Continuous developmental change explains discontinuities in word learning.

Cognitive development is often characterized in terms of discontinuities, but these discontinuities can sometimes be apparent rather than actual and can arise from continuous developmental change. To explore this idea, we use as a case study the finding by Stager and Werker (1997) that children's early ability to distinguish similar sounds does not automatically translate into word learning skills. Early explanations proposed that children may not be able to encode subtle phonetic contrasts when learning novel word meanings, thus suggesting a discontinuous/stage-like pattern of development. However, later work has revealed (e.g., through using more precise testing methods) that children do encode such contrasts, thus favoring a continuous pattern of development. Here, we propose a probabilistic model that represents word knowledge in a graded fashion and characterizes developmental change as improvement in the precision of this graded knowledge. Our model explained previous findings in the literature and provided a new prediction - the referents' visual similarity modulates word learning accuracy. The models' predictions were corroborated by human data collected from both preschool children and adults. The broader impact of this work is to show that computational models, such as ours, can help us explore the extent to which episodes of cognitive development that are typically thought of as discontinuities may emerge from simpler, continuous mechanisms.

Mathematical modelling of a liver hollow fibre bioreactor.

A mathematical model has been developed to assist with the development of a hollow fibre bioreactor (HFB) for hepatotoxicity testing of xenobiotics; specifically, to inform the HFB operating set-up, interpret data from HFB outputs and aid in optimizing HFB design to mimic certain hepatic physiological conditions. Additionally, the mathematical model has been used to identify the key HFB and compound parameters that will affect xenobiotic clearance. The analysis of this model has produced novel results that allow the operating set-up to be calculated, and predictions of compound clearance to be generated. The mathematical model predicts the inlet oxygen concentration and volumetric flow rate that gives a physiological oxygen gradient in the HFB to mimic a liver sinusoid. It has also been used to predict the concentration gradients and clearance of a test drug and paradigm hepatotoxin, paracetamol (APAP). The effect of altering the HFB dimensions and fibre properties on APAP clearance under the condition of a physiological oxygen gradient is analysed. These theoretical predictions can be used to design the most appropriate experimental set up and data analysis to quantitatively compare the functionality of cell types that are cultured within the HFB to those in other systems.

Integrated in vitro models for hepatic safety and metabolism: evaluation of a human Liver-Chip and liver spheroid.

Drug-induced liver injury remains a frequent reason for drug withdrawal. Accordingly, more predictive and translational models are required to assess human hepatotoxicity risk. This study presents a comprehensive evaluation of two promising models to assess mechanistic hepatotoxicity, microengineered Organ-Chips and 3D hepatic spheroids, which have enhanced liver phenotype, metabolic activity and stability in culture not attainable with conventional 2D models. Sensitivity of the models to two hepatotoxins, acetaminophen (APAP) and fialuridine (FIAU), was assessed across a range of cytotoxicity biomarkers (ATP, albumin, miR-122, α-GST) as well as their metabolic functionality by quantifying APAP, FIAU and CYP probe substrate metabolites. APAP and FIAU produced dose- and time-dependent increases in miR-122 and α-GST release as well as decreases in albumin secretion in both Liver-Chips and hepatic spheroids. Metabolic turnover of CYP probe substrates, APAP and FIAU, was maintained over the 10-day exposure period at concentrations where no cytotoxicity was detected and APAP turnover decreased at concentrations where cytotoxicity was detected. With APAP, the most sensitive biomarkers were albumin in the Liver-Chips (EC50 5.6 mM, day 1) and miR-122 and ATP in the liver spheroids (14-fold and EC50 2.9 mM, respectively, day 3). With FIAU, the most sensitive biomarkers were albumin in the Liver-Chip (EC50 126 µM) and miR-122 (15-fold) in the liver spheroids, both on day 7. In conclusion, both models exhibited integrated toxicity and metabolism, and broadly similar sensitivity to the hepatotoxicants at relevant clinical concentrations, demonstrating the utility of these models for improved hepatotoxicity risk assessment.

Timescale analysis of a mathematical model of acetaminophen metabolism and toxicity.

Acetaminophen is a widespread and commonly used painkiller all over the world. However, it can cause liver damage when taken in large doses or at repeated chronic doses. Current models of acetaminophen metabolism are complex, and limited to numerical investigation though provide results that represent clinical investigation well. We derive a mathematical model based on mass action laws aimed at capturing the main dynamics of acetaminophen metabolism, in particular the contrast between normal and overdose cases, whilst remaining simple enough for detailed mathematical analysis that can identify key parameters and quantify their role in liver toxicity. We use singular perturbation analysis to separate the different timescales describing the sequence of events in acetaminophen metabolism, systematically identifying which parameters dominate during each of the successive stages. Using this approach we determined, in terms of the model parameters, the critical dose between safe and overdose cases, timescales for exhaustion and regeneration of important cofactors for acetaminophen metabolism and total toxin accumulation as a fraction of initial dose.

Mass spectrometric characterization of circulating covalent protein adducts derived from a drug acyl glucuronide metabolite: multiple albumin adductions in diclofenac patients.

Covalent protein modifications by electrophilic acyl glucuronide (AG) metabolites are hypothetical causes of hypersensitivity reactions associated with certain carboxylate drugs. The complex rearrangements and reactivities of drug AG have been defined in great detail, and protein adducts of carboxylate drugs, such as diclofenac, have been found in liver and plasma of experimental animals and humans. However, in the absence of definitive molecular characterization, and specifically, identification of signature glycation conjugates retaining the glucuronyl and carboxyl residues, it cannot be assumed any of these adducts is derived uniquely or even fractionally from AG metabolites. We have therefore undertaken targeted mass spectrometric analyses of human serum albumin (HSA) isolated from diclofenac patients to characterize drug-: derived structures and, thereby, for the first time, have deconstructed conclusively the pathways of adduct formation from a drug AG and its isomeric rearrangement products in vivo. These analyses were informed by a thorough understanding of the reactions of HSA with diclofenac AG in vitro. HSA from six patients without drug-: related hypersensitivities had either a single drug-: derived adduct or one of five combinations of 2-8 adducts from among seven diclofenac N-acylations and three AG glycations on seven of the protein's 59 lysines. Only acylations were found in every patient. We present evidence that HSA modifications by diclofenac in vivo are complicated and variable, that at least a fraction of these modifications are derived from the drug's AG metabolite, and that albumin adduction is not inevitably a causation of hypersensitivity to carboxylate drugs or a coincidental association.

The generation, detection, and effects of reactive drug metabolites.

The decline in approval of new drugs during the past decade has led to a close analysis of the drug discovery process. One of the main reasons for attrition is preclinical toxicity, frequently attributed to the generation of protein-reactive drug metabolites. In this review, we present a critique of such reactive metabolites and evaluate the evidence linking them to observed toxic effects. Methodology for the characterization of reactive metabolites has advanced greatly in recent years, and is summarized first. Next, we consider the inhibition of key metabolic enzymes by electrophilic metabolites, as well as unfavorable drug-drug interactions that may ensue. One important class of protein-reactive metabolites, not linked conclusively to a toxic event, is acyl glucuronides. Their properties are discussed in light of the safety characteristics of carboxylic acid containing drugs. Many adverse drug reactions (ADRs) are known collectively as idiosyncratic events, that is, not predictable from knowledge of the pharmacology and pharmacokinetics of the parent compound. Observed ADRs may take various forms. Specific organ injury, particularly of the liver, is the most direct: we examine this in some detail. Moving to the cellular level, we also consider the upregulation of induced cellular processes. The related, but distinct, issue of hypersensitivity or allergic reactions to drugs and their metabolites, possibly via the immune system, is considered next. Finally, we discuss the impact of such data on the drug discovery process, both through early detection of reactive metabolites and informed synthetic design, which eliminates unfavorable functionality from drug candidates.

The impact of early postoperative pain on health-related quality of life.

OBJECTIVES: To examine how the severity of postoperative pain affects patient's health-related quality of life (HRQoL) at 1 week following surgery and to compare two generic validated HRQoL instruments. METHODS: Patients undergoing general or orthopaedic surgery at the Royal London Hospital were randomly sampled. The following patient outcome data were collected EQ-5D (EuroQoL) pre-operatively and the Revised American Pain Society Patient Outcome Questionnaire (APS-POQ-R) at 24 hours postoperation; and EQ-5D, Short-Form-12 (SF-12) and APS-POQ-R at 7 days postoperation. The degree of association between pain and HRQoL was assessed using Pearson's correlation coefficient and multivariate generalized linear regression models. RESULTS: Of the 228 patients included, 166 patients provided data at 7 days. Sixteen percent reported severe pain ≥ 50% of the day at 7 days. The severity of pain on both the APS-POQ-R pain severity and interference and affective impairment domains at 7 days was highly correlated with a decrease in HRQoL as assessed by the SF-12 Physical Component Score (PCS), SF-12 Mental Component Score (MCS), and EQ-5D scores (r = -0.34 to -0.61, P < 0.0001). Multivariate regression analyses showed that irrespective of confounding factors (eg, age, gender, and pre-operative HRQoL) patients with severe postoperative pain experience important reductions in both physical and mental well-being domains of their HRQoL. CONCLUSIONS: A proportion of patients continue to experience severe pain at 7 days postoperatively, even after minor surgery. HRQoL is strongly associated with the level of pain and provides additional data on the impact of postsurgery pain on patient's function and well-being. Additional studies are needed to elucidate the interaction between pain severity and HRQoL during the peri-operative period.

Acyl glucuronides: the good, the bad and the ugly.

Acyl glucuronidation is the major metabolic conjugation reaction of most carboxylic acid drugs in mammals. The physiological consequences of this biotransformation have been investigated incompletely but include effects on drug metabolism, protein binding, distribution and clearance that impact upon pharmacological and toxicological outcomes. In marked contrast, the exceptional but widely disparate chemical reactivity of acyl glucuronides has attracted far greater attention. Specifically, the complex transacylation and glycation reactions with proteins have provoked much inconclusive debate over the safety of drugs metabolised to acyl glucuronides. It has been hypothesised that these covalent modifications could initiate idiosyncratic adverse drug reactions. However, despite a large body of in vitro data on the reactions of acyl glucuronides with protein, evidence for adduct formation from acyl glucuronides in vivo is limited and potentially ambiguous. The causal connection of protein adduction to adverse drug reactions remains uncertain. This review has assessed the intrinsic reactivity, metabolic stability and pharmacokinetic properties of acyl glucuronides in the context of physiological, pharmacological and toxicological perspectives. Although numerous experiments have characterised the reactions of acyl glucuronides with proteins, these might be attenuated substantially in vivo by rapid clearance of the conjugates. Consequently, to delineate a relationship between acyl glucuronide formation and toxicological phenomena, detailed pharmacokinetic analysis of systemic exposure to the acyl glucuronide should be undertaken adjacent to determining protein adduct concentrations in vivo. Further investigation is required to ascertain whether acyl glucuronide clearance is sufficient to prevent covalent modification of endogenous proteins and consequentially a potential immunological response.

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.

Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study.

Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture.

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Haloarene derivatives of carbamazepine with reduced bioactivation liabilities: 2-monohalo and 2,8-dihalo derivatives.

The anticonvulsant carbamazepine 1 is associated with adverse drug reactions (ADRs), including hepatotoxicity; oxidative metabolism of 1 has been implicated in the pathogenesis of the ADRs. We report the synthesis and evaluation of 2-monohalo and 2,8-dihalo analogues of 1 that were intended to minimize reactive metabolite formation via arene oxidation and 10,11-epoxidation. Halo analogues were obtained either by rearrangement of halogenated N-arylindoles or from specifically halogenated iminodibenzyl derivatives. In rat hepatocytes, none of the analogues underwent oxidative dehalogenation or glutathione adduction. Some formation of the 10,11-epoxide still occurred, but aromatic hydroxylation was not seen with the exception of 2-fluoro, which allowed minor monohydroxylation. Complete inhibition of aromatic hydroxylation required at least monochlorination or difluorination of 1. In human liver microsomes, difluoro analogue 5b underwent 10,11-epoxidation but gave no arene oxidation.

The genotoxic potential of methapyrilene using the alkaline Comet assay in vitro and in vivo.

The genotoxicity of methapyrilne (MP) has been evaluated in a number of assays since it was found to be a rat hepatocarcinogen with subsequent withdrawal as an over-the-counter antihistamine. Whilst it has not been classified as a genotoxin, there are reports of positive findings from mammalian cell gene mutation and transformation assays. To investigate further the genotoxic potential of MP, the alkaline Comet assay was used to evaluate DNA damage both in primary hepatocytes in culture and in vivo in the rat. To confirm bioactivation was required to induce the hepatotoxic mechanism, aminobenzotriazole, a broad spectrum cytochrome P450 enzyme inhibitor was used as a pre-treatment. The levels of glutathione and glutathione disulfide were determined in both hepatocytes in culture and in the liver following in vivo exposure. MP showed significant increases in DNA damage in freshly isolated male rat hepatocyte suspensions that could be significantly reduced by pre-incubation of aminobenzotriazole (ABT). DNA damage showed a marked sex difference, with male hepatocytes being more susceptible, and showing a concurrent depletion of glutathione (GSH) compared with female hepatocytes. Modulation of the GSH levels by diethylmaleate and γ-glutamylcysteinylethyl ester, elevated and reduced the levels of DNA damage, respectively. In the in vivo Comet assay, there was no evidence of DNA damage following MP (150mg/kg p.o) treatment for three consecutive days, although histological and liver enzyme changes were seen. Total protein GSH content was elevated in MP-treated animals and superoxide dismutase levels were increased specifically in periportal regions. Taken together, these data support the potential for MP to induce oxidative stress. The differences in DNA damage detected by the Comet assay in vitro, and in rat liver in vivo, could be attributed to differences in metabolism and response to oxidant insult or the inability of the assay to discriminate damage in a small number of individual cells in the whole liver.

Convenient syntheses of benzo-fluorinated dibenz[b,f]azepines: rearrangements of isatins, acridines, and indoles.

Efficient procedures for the synthesis of benzo-fluorinated dibenz[b,f]azepines (iminostilbenes) from fluorinated isatins or indoles using a number of ring-expansion reactions are described. A range of mono- and difluorinated analogues is accessible, and the syntheses can deliver gram quantities of the final products, which are precursors of fluoro analogues of the important anticonvulsant carbamazepine.

High-mobility group box-1 protein and keratin-18, circulating serum proteins informative of acetaminophen-induced necrosis and apoptosis in vivo.

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.