Enhanced hepatic respiratory capacity and altered lipid metabolism support metabolic homeostasis during short-term hypoxic stress.

BACKGROUND: Tissue hypoxia is a key feature of several endemic hepatic diseases, including alcoholic and non-alcoholic fatty liver disease, and organ failure. Hypoxia imposes a severe metabolic challenge on the liver, potentially disrupting its capacity to carry out essential functions including fuel storage and the integration of lipid metabolism at the whole-body level. Mitochondrial respiratory function is understood to be critical in mediating the hepatic hypoxic response, yet the time-dependent nature of this response and the role of the respiratory chain in this remain unclear. RESULTS: Here, we report that hepatic respiratory capacity is enhanced following short-term exposure to hypoxia (2 days, 10% O2) and is associated with increased abundance of the respiratory chain supercomplex III2+IV and increased cardiolipin levels. Suppression of this enhanced respiratory capacity, achieved via mild inhibition of mitochondrial complex III, disrupted metabolic homeostasis. Hypoxic exposure for 2 days led to accumulation of plasma and hepatic long chain acyl-carnitines. This was observed alongside depletion of hepatic triacylglycerol species with total chain lengths of 39-53 carbons, containing palmitic, palmitoleic, stearic, and oleic acids, which are associated with de novo lipogenesis. The changes to hepatic respiratory capacity and lipid metabolism following 2 days hypoxic exposure were transient, becoming resolved after 14 days in line with systemic acclimation to hypoxia and elevated circulating haemoglobin concentrations. CONCLUSIONS: The liver maintains metabolic homeostasis in response to shorter term hypoxic exposure through transient enhancement of respiratory chain capacity and alterations to lipid metabolism. These findings may have implications in understanding and treating hepatic pathologies associated with hypoxia.

Expression of Concern to: Diet restriction inhibits apoptosis and HMGB1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatoxicity.

The Editors-in-Chief would like to alert readers that this article [1] is part of an investigation being conducted by the journal following the conclusions of an institutional enquiry at the University of Liverpool with respect to the quantitative mass spectrometry-generated results regarding acetylated and redox-modified HMGB1.

HepG2 (C3A) spheroids show higher sensitivity compared to HepaRG spheroids for drug-induced liver injury (DILI).

High-throughput, automation-friendly and therapeutically-predictive assays are needed in early drug discovery in order to prioritise compounds and reduce the risk of new drugs causing Drug-Induced Liver Injury (DILI). We evaluated the suitability of high-throughput 3D liver spheroid models of HepG2 (C3A clone) and HepaRG cell lines to predict DILI in early drug development. Spheroids were formed in 384-well ultra-low-attachment plates and dosed via direct acoustic droplet ejection at nine half-log spaced concentrations per compound. Spheroid viability was quantified with an ATP endpoint after a 4-day incubation with 150 drugs with known DILI liability. We derived a margin of safety for each cell line defined as the ratio between the IC50 values generated for each compound to their maximum plasma concentration Cmax which resulted in optimal classification accuracy. The margin of safety can be used to estimate a maximum safe Cmax for compounds in early drug discovery for which Cmax is not yet known. Both cell lines had similar level of accuracy in predicting DILI, with HepG2 spheroids being more sensitive. HepG2 spheroids had a sensitivity of 58% and a specificity of 83%, while HepaRG spheroids had a sensitivity of 47% and specificity of 86%. Ninety-nine of the 150 compounds were used to compare the relative sensitivities of HepG2 and HepaRG spheroids. HepaRG spheroids were more sensitive to 7 compounds and HepG2 spheroids were more sensitive to 34 compounds. In conclusion, across a diverse group of drugs HepG2 spheroids were more predictive of DILI compared to HepaRG spheroids.

The utility of a differentiated preclinical liver model, HepaRG cells, in investigating delayed toxicity via inhibition of mitochondrial-replication induced by fialuridine.

During its clinical development fialuridine caused liver toxicity and the death of five patients. This case remains relevant due to the continued development of mechanistically-related compounds against a back-drop of simple in vitro models which remain limited for the preclinical detection of such delayed toxicity. Here, proteomic investigation of a differentiated, HepaRG, and proliferating, HepG2 cell model was utilised to confirm the presence of the hENT1 transporter, thymidine kinase-1 and -2 (TK1, TK2) and thymidylate kinase, all essential in order to reproduce the cellular activation and disposition of fialuridine in the clinic. Acute metabolic modification assays could only identify mitochondrial toxicity in HepaRG cells following extended dosing, 2 weeks. Toxic effects were observed around 10 µM, which is within a range of 10-15 X approximate Cmax. HepaRG cell death was accompanied by a significant decrease in mitochondrial DNA content, indicative of inhibition of mitochondrial replication, and a subsequent reduction in mitochondrial respiration and the activity of mitochondrial respiratory complexes, not replicated in HepG2 cells. The structural epimer of fialuridine, included as a pharmacological negative control, was shown to have no cytotoxic effects in HepaRG cells up to 4 weeks. Overall, these comparative studies demonstrate the HepaRG model has translational relevance for fialuridine toxicity and therefore may have potential in investigating the inhibition of mitochondrial replication over prolonged exposure for other toxicants.

Predicting Drug-Induced Liver Injury with Bayesian Machine Learning.

Drug induced liver injury (DILI) can require significant risk management in drug development and on occasion can cause morbidity or mortality, leading to drug attrition. Optimizing candidates preclinically can minimize hepatotoxicity risk, but it is difficult to predict due to multiple etiologies encompassing DILI, often with multifactorial and overlapping mechanisms. In addition to epidemiological risk factors, physicochemical properties, dose, disposition, lipophilicity, and hepatic metabolic function are also relevant for DILI risk. Better human-relevant, predictive models are required to improve hepatotoxicity risk assessment in drug discovery. Our hypothesis is that integrating mechanistically relevant hepatic safety assays with Bayesian machine learning will improve hepatic safety risk prediction. We present a quantitative and mechanistic risk assessment for candidate nomination using data from in vitro assays (hepatic spheroids, BSEP, mitochondrial toxicity, and bioactivation), together with physicochemical (cLogP) and exposure (Cmaxtotal) variables from a chemically diverse compound set (33 no/low-, 40 medium-, and 23 high-severity DILI compounds). The Bayesian model predicts the continuous underlying DILI severity and uses a data-driven prior distribution over the parameters to prevent overfitting. The model quantifies the probability that a compound falls into either no/low-, medium-, or high-severity categories, with a balanced accuracy of 63% on held-out samples, and a continuous prediction of DILI severity along with uncertainty in the prediction. For a binary yes/no DILI prediction, the model has a balanced accuracy of 86%, a sensitivity of 87%, a specificity of 85%, a positive predictive value of 92%, and a negative predictive value of 78%. Combining physiologically relevant assays, improved alignment with FDA recommendations, and optimal statistical integration of assay data leads to improved DILI risk prediction.

Functionality of primary hepatic non-parenchymal cells in a 3D spheroid model and contribution to acetaminophen hepatotoxicity.

In addition to hepatocytes, the liver comprises a host of specialised non-parenchymal cells which are important to consider in the development of in vitro models which are both physiologically and toxicologically relevant. We have characterized a 3D co-culture system comprising primary human hepatocytes (PHH) and non-parenchymal cells (NPC) and applied it to the investigation of acetaminophen-induced toxicity. Firstly, we titrated ratios of PHH:NPC and confirmed the presence of functional NPCs via both immunohistochemistry and activation with both LPS and TGF-ß. Based on these data we selected a ratio of 2:1 PHH:NPC for further studies. We observed that spheroids supplemented with NPCs were protected against acetaminophen (APAP) toxicity as determined by ATP (up to threefold difference in EC50 at day 14 compared to hepatocytes alone) and glutathione depletion, as well as miR-122 release. APAP metabolism was also altered in the presence of NPCs, with significantly lower levels of APAP-GSH detected. Expression of several CYP450 enzymes involved in the bioactivation of APAP was also lower in NPC-containing spheroids. Spheroids containing NPCs also expressed higher levels of miRNAs which have been implicated in APAP-induced hepatotoxicity, including miR-382 and miR-155 which have potential roles in liver regeneration and inflammation, respectively. These data indicate that the interaction between hepatocytes and NPCs can have significant metabolic and toxicological consequences important for the correct elucidation of hepatic safety mechanisms.

Reactive Metabolites: Current and Emerging Risk and Hazard Assessments.

Although idiosyncratic adverse drug reactions are rare, they are still a major concern to patient safety. Reactive metabolites are widely accepted as playing a pivotal role in the pathogenesis of idiosyncratic adverse drug reactions. While there are today well established strategies for the risk assessment of stable metabolites within the pharmaceutical industry, there is still no consensus on reactive metabolite risk assessment strategies. This is due to the complexity of the mechanisms of these toxicities as well as the difficulty in identifying and quantifying short-lived reactive intermediates such as reactive metabolites. In this review, reactive metabolite risk and hazard assessment approaches are discussed, and their pros and cons highlighted. We also discuss the nature of idiosyncratic adverse drug reactions, using acetaminophen and nefazodone to exemplify the complexity of the underlying mechanisms of reactive metabolite mediated hepatotoxicity. One of the key gaps moving forward is our understanding of and ability to predict the contribution of immune activation in idiosyncratic adverse drug reactions. Sections are included on the clinical phenotypes of immune mediated idiosyncratic adverse drug reactions and on the present understanding of immune activation by reactive metabolites. The advances being made in microphysiological systems have a great potential to transform our ability to risk assess reactive metabolites, and an overview of the key components of these systems is presented. Finally, the potential impact of systems pharmacology approaches in reactive metabolite risk assessments is highlighted.

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.

Ochronotic osteoarthropathy in a mouse model of alkaptonuria, and its inhibition by nitisinone.

BACKGROUND: Alkaptonuria (AKU) is a rare metabolic disease caused by deficiency of homogentisate 1,2 dioxygenase, an enzyme involved in tyrosine catabolism, resulting in increased circulating homogentisic acid (HGA). Over time HGA is progressively deposited as a polymer (termed ochronotic pigment) in collagenous tissues, especially the cartilages of weight bearing joints, leading to severe joint disease. OBJECTIVES: To characterise blood biochemistry and arthropathy in the AKU mouse model (Hgd-/-). To examine the therapeutic effect of long-term treatment with nitisinone, a potent inhibitor of the enzyme that produces HGA. METHODS: Lifetime levels of plasma HGA from AKU mice were measured by high-performance liquid chromatography (HPLC). Histological sections of the knee joint were examined for pigmentation. The effect of nitisinone treatment in both tissues was examined. RESULTS: Mean (±SE) plasma HGA levels were 3- to 4-fold higher (0.148±0.019 mM) than those recorded in human AKU. Chondrocyte pigmentation within the articular cartilage was first observed at 15 weeks, and found to increase steadily with mouse age. Nitisinone treatment reduced plasma HGA in AKU mice throughout their lifetime, and completely prevented pigment deposition. CONCLUSIONS: The AKU mouse was established as a model of both the plasma biochemistry of AKU and its associated arthropathy. Early-stage treatment of AKU patients with nitisinone could prevent the development of associated joint arthropathies. The cellular pathology of ochronosis in AKU mice is identical to that observed in early human ochronosis and thus is a model in which the early stages of joint pathology can be studied and novel interventions evaluated.

Improved Detection of Drug-Induced Liver Injury by Integrating Predicted in vivo and in vitro Data.

Drug-induced liver injury (DILI) has been significant challenge in drug discovery, often leading to clinical trial failures and necessitating drug withdrawals. The existing suite of in vitro proxy-DILI assays is generally effective at identifying compounds with hepatotoxicity. However, there is considerable interest in enhancing in silico prediction of DILI because it allows for the evaluation of large sets of compounds more quickly and cost-effectively, particularly in the early stages of projects. In this study, we aim to study ML models for DILI prediction that first predicts nine proxy-DILI labels and then uses them as features in addition to chemical structural features to predict DILI. The features include in vitro (e.g., mitochondrial toxicity, bile salt export pump inhibition) data, in vivo (e.g., preclinical rat hepatotoxicity studies) data, pharmacokinetic parameters of maximum concentration, structural fingerprints, and physicochemical parameters. We trained DILI-prediction models on 888 compounds from the DILIst dataset and tested on a held-out external test set of 223 compounds from DILIst dataset. The best model, DILIPredictor, attained an AUC-ROC of 0.79. This model enabled the detection of top 25 toxic compounds compared to models using only structural features (2.68 LR+ score). Using feature interpretation from DILIPredictor, we were able to identify the chemical substructures causing DILI as well as differentiate cases DILI is caused by compounds in animals but not in humans. For example, DILIPredictor correctly recognized 2-butoxyethanol as non-toxic in humans despite its hepatotoxicity in mice models. Overall, the DILIPredictor model improves the detection of compounds causing DILI with an improved differentiation between animal and human sensitivity as well as the potential for mechanism evaluation. DILIPredictor is publicly available at https://broad.io/DILIPredictor for use via web interface and with all code available for download and local implementation via https://pypi.org/project/dilipred/.

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.

RETRACTED: Molecular forms of HMGB1 and keratin-18 as mechanistic biomarkers for mode of cell death and prognosis during clinical acetaminophen hepatotoxicity.

BACKGROUND & AIMS: Full length keratin-18 (FL-K18) and High Mobility Group Box-1 (HMGB1) represent circulating indicators of necrosis during acetaminophen (APAP) hepatotoxicity in vivo. In addition, the caspase-cleaved fragment of K18 (cK18) and hyper-acetylated HMGB1 represent serum indicators of apoptosis and immune cell activation, respectively. The study aim was to assess their mechanistic utility to establish the balance between apoptosis, necrosis, and immune cell activation throughout the time course of clinical APAP hepatotoxicity. METHODS: HMGB1 (total, acetylated) and K18 (apoptotic, necrotic) were identified and quantified by novel LC-MS/MS assays in APAP overdose patients (n=78). RESULTS: HMGB1 (total; 15.4±1.9ng/ml, p<0.01, acetylated; 5.4±2.6ng/ml, p<0.001), cK18 (5649.8±721.0U/L, p<0.01), and FL-K18 (54770.2±6717.0U/L, p<0.005) were elevated in the sera of APAP overdose patients with liver injury compared to overdose patients without liver injury and healthy volunteers. HMGB1 and FL-K18 correlated with alanine aminotransferase (ALT) activity (R(2)=0.60 and 0.58, respectively, p<0.0001) and prothrombin time (R(2)=0.62 and 0.71, respectively, p<0.0001). Increased total and acetylated HMGB1 and FL-K18 were associated with worse prognosis (King's College Criteria) or patients that died/required liver transplant compared to spontaneous survivors (all p<0.05-0.001), a finding not reflected by ALT and supported by ROC analysis. Acetylated HMGB1 was a better predictor of outcome than the other markers of cell death. CONCLUSIONS: K18 and HMGB1 represent blood-based tools to investigate the cell death balance clinical APAP hepatotoxicity. Activation of the immune response was seen later in the time course as shown by the distinct profile of acetylated HMGB1 and was associated with worse outcome.

The hepatocyte export carrier inhibition assay improves the separation of hepatotoxic from non-hepatotoxic compounds.

An in vitro/in silico method that determines the risk of human drug induced liver injury in relation to oral doses and blood concentrations of drugs was recently introduced. This method utilizes information on the maximal blood concentration (Cmax) for a specific dose of a test compound, which can be estimated using physiologically-based pharmacokinetic modelling, and a cytotoxicity test in cultured human hepatocytes. In the present study, we analyzed if the addition of an assay that measures the inhibition of bile acid export carriers, like BSEP and/or MRP2, to the existing method improves the differentiation of hepatotoxic and non-hepatotoxic compounds. Therefore, an export assay for 5-chloromethylfluorescein diacetate (CMFDA) was established. We tested 36 compounds in a concentration-dependent manner for which the risk of hepatotoxicity for specific oral doses and the capacity to inhibit hepatocyte export carriers are known. Compared to the CTB cytotoxicity test, substantially lower EC10 values were obtained using the CMFDA assay for several known BSEP and/or MRP2 inhibitors. To quantify if the addition of the CMFDA assay to our test system improves the overall separation of hepatotoxic from non-hepatotoxic compounds, the toxicity separation index (TSI) was calculated. We obtained a better TSI using the lower alert concentration from either the CMFDA or the CTB test (TSI: 0.886) compared to considering the CTB test alone (TSI: 0.775). In conclusion, the data show that integration of the CMFDA assay with an in vitro test battery improves the differentiation of hepatotoxic and non-hepatotoxic compounds in a set of compounds that includes bile acid export carrier inhibitors.

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.

Diet restriction inhibits apoptosis and HMGB1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatotoxicity.

Acetaminophen (APAP) overdose is a major cause of acute liver failure and serves as a paradigm to elucidate mechanisms, predisposing factors and therapeutic interventions. The roles of apoptosis and inflammation during APAP hepatotoxicity remain controversial. We investigated whether fasting of mice for 24 h can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP. We also investigated in fasted mice the critical role played by inhibition of caspase-dependent cysteine 106 oxidation within high mobility group box-1 protein (HMGB1) released by ATP depletion in dying cells as a mechanism of immune activation. In fed mice treated with APAP, necrosis was the dominant form of hepatocyte death. However, apoptosis was also observed, indicated by K18 cleavage, DNA laddering and procaspase-3 processing. In fasted mice treated with APAP, only necrosis was observed. Inflammatory cell recruitment as a consequence of hepatocyte death was observed only in fasted mice treated with APAP or fed mice cotreated with a caspase inhibitor. Hepatic inflammation was also associated with loss in detection of serum oxidized-HMGB1. A significant role of HMGB1 in the induction of inflammation was confirmed with an HMGB1-neutralizing antibody. The differential response between fasted and fed mice was a consequence of a significant reduction in basal hepatic ATP, which prevented caspase processing, rather than glutathione depletion or altered APAP metabolism. Thus, the inhibition of caspase-driven apoptosis and HMGB1 oxidation by ATP depletion from fasting promotes an inflammatory response during drug-induced hepatotoxicity/liver pathology.

Quantifying the metabolic activation of nevirapine in patients by integrated applications of NMR and mass spectrometries.

Nevirapine (NVP), an antiretroviral drug, is associated with idiosyncratic hepatotoxicity and skin reactions. Metabolic pathways of haptenation and immunotoxicity mechanisms have been proposed. NVP is metabolized by liver microsomes to a reactive intermediate that binds irreversibly to protein and forms a GSH adduct. However, no reactive metabolite of NVP, trapped as stable thioether conjugates, has hitherto been identified in vivo. This study has defined the metabolism of NVP with respect to reactive intermediate formation in patients and a rat model of NVP-induced skin reactions. An integrated NMR and mass spectrometry approach has been developed to discover and quantify stable urinary metabolite biomarkers indicative of NVP bioactivation in patients. Two isomeric NVP mercapturates were identified in the urine of HIV-positive patients undergoing standard antiretroviral chemotherapy. The same conjugates were found in rat bile and urine. The mercapturates were isolated from rat bile and characterized definitively by NMR as thioethers substituted at the C-3 and exocyclic C-12 positions of the methylpyrido ring of NVP. It is proposed that NVP undergoes bioactivation to arene oxide and quinone methide intermediates. The purified major mercapturate was quantified by NMR and used to calibrate a mass spectrometric assay of the corresponding metabolite in patient urine. This is the first evidence for metabolic activation of NVP in humans, and only the second minimum estimate in patients of bioactivation of a widely prescribed drug associated with idiosyncratic toxicities. The method can be used as a template for comparative estimations of bioactivation of any drug in patients.

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.

Determining organ weight toxicity with Bayesian causal models: Improving on the analysis of relative organ weights.

Regulatory authorities require animal toxicity tests for new chemical entities. Organ weight changes are accepted as a sensitive indicator of chemically induced organ damage, but can be difficult to interpret because changes in organ weight might reflect chemically-induced changes in overall body weight. A common solution is to calculate the relative organ weight (organ to body weight ratio), but this inadequately controls for the dependence on body weight - a point made by statisticians for decades, but which has not been widely adopted. The recommended solution is an analysis of covariance (ANCOVA), but it is rarely used, possibly because both the method of statistical correction and the interpretation of the output may be unclear to those with minimal statistical training. Using relative organ weights can easily lead to incorrect conclusions, resulting in poor decisions, wasted resources, and an ethically questionable use of animals. We propose to cast the problem into a causal modelling framework as it directly assesses questions of scientific interest, the results are easy to interpret, and the analysis is simple to perform with freely available software. Furthermore, by taking a Bayesian approach we can model unequal variances, control for multiple testing, and directly provide evidence of safety.