutation assays based on transgenic reporters: A report of the International Workshop on Genotoxicity Testing (IWGT).

Chemical safety evaluations require assessment of genetic toxicity. Transgenic rodent (TGR) assays permit enumeration of mutations in chromosomally-integrated targets contained in shuttle vectors. In order to improve in vitro mutagenicity assessment, and to substantially reduce animal use, in vitro assays using transgenic reporters have been developed. These assays are based on cells derived from TGRs, or cells transfected with transgenic shuttle vectors containing a mutation target. As part of the 7th International Workshop on Genotoxicity Testing, an In Vitro Mammalian Cell Gene Mutation Assay working group reviewed all published information pertaining to in vitro transgene mutagenicity assays; the utility, advantages and disadvantages of the assays were evaluated and discussed. The review revealed that over 20 TGR-based in vitro assays have been used to assess the mutagenic activity of over 150 agents. Overall, the Working Group considered in vitro transgene mutagenicity assays pragmatic tools for the safety evaluation of new and existing substances. A formal SWOT (strengths, weaknesses, opportunities, threats) analysis revealed advantages including the use of established scoring protocols, avoidance of laborious clone isolation and enumeration, ability to use metabolically competent primary cells, ability to detect different types of genetic damage, large dynamic range, and complementarity to in vivo TGR endpoints. Disadvantages include lack of validation and little consistency in protocols, the use of specialised reagents, the time and effort required for mutant enumeration, the use of some cell lines that lack metabolic capacity, and the need for multiple assays to cover all mutational mechanisms. Several assays have been partially validated, indicating promising reliability, reproducibility and applicability domain. Once in vitro transgene mutagenicity assays have been more thoroughly validated, they are well placed to augment or replace existing in vitro mammalian cell mutagenicity assays, particularly in cases where the in vivo TGR mutation assay is intended for follow-up.

Discovery of Pyrazolocarboxamides as Potent and Selective Receptor Interacting Protein 2 (RIP2) Kinase Inhibitors.

Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.

Identification of a RIP1 Kinase Inhibitor Clinical Candidate (GSK3145095) for the Treatment of Pancreatic Cancer.

RIP1 regulates cell death and inflammation and is believed to play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases and cancer. While small-molecule inhibitors of RIP1 kinase have been advanced to the clinic for inflammatory diseases and CNS indications, RIP1 inhibitors for oncology indications have yet to be described. Herein we report on the discovery and profile of GSK3145095 (compound 6). Compound 6 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking RIP1 kinase-dependent cellular responses. Highlighting its potential as a novel cancer therapy, the inhibitor was also able to promote a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures. Compound 6 is currently in phase 1 clinical studies for pancreatic adenocarcinoma and other selected solid tumors.

Discovery and Lead-Optimization of 4,5-Dihydropyrazoles as Mono-Kinase Selective, Orally Bioavailable and Efficacious Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase.

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.

Discovery of a First-in-Class Receptor Interacting Protein 1 (RIP1) Kinase Specific Clinical Candidate (GSK2982772) for the Treatment of Inflammatory Diseases.

RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.

Evaluation of the LacZ reporter assay in cryopreserved primary hepatocytes for In vitro genotoxicity testing.

Assessment of genotoxic potential is an important step in the safety evaluation of chemical substances. Under most regulatory jurisdictions, the first tier of testing comprises a standard battery of in vitro genotoxicity tests in bacterial and mammalian cells. However, the mammalian cell tests commonly used exhibit a relatively high rate of misleading positive results, which may lead to unnecessary in vivo testing. We previously established a proof-of-concept for the LacZ reporter assay in proliferating primary hepatocytes as a promising alternative genotoxicity test. Here, cryopreserved instead of freshly isolated hepatocytes were used and the assay was evaluated in more detail. We examined the effect of cryopreservation on phenotype and metabolic capacity of the LacZ hepatocytes, and assessed the predictive performance of the assay by testing a set of substances comprising true positive, true negative, and misleading positive substances. Additionally, a historical negative control database was created and the type of mutations induced was analyzed for two of the substances tested. Our findings indicate that proliferating cryopreserved primary hepatocytes derived from LacZ plasmid mice retain their hepatocyte-specific characteristics and metabolic competence. Furthermore, we demonstrate that both gene mutations and genome rearrangements due to large deletions can be detected with the LacZ reporter assay. The assay seems to have a lower rate of misleading positive test results compared to the assays currently used. Together, our findings strongly support the use of the LacZ reporter assay in cryopreserved primary hepatocytes as follow-up to the standard in vitro test battery for genotoxicity testing. Environ. Mol. Mutagen. 57:643-655, 2016. © 2016 Wiley Periodicals, Inc.

DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

Evidence-based guideline summary: evaluation, diagnosis, and management of congenital muscular dystrophy: Report of the Guideline Development Subcommittee of the American Academy of Neurology and the Practice Issues Review Panel of the American Association of Neuromuscular & Electrodiagnostic Medicine.

OBJECTIVE: To delineate optimal diagnostic and therapeutic approaches to congenital muscular dystrophy (CMD) through a systematic review and analysis of the currently available literature. METHODS: Relevant, peer-reviewed research articles were identified using a literature search of the MEDLINE, EMBASE, and Scopus databases. Diagnostic and therapeutic data from these articles were extracted and analyzed in accordance with the American Academy of Neurology classification of evidence schemes for diagnostic, prognostic, and therapeutic studies. Recommendations were linked to the strength of the evidence, other related literature, and general principles of care. RESULTS: The geographic and ethnic backgrounds, clinical features, brain imaging studies, muscle imaging studies, and muscle biopsies of children with suspected CMD help predict subtype-specific diagnoses. Genetic testing can confirm some subtype-specific diagnoses, but not all causative genes for CMD have been described. Seizures and respiratory complications occur in specific subtypes. There is insufficient evidence to determine the efficacy of various treatment interventions to optimize respiratory, orthopedic, and nutritional outcomes, and more data are needed regarding complications. RECOMMENDATIONS: Multidisciplinary care by experienced teams is important for diagnosing and promoting the health of children with CMD. Accurate assessment of clinical presentations and genetic data will help in identifying the correct subtype-specific diagnosis in many cases. Multiorgan system complications occur frequently; surveillance and prompt interventions are likely to be beneficial for affected children. More research is needed to fill gaps in knowledge regarding this category of muscular dystrophies.

Discovery of Small Molecule RIP1 Kinase Inhibitors for the Treatment of Pathologies Associated with Necroptosis.

Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.