Proteomic profiling of centrosomes across multiple mammalian cell and tissue types by an affinity capture method.

Centrosomes are the major microtubule-organizing centers in animals and play fundamental roles in many cellular processes. Understanding how their composition varies across diverse cell types and how it is altered in disease are major unresolved questions, yet currently available centrosome isolation protocols are cumbersome and time-consuming, and they lack scalability. Here, we report the development of centrosome affinity capture (CAPture)-mass spectrometry (MS), a powerful one-step purification method to obtain high-resolution centrosome proteomes from mammalian cells. Utilizing a synthetic peptide derived from CCDC61 protein, CAPture specifically isolates intact centrosomes. Importantly, as a bead-based affinity method, it enables rapid sample processing and multiplexing unlike conventional approaches. Our study demonstrates the power of CAPture-MS to elucidate cell-type-dependent heterogeneity in centrosome composition, dissect hierarchical interactions, and identify previously unknown centrosome components. Overall, CAPture-MS represents a transformative tool to unveil temporal, regulatory, cell-type- and tissue-specific changes in centrosome proteomes in health and disease.

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

Ordered dephosphorylation initiated by the selective proteolysis of cyclin B drives mitotic exit.

APC/C-mediated proteolysis of cyclin B and securin promotes anaphase entry, inactivating CDK1 and permitting chromosome segregation, respectively. Reduction of CDK1 activity relieves inhibition of the CDK1-counteracting phosphatases PP1 and PP2A-B55, allowing wide-spread dephosphorylation of substrates. Meanwhile, continued APC/C activity promotes proteolysis of other mitotic regulators. Together, these activities orchestrate a complex series of events during mitotic exit. However, the relative importance of regulated proteolysis and dephosphorylation in dictating the order and timing of these events remains unclear. Using high temporal-resolution proteomics, we compare the relative extent of proteolysis and protein dephosphorylation. This reveals highly-selective rapid proteolysis of cyclin B, securin and geminin at the metaphase-anaphase transition, followed by slow proteolysis of other substrates. Dephosphorylation requires APC/C-dependent destruction of cyclin B and was resolved into PP1-dependent categories with unique sequence motifs. We conclude that dephosphorylation initiated by selective proteolysis of cyclin B drives the bulk of changes observed during mitotic exit.

New cells are made when a single cell duplicates its DNA and divides into two cells, distributing the DNA equally between them, in a process called mitosis. The splitting of the two copies of DNA happens through a series of controlled events known as mitotic exit. Previous research has suggested that mitotic exit relies on both the destruction of specific proteins and the removal of tags called phosphate groups from other proteins. Phosphate groups modify how proteins behave and their removal can trigger changes in a protein's activity. Although protein destruction and phosphate group removal were known to be important to mitotic exit, it was not understood how they are coordinated in the cell to ensure the correct order of events. Holder et al. have used a technique called mass spectrometry to monitor the level of thousands of proteins, and any tags attached to them, during mitotic exit in human cells grown in the laboratory. The experiments revealed that the destruction of a single protein, known as cyclin B, plays a major role in triggering subsequent events. The removal of cyclin B activates enzymes known as phosphatases, which remove phosphate groups from proteins. Phosphatases then act on a wide range of proteins in a specific order that depends on the environment surrounding the phosphate group. This 'chain' of phosphatase activity determines the order of events during mitotic exit. The findings of Holder et al. contribute to the basic understanding of how mitotic exit works. Errors in the process can affect the stability of a cell's genome, contributing to diseases such as cancer. In the future, this may help to identify what goes wrong in these cases and potential avenues for developing treatments.

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20.

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/CCDC20) form the main ubiquitin E3 ligase for these two proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation-defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

The sixth international RASopathies symposium: Precision medicine-From promise to practice.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.

Getting out of mitosis: spatial and temporal control of mitotic exit and cytokinesis by PP1 and PP2A.

Here, we will review the evidence showing that mitotic exit is initiated by regulated proteolysis and then driven by the PPP family of phosphoserine/threonine phosphatases. Rapid APC/CCDC20 and ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid separation, the first step of mitotic exit. Because proteolysis of Aurora and Polo family kinases dependent on APC/CCDH1 is relatively slow, this creates a new regulatory state, anaphase, different to G2 and M-phase. We will discuss how the CDK1-counteracting phosphatases PP1 and PP2A-B55, together with Aurora and Polo kinases, contribute to the temporal regulation and order of events in the different stages of mitotic exit from anaphase to cytokinesis. For PP2A-B55, these timing properties are created by the ENSA-dependent inhibitory pathway and differential recognition of phosphoserine and phosphothreonine. Finally, we will discuss how Aurora B and PP2A-B56 are needed for the spatial regulation of anaphase spindle formation and how APC/C-dependent destruction of PLK1 acts as a timer for abscission, the final event of cytokinesis.

CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization.

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.

MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling.

Cyclin B-dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.

Glass ionomer cements with milled, dry chlorhexidine hexametaphosphate filler particles to provide long-term antimicrobial properties with recharge capacity.

OBJECTIVE: Glass ionomer cements (GICs) are a versatile material, offering the opportunity for ion exchange with the oral environment. The aim of this study was to develop a GIC that delivers a controlled, rechargeable dose of chlorhexidine (CHX) over an extended period without compromising mechanical properties. METHODS: GICs were supplemented with finely milled particles of chlorhexidine hexametaphosphate (CHX-HMP). CHX release into artificial saliva was measured over 660 days, and recharge with CHX and CHX-HMP was investigated. Mechanical properties were investigated, and an agar diffusion test was carried out to assess antimicrobial properties using Streptococcus mutans and Scardovia wiggsiae. RESULTS: Dose-dependent CHX release was observed, and this was ongoing at 660 days. Compared with related studies of GICs containing CHX-HMP, the fine, dry particles resulted in fewer adverse effects on mechanical properties, including tensile, compressive and biaxial flexural strength, with 1% CHX-HMP GICs indistinguishable from control specimens. The GICs could be recharged with CHX using both a conventional CHX digluconate solution comparable to commercial mouthrinses, and a suspension of CHX-HMP of equivalent concentration. Recharging with CHX digluconate increased subsequent CHX release by 50% compared with no recharge, and recharging with CHX-HMP increased subsequent CHX release by 100% compared with no recharge. The GICs inhibited growth of St. mutans and Sc. wiggsiae in a simple agar diffusion model. SIGNIFICANCE: These materials, which provide sustained CHX release over clinically relevant timescales, may find application as a restorative material intended to inhibit secondary caries as well as in temporary restorations and fissure sealants.

A PP2A-B55 recognition signal controls substrate dephosphorylation kinetics during mitotic exit.

PP2A-B55 is one of the major phosphatases regulating cell division. Despite its importance for temporal control during mitotic exit, how B55 substrates are recognized and differentially dephosphorylated is unclear. Using phosphoproteomics combined with kinetic modeling to extract B55-dependent rate constants, we have systematically identified B55 substrates and assigned their temporal order in mitotic exit. These substrates share a bipartite polybasic recognition determinant (BPR) flanking a Cdk1 phosphorylation site. Experiments and modeling show that dephosphorylation rate is encoded into B55 substrates, including its inhibitor ENSA, by cooperative action of basic residues within the BPR. A complementary acidic surface on B55 decodes this signal, supporting a cooperative electrostatic mechanism for substrate selection. A further level of specificity is encoded into B55 substrates because B55 displays selectivity for phosphothreonine. These simple biochemical properties, combined with feedback control of B55 activity by the phosphoserine-containing substrate/inhibitor ENSA, can help explain the temporal sequence of events during exit from mitosis.

Glass ionomer cements functionalised with a concentrated paste of chlorhexidine hexametaphosphate provides dose-dependent chlorhexidine release over at least 14 months.

OBJECTIVES: The aim of this study was to create prototype glass ionomer cements (GICs) incorporating a concentrated paste of chlorhexidine-hexametaphosphate (CHX-HMP), and to investigate the long-term release of soluble chlorhexidine and the mechanical properties of the cements. The purpose is the design of a glass ionomer with sustained anticaries efficacy. METHODS: CHX-HMP paste was prepared by mixing equimolar solutions of chlorhexidine digluconate and sodium hexametaphosphate, adjusting ionic strength, decanting and centrifuging. CHX-HMP paste was incorporated into a commercial GIC in substitution for glass powder at 0.00, 0.17, 0.34, 0.85 and 1.70% by mass CHX-HMP. Soluble chlorhexidine release into artificial saliva was observed over 436 days using absorbance at 255nm. Diametral tensile and compressive strength were measured after 7 days' setting (37°C, 100% humidity) and tensile strength after 436 days' aging in artificial saliva. 0.34% CHX-HMP GICs were tested for their ability to inhibit growth of Streptococcus mutans in vitro. RESULTS: GICs supplemented with CHX-HMP exhibited a sustained dose-dependent release of soluble chlorhexidine. Diametral tensile strength of new specimens was unaffected up to and including 0.85% CHX-HMP, and individual values of tensile strength were unaffected by aging, but the proportion of CHX-HMP required to adversely affect tensile strength was lower after aging, at 0.34%. Compressive strength was adversely affected by CHX-HMP at substitutions of 0.85% CHX-HMP and above. CONCLUSIONS: Supplementing a GIC with CHX-HMP paste resulted in a cement which released soluble chlorhexidine for over 14 months in a dose dependent manner. 0.17% and 0.34% CHX-HMP did not adversely affect strength at baseline, and 0.17% CHX-HMP did not affect strength after aging. 0.34% CHX-HMP GICs inhibited growth of S. mutans at a mean distance of 2.34mm from the specimen, whereas control (0%) GICs did not inhibit bacterial growth. CLINICAL SIGNIFICANCE: Although GICs release fluoride in vivo, there is inconclusive evidence regarding any clinical anticaries effect. In this study, GICs supplemented with a paste of chlorhexidine-hexametaphosphate (CHX-HMP) exhibited a sustained release of chlorhexidine over at least 14 months, and small additions of CHX-HMP did not adversely affect strength.

A molecular mechanism of mitotic centrosome assembly in Drosophila.

Centrosomes comprise a pair of centrioles surrounded by pericentriolar material (PCM). The PCM expands dramatically as cells enter mitosis, but it is unclear how this occurs. In this study, we show that the centriole protein Asl initiates the recruitment of DSpd-2 and Cnn to mother centrioles; both proteins then assemble into co-dependent scaffold-like structures that spread outwards from the mother centriole and recruit most, if not all, other PCM components. In the absence of either DSpd-2 or Cnn, mitotic PCM assembly is diminished; in the absence of both proteins, it appears to be abolished. We show that DSpd-2 helps incorporate Cnn into the PCM and that Cnn then helps maintain DSpd-2 within the PCM, creating a positive feedback loop that promotes robust PCM expansion around the mother centriole during mitosis. These observations suggest a surprisingly simple mechanism of mitotic PCM assembly in flies.

Development of a novel antimicrobial-releasing glass ionomer cement functionalized with chlorhexidine hexametaphosphate nanoparticles.

BACKGROUND: Glass ionomer cements (GICs) are a class of dental biomaterials. They have a wide range of uses including permanent restorations (fillings), cavity linings, fissure sealants and adhesives. One of the most common reasons for replacing a dental restoration is recurrent bacterial tooth decay around the margins of the biomaterial. Therefore, a dental biomaterial which creates a sustained antimicrobial environment around the restoration would be of considerable clinical benefit. In this manuscript, the formulation of a GIC containing novel antimicrobial nanoparticles composed of chlorhexidine hexametaphosphate at 1, 2, 5, 10 and 20% powder substitution by mass is reported. The aim is to create GICs which contain chlorhexidine-hexametaphosphate nanoparticles and characterize the nanoparticle size, morphology and charge and the release of chlorhexidine and fluoride, tensile strength and morphology of the GICs. RESULTS: The GICs released chlorhexidine, which is a broad spectrum antimicrobial agent effective against a wide range of oral bacteria, over the duration of the experiment in a dose-dependent manner. This was not at the expense of other properties; fluoride release was not significantly affected by the substitution of antimicrobial nanoparticles in most formulations and internal structure appeared unaffected up to and including 10% substitution. Diametral tensile strength decreased numerically with substitutions of 10 and 20% nanoparticles but this difference was not statistically significant. CONCLUSION: A series of GICs functionalized with chlorhexidine-hexametaphosphate nanoparticles were created for the first time. These released chlorhexidine in a dose-dependent manner. These materials may find application in the development of a new generation of antimicrobial dental nanomaterials.

The BEG (PP2A-B55/ENSA/Greatwall) pathway ensures cytokinesis follows chromosome separation.

Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55α/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway.

Physical and physicochemical factors effecting transport of chlorohydrocarbon gases from lung alveolar air to blood as measured by the causation of narcosis.

This systematic investigation examines gas transport in the lung for two sets of chlorohydrocarbons (CHCs): the chloromethanes (C1) and chloroethanes (C2). The C1 series includes chloromethane, methylene chloride, chloroform, and carbon tetrachloride, and the C2 series includes chloroethane, 1,2-dichloroethane, 1, 1, 2-trichloroethane, and 1, 1, 2, 2-tetrachloroethane. Most CHC gases cause narcosis. The comprehensive narcosis work of Lehmann and colleagues on CHCs was used as a basis for the narcosis endpoint in the present examination. The sites for narcosis are located in the brain (midline cortex and posterior parietal area), the spine, and at many peripheral nerve sites. Central nervous system (CNS) exposure executes a multisite, neural transmission set of inhibitions that promotes rapid loss of consciousness, sensory feeling, and current and stored memory while providing temporary amnesia. Absorption into the system requires dissolution into many lipid membranes and binding to lipoproteins. Lipophilicity is a CHC property shared with many anesthetics according to the Meyer-Overton Rule. Many structurally different lipid chemicals produce the narcosis response when the lipid concentration exceeds -67 mM. This suggests narcotic or anesthetic dissolution into CNS membranes until the lipid organization is disrupted or perturbed. This perturbation includes loading of Na(+)- and K(+)-channel transmembrane lipoprotein complexes and disrupting their respective channel functional organizations. The channel functions become attenuated or abrogated until the CHC exposure ceases and CHC loading reverses. This investigation demonstrates how the CHC physical and chemical properties influence the absorption of these CHCs via the lung and the alveolar system on route to the blood. Narcosis in test animals was used here as an objective biological endpoint to study the effects of the physical factors Bp, Vp, Kd (oil: gas) partition, Henry's constant (HK), and water solubility (S%) on gas transport. Narcosis is immediate after gas exposure and requires no chemical activation only absorption into the blood and circulation to CNS narcotic sites. The three physical factors Bp, K(d) (oil: air), and S% vary directly with unitary narcosis (UN) whereas Vp and HK vary inversely with UN in linear log-log relationships for the C2 series but not for the C1 series. Physicochemical properties of C1 series gases indicate why they depart from what is usually assumed to be an Ideal Gas. An essential discriminating process in the distal lung is the limiting alveolar film layer (AFL) and the membrane layer of the alveolar acini. The AFL step influences gas uptake by physically limiting the absorption process. Interaction with and dissolution into aqueous solvent of the AFL is required for transport and narcotic activity. Narcotics or anesthetics must engage the aqueous AFL with sufficient strength to allow transport and absorption for downstream CNS binding. CHCs that do not engage well with the AFL are not narcotic. Lipophilicity and amphipathicity are also essential solvency properties driving narcotics' transport through the alveolar layer, delivery to the blood fats and lipoproteins, and into critical CNS lipids, lipoproteins, and receptor sites that actuate narcosis. AFL disruption is thought to be strongly related to a number of serious pulmonary diseases such acute respiratory distress syndrome, infant respiratory distress syndrome, emphysema, chronic obstructive pulmonary disease, asthma, chronic bronchitis, pneumonia, pulmonary infections, and idiopathic pulmonary fibrosis. The physical factors (Bp, Vp, Kd [oil: gas] partition, Henry's constant, and water solubility [S%]) combine to affect a specific transport through the AFL if lung C > C(0) (threshold concentration for narcosis). The degree of blood CHC absorption depends on dose, lipophilicity, and lung residence time. AFL passage can be manipulated by physical factors of increased pressure (kPa) or increased gas exposure (moles). Molecular lipophilicity facilitates narcosis but lipophilicity alone does not explain narcosis. Vapor pressure is also required for narcosis. Narcotic activity apparently requires stereospecific processing in the AFL and/or down-stream inhibition at stereospecific lipoproteins at CNS inhibitory sites. It is proposed that CHCs likely cannot proceed through the AFL without perturbation or disruption of the integrity of the AFL at the alveoli. CHC physicochemical properties are not expected to allow their transport through the AFL as physiological CO(2) and O(2) naturally do in respiration. This work considers CHC inspiration and systemic absorption into the blood with special emphasis on the CHC potential perturbation effects on the lipid, protein liquid layer supra to the alveolar membrane (AFL). A heuristic gas transport model for the CHCs is presented as guidance for this examination. The gas transport model can be used to study absorption for other gas delivery endpoints of environmental concern such as carcinogens.

Physiologically based pharmacokinetic modeling of chloroethane disposition in mice, rats, and women.

Chloroethane was observed in a chronic cancer bioassay to be a mouse-specific uterine carcinogen at a single high inhaled concentration (15,000 ppm). Although high incidence occurred in the female mouse (86%), no uterine tumor increases were observed in female rats. Chloroethane is a weak alkylating agent and has low acute toxicity. No genotoxicity potential has been observed below 40,000 ppm. Chloroethane is eliminated from the body by pulmonary exhalation and metabolically by oxidation via cytochrome P-450 (likely producing acetaldehyde) and conjugation with glutathione (GSH). The mode of action for the mouse-specific uterine tumors is not definitively known and could involve parent chemical and/or metabolite(s). A physiologically based pharmacokinetic (PBPK) model for chloroethane disposition in the rat was developed previously, but no such models have been described for mice or humans. For the work reported here, the existing PBPK model for chloroethane in rats was expanded and refined, and PBPK models for chloroethane disposition in mice and humans were developed to allow species comparisons of internal dosimetry and for possible insights into the carcinogenic mode of action. The amounts metabolized via glutathione-S-transferase (GST) versus cytochrome P-450, and the total amount of chloroethane absorbed, were most consistent with the observations made concerning uterine tumors, with amounts metabolized via GST providing the larger quantitative difference between the two rodent species. Choice of the most relevant dose metric for risk assessments involving uterine tumors in mice will require pharmacodynamic considerations in the mode of action in addition to the pharmacokinetic differences reported here.

Framework analysis for the carcinogenic mode of action of nitrobenzene.

Nitrobenzene (CASRN: 98-95-3) has been shown to induce cancers in many tissues including kidney, liver, and thyroid, following chronic inhalation in animals. However, with a few exceptions, genotoxicity assays using nitrobenzene have given negative results. Some DNA binding/adduct studies have brought forth questionable results and, considering the available weight of evidence, it does not appear that nitrobenzene causes cancer via a genotoxic mode of action. Nitrobenzene produces a number of free radicals during its reductive metabolism, in the gut as well as at the cellular level, and generates superoxide anion as a by-product during oxidative melabolism. The reactive species generated during nitrobenzene metabolism are considered candidates for carcinogenicity. Furthermore, several lines of evidence suggest that nitrobenzene exerts its carcinogenicity through a non-DNA reactive (epigenetic) fashion, such as a strong temporal relationship between non-, pre-, and neoplastic lesions leading to carcinogenesis. In this report, we first describe the absorption, distribution, metabolism, and excretion of nitrobenzene followed by a summary of the available genotoxicity studies and the only available cancer bioassay. We subsequently refer to the mode of action framework of the U.S. Environmental Protection Agency's 2005 Guidelines for Carcinogen Risk Assessment as a basis for presenting possible modes of action for nitrobenzene-induced cancers of the liver, thyroid, and kidney, as supported by the available experimental data. The rationale(s) regarding human relevance of each mode of action is also presented. Finally, we hypothesize that the carcinogenic mode of action for nitrobenzene is multifactorial in nature and reflective of free radicals, inflammation, and/or altered methylation.

Bromoethane, chloroethane and ethylene oxide induced uterine neoplasms in B6C3F1 mice from 2-year NTP inhalation bioassays: pathology and incidence data revisited.

Chloroethane, bromoethane and ethylene oxide represent a unique set of chemicals that induce endometrial neoplasms in the uterus of B6C3F1 mice following an inhalation route of exposure. The results of the NTP's chronic bioassays with these three compounds resulted in an unusually high incidence of uterine epithelial neoplasms in B6C3F1 mice (chloroethane 86%, bromoethane 56%) and a lower incidence for ethylene oxide (10%). The uterine neoplasms were classified as adenomas, adenocarcinomas, and squamous cell carcinomas for bromoethane, and as adenocarcinomas for both chloroethane and ethylene oxide. The adenocarcinomas and squamous cell carcinomas were invasive into the myometrium and the serosa, and metastasized to a wide variety of organs. Metastatic sites included most commonly the lung, lymph nodes, and ovary at unusually high rates of metastases (79% for chloroethane and 38% for bromoethane). Because of the dramatically high rates of uterine neoplasms (induced by chemicals given by the inhalation route) and metastases, a re-evaluation of the pathology and incidence data was undertaken. The earlier results were confirmed. The mechanism of uterine carcinogenesis by chloroethane, bromoethane and ethylene oxide is unclear.