BIVV001 Fusion Protein as Factor VIII Replacement Therapy for Hemophilia A.

BACKGROUND: Factor VIII replacement products have improved the care of patients with hemophilia A, but the short half-life of these products affects the patients' quality of life. The half-life of recombinant factor VIII ranges from 15 to 19 hours because of the von Willebrand factor chaperone effect. BIVV001 (rFVIIIFc-VWF-XTEN) is a novel fusion protein designed to overcome this half-life ceiling and maintain high sustained factor VIII activity levels. Data are lacking on the safety and pharmacokinetics of single-dose BIVV001. METHODS: In this phase 1-2a open-label trial, we consecutively assigned 16 previously treated men (18 to 65 years of age) with severe hemophilia A (factor VIII activity, <1%) to receive a single intravenous injection of recombinant factor VIII at a dose of 25 IU per kilogram of body weight (lower-dose group) or 65 IU per kilogram (higher-dose group). This injection was followed by a washout period of at least 3 days. The patients then received a single intravenous injection of BIVV001 at the same corresponding dose of either 25 IU or 65 IU per kilogram. Adverse events and pharmacokinetic measurements were assessed. RESULTS: No inhibitors to factor VIII were detected and no hypersensitivity or anaphylaxis events were reported up to 28 days after the injection of single-dose BIVV001. The geometric mean half-life of BIVV001 was three to four times as long as that of recombinant factor VIII (37.6 hours vs. 9.1 hours in the lower-dose group and 42.5 vs. 13.2 hours in the higher-dose group); the area under the curve (AUC) for product exposure was six to seven times as great in the two dose groups (4470 hours vs. 638 hours × IU per deciliter in the lower-dose group and 12,800 hours vs. 1960 hours × IU per deciliter in the higher-dose group). After the injection of BIVV001 in the higher-dose group, the mean factor VIII level was in the normal range (≥51%) for 4 days and 17% at day 7, which suggested the possibility of a weekly interval between treatments. CONCLUSIONS: In a small, early-phase study involving men with severe hemophilia A, a single intravenous injection of BIVV001 resulted in high sustained factor VIII activity levels, with a half-life that was up to four times the half-life associated with recombinant factor VIII, an increase that could signal a new class of factor VIII replacement therapy with a weekly treatment interval. No safety concerns were reported during the 28-day period after administration. (Funded by Sanofi and Sobi; ClinicalTrials.gov number, NCT03205163.).

Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency.

[Figure: see text].

Heat Generation and Accumulation in Municipal Solid Waste Landfills.

There have been reports of North American landfills that are experiencing temperatures in excess of 80-100 °C. However, the processes causing elevated temperatures are not well understood. The objectives of this study were to develop a model to describe the generation, consumption and release of heat from landfills, to predict landfill temperatures, and to understand the relative importance of factors that contribute to heat generation and accumulation. Modeled heat sources include energy from aerobic and anaerobic biodegradation, anaerobic metal corrosion, ash hydration and carbonation, and acid-base neutralization. Heat removal processes include landfill gas convection, infiltration, leachate collection, and evaporation. The landfill was treated as a perfectly mixed batch reactor. Model predictions indicate that both anaerobic metal corrosion and ash hydration/carbonation contribute to landfill temperatures above those estimated from biological reactions alone. Exothermic pyrolysis of refuse, which is hypothesized to be initiated due to a local accumulation of heat, was modeled empirically to illustrate its potential impact on heat generation.

Evaluation of Soil Manipulation to Prepare Engineered Earthen Waste Covers for Revegetation.

Seven ripping treatments designed to improve soil physical conditions for revegetation were compared on a test pad simulating an earthen cover for a waste disposal cell. The field test was part of study of methods to convert compacted-soil waste covers into evapotranspiration covers. The test pad consisted of a compacted layer of fine-textured soil simulating a barrier protection layer overlain by a gravelly sand bedding layer and a cobble armor layer. Treatments included combinations of soil-ripping implements (conventional shank [CS], wing-tipped shank [WTS], and parabolic oscillating shank with wings [POS]), ripping depths, and number of passes. Dimensions, dry density, moisture content, and particle size distribution of disturbance zones were determined in two trenches excavated across rip rows. The goal was to create a root-zone dry density between 1.2 and 1.6 Mg m and a seedbed soil texture ranging from clay loam to sandy loam with low rock content. All treatments created V-shaped disturbance zones as measured on trench faces. Disturbance zone size was most influenced by ripping depth. Winged implements created larger disturbance zones. All treatments lifted fines into the bedding layer, moved gravel and cobble down into the fine-textured protection layer, and thereby disrupted the capillary barrier at the interface. Changes in dry density within disturbance zones were comparable for the CS and WTS treatments but were highly variable among POS treatments. Water content increased in the bedding layer and decreased in the protection layer after ripping. The POS at 1.2-m depth and two passes created the largest zone with a low dry density (1.24 Mg m) and the most favorable seedbed soil texture (gravely silt loam). However, ripping also created large soil aggregates and voids in the protection layer that may produce preferential flow paths and reduce water storage capacity.

Techno-Economic and life cycle assessment of standalone Single-Stream material recovery facilities in the United states.

Material Recovery Facilities (MRFs) are crucial players in achieving a circular economy. MRFs receive complex waste streams and separate valuable recyclables from these mixtures. This study conducts techno-economic analysis (TEA) to estimate the net present value (NPV) and life cycle assessment (LCA) to estimate different environmental impacts of a commercial scale standalone, single-stream MRF to assess the economic feasibility and environmental impacts of recovering valuable recyclables from an MRF processing 120,000 tonnes per year (t/y). The TEA employs a discounted cash flow rate of return (DCFROR) analysis over a 20-year facility lifetime, along with a sensitivity analysis on the impact of different operating and economic parameters. Results show that the total fixed cost of building the MRF facility is $23 MM, and the operating cost is $45.48/tonne. The NPV of the MRF can vary from $3.57 MM to $60 MM, while 100-year global warming potential can range from 5.98 to 8.53 kg carbon dioxide equivalents (CO2-eq) per tonne of MSW. We have also found that MSW composition (arising from regional effects) significantly impacts costs, 100-year global warming potential, and other impact categories such as acidification potential, eutrophication potential, ecotoxicity, ozone depletion, photochemical oxidation, carcinogenic effects, and non-carcinogenic effects. Sensitivity and uncertainty analysis indicate that waste composition and market prices significantly impact the profitability of the MRF, and the waste composition mostly impacts global warming potential. Our analysis also indicates that facility capacity, fixed capital cost, and waste tipping fees are vital parameters that affect the economic viability of MRF operations.

Evolving radon diffusion through earthen barriers at uranium waste disposal sites.

Field measurements of Rn-222 fluxes from the tops and bottoms of compacted clay radon barriers were used to calculate effective Rn diffusion coefficients (DRn) at four uranium waste disposal sites in the western United States to assess cover performance after more than 20 years of service. Values of DRn ranged from 7.4 × 10-7 to 6.0 × 10-9 m2/s, averaging 1.42 × 10-7. Water saturation (SW) from soil cores indicated that there was relatively little control of DRn by SW, especially at higher moisture levels, in contrast to estimates from most steady-state diffusion models. This is attributed to preferential pathways intrinsic to construction of the barriers or to natural process that have developed over time including desiccation cracks, root channels, and insect burrows in the engineered earthen barriers. A modification to some models in which fast and slow pathway DRn values are partitioned appears to give a good representation of the data; 4% of the fast pathway was needed to fit the data regression. For locations with high Sw and highest DRn (and fluxes) at each site, the proportion of fast pathway ranged from 1.7% to 34%, but for many locations with lower fluxes, little if any fast pathway was needed.

Nuclear waste Educator's workshop: What and how do we teach about nuclear waste?

A workshop was held at the Massachusetts Institute of Technology (MIT) on July 25th and 26th, 2022. The objective was to develop a blueprint for educating next-generation engineers and scientists about nuclear waste management and disposal, which requires knowledge from diverse disciplines, including nuclear, chemical, civil, environmental, and geological science and engineering. The 49 participants included university professors, researchers, industry experts, and government officials from different areas. First, we have developed a list of key fundamental knowledge on waste management and disposal across the nuclear fuel cycle. In addition, we discussed strategies on how to teach students with diverse backgrounds through innovative teaching strategies as well as how to attract students into this area. Through the workshop, we identified the critical needs to (1) develop community resources for nuclear waste education; (2) synthesize historical perspectives, including past contamination and the management of general hazardous waste; (3) emphasize a complete life-cycle perspective, including proper waste management as the key component for energy sustainability; (4) teach students how to communicate about the key facts and risks to technical and non-technical audiences; and (5) accelerate the use of the state-of-art-technologies to attract and retain a young workforce. Furthermore, we aim to build a diverse, inclusive community that supports students in developing their own narratives about nuclear waste, particularly in recognizing that antagonistic views have been important to improving safety and protecting public health and the environment.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities to transform the care of people with hemophilia.

BACKGROUND: Decades of research have transformed hemophilia from severely limiting children's lives to a manageable disorder compatible with a full, active life, for many in high-income countries. The direction of future research will determine whether exciting developments truly advance health equity for all people with hemophilia (PWH). National Hemophilia Foundation (NHF) and American Thrombosis and Hemostasis Network conducted extensive inclusive all-stakeholder consultations to identify the priorities of people with inherited bleeding disorders and those who care for them. RESEARCH DESIGN AND METHODS: Working group (WG) 1 of the NHF State of the Science Research Summit distilled the community-identified priorities for hemophilia A and B into concrete research questions and scored their feasibility, impact, and risk. RESULTS: WG1 defined 63 top priority research questions concerning arthropathy/pain/bone health, inhibitors, diagnostics, gene therapy, the pediatric to adult transition of care, disparities faced by the community, and cardiovascular disease. This research has the potential to empower PWH to thrive despite lifelong comorbidities and achieve new standards of wellbeing, including psychosocial. CONCLUSIONS: Collaborative research and care delivery will be key to capitalizing on current and horizon treatments and harnessing technical advances to improve diagnostics and testing, to advance health equity for all PWH.

Hemophilia is the best known of the inherited bleeding disorders (BD). This is a rare condition that causes disproportionate bleeding, often into joints and vital organs. Factor replacement, injecting recombinant or plasma-based clotting factor products directly into the vein, became commonplace to control the disorder in the 1990s and 2000s. Prophylaxis, or injecting replacement factor every few days into people with hemophilia (PWH), has revolutionized patients' lives. In the last few years, other advances in new therapies have entered this space, such as non-factor replacement therapies and gene therapy. With many more research advances on the horizon, the National Hemophilia Foundation (NHF) initiated a State of the Science Research Summit in 2020. This event was attended by over 880 interested parties to help design an agenda of research priorities for inherited BDs for the next decade, based on community consultations. NHF formed multiple Working Groups (WG), each exploring a theme resulting from the community consultations, and presenting their results at the Summit. Led by 2 hematologists who manage and treat PWH daily, the 21-community member WG1 assigned to hemophilia A and B divided into 7 subgroups to identify and organize research priorities for different topic areas. The outcomes focused on prioritizing patients' needs, technological advances, and research in the areas of greatest potential for PWH and those who care for them. The results are a roadmap for the future execution of a research plan that truly serves the community.

Efanesoctocog alfa for hemophilia A: results from a phase 1 repeat-dose study.

Efanesoctocog alfa (rFVIIIFc-VWF-XTEN; BIVV001) is a new class of factor VIII (FVIII) replacement that breaks the von Willebrand factor-imposed FVIII half-life ceiling. In a phase 1/2a study, single-dose efanesoctocog alfa was well tolerated, and no safety concerns were identified. We evaluated the safety, tolerability, and pharmacokinetics of repeat-dose efanesoctocog alfa in a phase 1 study in previously treated adults (≥150 exposure days) with severe hemophilia A. Participants received 4 once weekly doses of efanesoctocog alfa (cohort 1, 50 IU/kg; cohort 2, 65 IU/kg). All enrolled participants (cohort 1, n = 10; cohort 2, n = 14) completed the study. Inhibitor development to FVIII was not detected. After the last dose of efanesoctocog alfa, geometric mean (range) FVIII activity half-life, area under the activity-time curve, and steady-state maximum concentration for cohort 1 and cohort 2 were 41.3 (34.2-50.1) and 37.3 (28.9-43.8) hours, 8290 (5810-10 300) and 11 200 (7040-15 800) hours × IU/dL, and 131 (96-191) and 171 (118-211) IU/dL, respectively. There was minimal accumulation after 4 doses. Mean FVIII activity for cohort 1 and cohort 2, respectively, was 46% and 69% on day 3 postdose and 10% and 12% on day 7 postdose. Overall, 4 once-weekly doses of efanesoctocog alfa were well tolerated, no safety concerns were identified, and no bleeds were reported during the treatment period. Once-weekly efanesoctocog alfa provided high sustained FVIII activity within the normal to near-normal range for 3 to 4 days postdose and may improve protection against bleeds in patients with hemophilia A. The trial is study 2018-001535-51 in the EU Clinical Trials Register.

Identifying functional single nucleotide polymorphisms in the human CArGome.

Regulatory SNPs (rSNPs) reside primarily within the nonprotein coding genome and are thought to disturb normal patterns of gene expression by altering DNA binding of transcription factors. Nevertheless, despite the explosive rise in SNP association studies, there is little information as to the function of rSNPs in human disease. Serum response factor (SRF) is a widely expressed DNA-binding transcription factor that has variable affinity to at least 1,216 permutations of a 10 bp transcription factor binding site (TFBS) known as the CArG box. We developed a robust in silico bioinformatics screening method to evaluate sequences around RefSeq genes for conserved CArG boxes. Utilizing a predetermined phastCons threshold score, we identified 8,252 strand-specific CArGs within an 8 kb window around the transcription start site of 5,213 genes, including all previously defined SRF target genes. We then interrogated this CArG dataset for the presence of previously annotated common polymorphisms. We found a total of 118 unique CArG boxes harboring a SNP within the 10 bp CArG sequence and 1,130 CArG boxes with SNPs located just outside the CArG element. Gel shift and luciferase reporter assays validated SRF binding and functional activity of several new CArG boxes. Importantly, SNPs within or just outside the CArG box often resulted in altered SRF binding and activity. Collectively, these findings demonstrate a powerful approach to computationally define rSNPs in the human CArGome and provide a foundation for similar analyses of other TFBS. Such information may find utility in genetic association studies of human disease where little insight is known regarding the functionality of rSNPs.

Role of the Nalp3 inflammasome in acetaminophen-induced sterile inflammation and liver injury.

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in the US and UK. Recent studies implied that APAP-induced injury is partially mediated by interleukin-1ß (IL-1ß), which can activate and recruit neutrophils, exacerbating injury. Mature IL-1ß is formed by caspase-1, dependent on inflammasome activation. The objective of this invetstigation was to evaluate the role of the Nalp3 inflammasome on release of damage associated molecular patterns (DAMPs), hepatic neutrophil accumulation and liver injury (ALT, necrosis) after APAP overdose. Mice deficient for each component of the Nalp3 inflammasome (caspase-1, ASC and Nalp3) were treated with 300mg/kg APAP for 24h; these mice had similar neutrophil recruitment and liver injury as APAP-treated C57Bl/6 wildtype animals. In addition, plasma levels of DAMPs (DNA fragments, keratin-18, hypo- and hyper-acetylated forms of high mobility group box-1 protein) were similarly elevated with no significant difference between wildtype and gene knockout mice. In addition, aspirin treatment, which has been postulated to attenuate cytokine formation and the activation of the Nalp3 inflammasome after APAP, had no effect on release of DAMPs, hepatic neutrophil accumulation or liver injury. Together, these data confirm the release of DAMPs and a sterile inflammatory response after APAP overdose. However, as previously reported minor endogenous formation of IL-1ß and the activation of the Nalp3 inflammasome have little impact on APAP hepatotoxicity. It appears that the Nalp3 inflammasome is not a promising therapeutic target to treat APAP overdose.

Radon fluxes at four uranium mill tailings disposal sites after about 20 years of service.

To evaluate the properties of earthen covers over uranium mill tailings disposal cells after about 20 years of service, we measured Rn-222 fluxes and radon barrier properties at the Falls City, TX, Bluewater, NM, Shirley Basin South, WY, and Lakeview, OR disposal sites in western USA. A total of 115 in-service Rn fluxes were obtained at 26 test pit locations from the top surface of the exposed Rn barrier (i.e., after protective layers were removed by excavation) and 24 measurements were obtained from the surface of the underlying waste after excavation through the Rn barrier layer. Rn-222 concentrations were determined in accumulation chambers using a continuously monitoring electronic radon monitor (ERM) equipped with a solid-state alpha particle detector. Effects of surface features on Rn flux including vegetation, seasonal ponding, and animal burrowing were quantified. Comparison of measured fluxes with values that were measured shortly after the Rn barriers were completed (as-built) show that most measurements fell within the range of the as-built fluxes, generally at very low fluxes. At two sites fluxes were measured that were greater than the highest as-built flux. High fluxes are typically caused by a combination of enhanced moisture removal and preferential pathways for Rn transport, often caused by deep-rooted plants. Such localized features result in a spatially heterogeneous distribution of fluxes that can vary substantially over only a meter or two.

Targeting of antithrombin in hemophilia A or B with investigational siRNA therapeutic fitusiran-Results of the phase 1 inhibitor cohort.

BACKGROUND: Fitusiran, an investigational small interfering RNA therapy, reduces antithrombin production to rebalance hemostasis in people with hemophilia A or B, with or without inhibitors. OBJECTIVES: To evaluate the safety and efficacy of fitusiran treatment for people with moderate/severe hemophilia A or B with inhibitors. PATIENTS/METHODS: In this open-label phase 1, part D study, 17 males with hemophilia A or B with inhibitors received three once-monthly subcutaneous injections of fitusiran 50 mg (n = 6) or 80 mg (n = 11); followed for up to 112 days. Endpoints included safety (primary), pharmacokinetics/pharmacodynamics (secondary), annualized bleeding rate, and patient-reported outcomes (exploratory). RESULTS: The most common adverse event was injection site erythema (n = 8). No thrombotic events were reported. At nadir, mean (standard error of the mean [SEM]) antithrombin activity decreased from baseline by 82.0% (2.2) and 87.4% (0.7) in the 50 mg and 80 mg groups, respectively. Antithrombin reduction was associated with increased thrombin generation. 11/17 (64.7%) participants had no bleeds during the observation period (mean [standard deviation] 69.4 [16.3] days). Mean (SEM) changes from baseline in Haemophilia Quality of Life Questionnaire for Adults total (-9.2 [2.9]) and physical health (-12.3 [3.9]) domain scores suggested clinically meaningful improvement. CONCLUSIONS: Monthly fitusiran was generally well tolerated, lowered antithrombin levels from baseline, and resulted in improved thrombin generation. These preliminary results suggest that monthly fitusiran treatment may reduce bleeding episodes and improve quality of life in participants with hemophilia A or B with inhibitors.

The potential of cytokines as safety biomarkers for drug-induced liver injury.

Drug-induced liver injury (DILI) is an event that has a detrimental impact on drug development and patient safety; therefore the identification of novel biomarkers that are both sensitive and specific to the liver would have great benefit. Inflammation is known to be associated with human cases of DILI, and given the role of cytokines in modulating the inflammatory response, changes in cytokine expression patterns certainly show promise as potential biomarkers of DILI. Cytokines are interesting candidates for novel biomarkers as they are relatively accessible (by blood sampling) and accurately quantifiable. In particular, recent interest has developed in mechanism-specific, rather than tissue-specific, biomarkers. However, without fully understanding the role of inflammation in DILI and the role of cytokines in modulating the inflammatory response, cytokines may be limited in their use, being either diagnostic of the type of injury that has occurred and/or prognostic of outcome (recovery from DILI, cirrhosis, acute liver failure). Intracellular components released by damaged hepatocytes, although inaccessible and currently difficult to quantify, may be better biomarkers for the prognosis of severity of injury. In both cases there is a pressing need for the development and validation of assays sensitive enough and with a sufficient dynamic range to detect changes upon drug treatment. Although promising candidates are appearing in the literature, much remains to be done to understand the role of inflammation in DILI and the role that a given cytokine has in the inflammatory cascade associated with DILI before cytokines are viewed as biomarkers for DILI.

Transport of the pathogenic prion protein through soils.

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases and include bovine spongiform encephalopathy of cattle, chronic wasting disease (CWD) of deer and elk, scrapie in sheep and goats, and Creutzfeldt-Jakob disease in humans. An abnormally folded form of the prion protein (designated PrP(TSE)) is typically associated with TSE infectivity and may constitute the major, if not sole, component of the infectious agent. Transmission of CWD and scrapie is mediated in part by an environmental reservoir of infectivity. Soil appears to be a plausible candidate for this reservoir. The transport of TSE agent through soil is expected to influence the accessibility of the pathogen to animals after deposition and must be understood to assess the risks associated with burial of infected carcasses. We report the results of saturated column experiments designed to evaluate PrP(TSE) transport through five soils with relatively high sand or silt contents and low organic carbon content. Protease-treated TSE-infected brain homogenate was used as a model for PrP(TSE) present in decomposing infected tissue. Synthetic rainwater was used as the eluent. All five soils retained PrP(TSE); no detectable PrP(TSE) was eluted over more than 40 pore volumes of flow. Lower bound apparent attachment coefficients were estimated for each soil. Our results suggest that TSE agent released from decomposing tissues to soils with low organic carbon content would remain near the site of initial deposition. In the case of infected carcasses deposited on the land surface, this may result in local sources of infectivity to other animals.

Hydraulic and mechanical behavior of municipal solid waste and high-moisture waste mixtures.

The objective of this study was to investigate how addition of high-moisture waste (HMW) affects the hydraulic and mechanical behavior of municipal solid waste (MSW). Direct shear and hydraulic conductivity tests were conducted on MSW, HMW, and MSW-HMW mixtures prepared with HMW contents ranging from 20% to 80% (by total mass). Direct shear tests were conducted at normal stress between 22 and 168 kPa and hydraulic conductivity tests were conducted at vertical effective stresses of approximately 50, 100, and 200 kPa. A threshold HMW content of 40% was identified corresponding to substantial change in friction angle and hydraulic conductivity of the mixtures. Municipal solid waste and MSW-HMW mixtures with less than 40% HMW had friction angles between 29° and 32° and hydraulic conductivities greater than or equal to 1.3 × 10-6 m/s. At HMW contents above 40%, the friction angle and hydraulic conductivity decreased with increasing HMW content. At 80% HMW, the hydraulic and mechanical behavior of the MSW-HMW mixture was comparable to HMW. The HMW had a friction angle of approximately 2° and hydraulic conductivity of 1.1 × 10-11 m/s at a vertical effective stress of 50 kPa. Additional direct shear tests conducted on MSW and MSW-HMW mixtures soaked in water to simulate subsequent wetting post disposal revealed a decrease in friction angle from approximately 29° to 24° for MSW mixed with 40% HMW.

The impact of pressure, moisture and temperature on pyrolysis of municipal solid waste under simulated landfill conditions and relevance to the field data from elevated temperature landfill.

Experiments were conducted with simulated Municipal Solid Waste (MSW) to understand the impact of pressure, moisture, and temperature on MSW decomposition under simulated landfill conditions. Three experimental phases were completed, where the first two phases provided baseline results and assisted in fine tuning parameters such as pressure, temperature, gas composition, and moisture content for phase three. The manuscript focuses on the results from third phase. In the third phase, the composition of the gases evolved from representative MSW samples was tested over time in two pressure conditions, 101 kilopascals (kPa) (atmospheric pressure) and 483 kPa, with varying moisture contents (38 to 55 wt%) and controlled temperatures (50 to 200 °C) in the presence of biological inhibitors. The headspace in the reactor in phase three was pressurized with gas mixture of 50/50 (vol%) of methane (CH4) and carbon dioxide (CO2) setting the initial CH4/CO2 gas composition ratio to 1.0 at time t = 0 days. The results established moisture ranges that affect hydrogen (H2) production and the CH4/CO2 ratio at different temperature and pressure conditions. Results show that at 85 °C, there was a change in the CH4/CO2 ratio from 1.0 to 0.3. Additionally, moisture contents from 47 to 43.5 wt% caused the CH4/CO2 ratio to increase from 1.0 to 1.2, yet from 43.5 to 38 wt%, the ratio reversed and declined to 0.3, returning to 1.0 for moisture levels below 38 wt%. Thus, moisture levels above 47 wt% and below 38 wt%, for the system tested, allow thermal reactions to proceed without a measured change in CH4/CO2 ratio. H2 generation rates follow a similar trend with moisture, yet definitively increase with increased pressure from 101 kPa to 483 kPa. The observed change in solid MSW and gas composition under controlled pressure, moisture, and temperature suggests the presence of thermal reactions in the absence of oxygen.

Functional and toxicological consequences of metabolic bioactivation of methapyrilene via thiophene S-oxidation: Induction of cell defence, apoptosis and hepatic necrosis.

UNLABELLED: Methapyrilene, [N,N-dimethyl-N'-pyridyl-N'(2-thienylmethyl)-1,2-ethanediamine] (MP) was withdrawn from, clinical use due to reported periportal hepatic necrosis and hepatocarcinogenicity in the rat, via S-oxidation of the thiophene group. In this study MP is used as a model hepatotoxin to further characterise the functional consequences of S-oxidation of the thiophene group in vivo, in rat models and in vitro, in freshly isolated rat hepatocyte suspensions. In vivo histological studies revealed the early depletion of glutathione (GSH), which was confined to the damaged periportal area, in contrast to an increase in GSH levels in the centrilobular region. Additionally, the induction of cell defence was demonstrated by an increase in the protein levels of heme-oxygenase 1 (HO-1) and glutamate cysteine ligase, catalytic subunit (GCLC) in vivo. Histological examination demonstrated that cytotoxicity progresses initially via apoptosis before an increase in necrosis over the 3-day administration. An apoptotic-like mechanism was observed in vitro via the measurement of cytochrome c release and caspase activation. CONCLUSION: This study provides evidence for a complex pathway of MP-induced hepatotoxicity which progresses through early adaptation, apoptosis, necrosis and inflammation, all underpinned by the zonal induction and depletion of GSH within the liver.

Investigation of the effect of a panel of model hepatotoxins on the Nrf2-Keap1 defence response pathway in CD-1 mice.

The Keap1-Nrf2-ARE signalling pathway has emerged as an important regulator of the mammalian defence system to enable detoxification and clearance of foreign chemicals. Recent studies by our group using paracetamol (APAP), diethylmaleate and buthionine sulphoximine have shown that for a given xenobiotic molecule, Nrf2 induction in the murine liver is associated with protein reactivity and glutathione depletion. Here, we have investigated, in vivo, whether the ability of four murine hepatotoxins, paracetamol, bromobenzene (BB), carbon tetrachloride (CCl4) and furosemide (FS) to deplete hepatic glutathione (GSH) is related to induction of hepatic Nrf2 nuclear translocation and Nrf2-dependent gene expression. Additionally, we studied whether hepatic Nrf2 nuclear translocation is a general response during the early stages of acute hepatic chemical stress in vivo. Male CD-1 mice were administered APAP (3.5 mmol/kg), FS (1.21 mmol/kg), BB (4.8 mmol/kg) and CCl4 (1 mmol/kg) for 1, 5 and 24h. Each compound elicited significant serum ALT increases after 24h (ALT U/L: APAP, 3036+/-1462; BB, 5308+/-2210; CCl4, 5089+/-1665; FS, 2301+/-1053), accompanied by centrilobular damage as assessed by histopathology. Treatment with APAP also elicited toxicity at a much earlier time point (5h) than the other hepatotoxins (ALT U/L: APAP, 1780+/-661; BB, 161+/-15; CCl4, 90+/-23; FS, 136+/-27). Significant GSH depletion was seen with APAP (9.6+/-1.7% of control levels) and BB (52.8+/-6.2% of control levels) 1h after administration, but not with FS and CCl4. Western Blot analysis revealed an increase in nuclear Nrf2, 1h after administration of BB (209+/-10% control), CCl4 (146+/-3% control) and FS (254+/-41% control), however this was significantly lower than the levels observed in the APAP-treated mice (462+/-36% control). The levels of Nrf2-dependent gene induction were also analysed by quantitative real-time PCR and Western blotting. Treatment with APAP for 1h caused a significant increase in the levels of haem oxygenase-1 (HO-1; 2.85-fold) and glutamate cysteine ligase (GCLC; 1.62-fold) mRNA. BB and FS did not affect the mRNA levels of either gene after 1h of treatment; however CCl4 significantly increased HO-1 mRNA at this time point. After 24h treatment with the hepatotoxins, there was evidence for the initiation of a late defence response. BB significantly increased both HO-1 and GCLC protein at this time point, CCl4 increased GCLC protein alone, although FS did not alter either of these proteins. In summary, we have demonstrated that the hepatotoxins BB, CCl4 and FS can induce a small but significant increase in Nrf2 accumulation in hepatic nuclei. However, this was associated with modest changes in hepatic GSH, a delayed development of toxicity and was insufficient to activate an early functional adaptive response to these hepatotoxins.