Neil R. Kitteringham

Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics

The conventional pipeline for biomarker development involves a discovery phase, typically conducted by mass spectrometry (MS), followed by validation and clinical application, usually on an alternative platform, such as immunoassay. Whilst this approach is suitable for the development of single biomarkers, with the current drive towards larger panels of multiplexed biomarkers, the process becomes inefficient and costly. Consequently, the emphasis is now shifting towards performing full biomarker discovery, qualification and quantification on the same technology platform. The ease of multiplexing and ability to determine protein modifications makes MS an attractive alternative to antibody-based technologies. In addition, developments in quantitative MS, through the application of stable isotope labelling and scanning techniques, such as multiple reaction monitoring (MRM), have greatly enhanced both the specificity and sensitivity of MS-based assays to the point that they can rival immunoassay for some analytes. This review focuses on the application of MRM for quantitative MS analysis, particularly with respect to proteins and peptides.

Fortnightly review: Adverse drug reactions

An adverse drug reaction is any undesirable effect of a drug beyond its anticipated therapeutic effects occurring during clinical use. In contrast, an adverse drug event is an untoward occurrence after exposure to a drug that is not necessarily caused by the drug.1 When a drug is marketed little is known about its safety in clinical use because only about 1500 patients are likely to have been exposed to it.1,2 Thus drug safety assessment should be considered an integral part of everyday clinical practice since detection and diagnosis often depend on clinical acumen. In this article we review the current status of adverse drug reactions, briefly describing the complexity of the more bizarre reactions and outlining a strategy to eliminate serious adverse drug reactions. Summary points Adverse drug reactions are a common clinical problem They are diagnosed on clinical grounds from the temporal relation between the start and finish of drug treatment and the onset and offset of the reaction Pharmacological adverse reactions are generally dose-dependent, related to the pharmacokinetic properties of the drug, and resolve when the dose is reduced Idiosyncratic adverse reactions are not related to the known pharmacology of the drug, do not show any simple dose-response relation, and resolve only when treatment is discontinued Vigilance by clinicians in detecting, diagnosing, and reporting adverse reactions is important for continued drug safety monitoring

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.

Activation of hepatic Nrf2 in vivo by acetaminophen in CD-1 mice

The transcription factor NF‐E2‐related factor 2 (Nrf2) plays an essential role in the mammalian response to chemical and oxidative stress through induction of hepatic phase II detoxification enzymes and regulation of glutathione (GSH). Enhanced liver damage in Nrf2‐deficient mice treated with acetaminophen suggests a critical role for Nrf2; however, direct evidence for Nrf2 activation following acetaminophen exposure was previously lacking. We show that acetaminophen can initiate nuclear translocation of Nrf2 in vivo, with maximum levels reached after 1 hour, in a dose dependent manner, at doses below those causing overt liver damage. Furthermore, Nrf2 was shown to be functionally active, as assessed by the induction of epoxide hydrolase, heme oxygenase‐1, and glutamate cysteine ligase gene expression. Increased nuclear Nrf2 was found to be associated with depletion of hepatic GSH. Activation of Nrf2 is considered to involve dissociation from a cytoplasmic inhibitor, Kelch‐like ECH‐associated protein 1 (Keap1), through a redox‐sensitive mechanism involving either GSH depletion or direct chemical interaction through Michael addition. To investigate acetaminophen‐induced Nrf2 activation we compared the actions of 2 other GSH depleters, diethyl maleate (DEM) and buthionine sulphoximine (BSO), only 1 of which (DEM) can function as a Michael acceptor. For each compound, greater than 60% depletion of GSH was achieved; however, in the case of BSO, this depletion did not cause nuclear translocation of Nrf2. In conclusion, GSH depletion alone is insufficient for Nrf2 activation: a more direct interaction is required, possibly involving chemical modification of Nrf2 or Keap1, which is facilitated by the prior loss of GSH. (HEPATOLOGY 2004;39:1267–1276.)

The role of cytochrome P450 enzymes in hepatic and extrahepatic human drug toxicity

The human cytochrome P450 enzyme system metabolises a wide array of xenobiotics to pharmacologically inactive metabolites, and occasionally, to toxicologically active metabolites. Impairment of cytochrome P450 activity, which may be either genetic or environmental, may lead to toxicity caused by the parent compound itself. In practise, this usually only applies to drugs that have a narrow therapeutic index and when their clearance is critically dependent upon the fraction normally metabolised by that pathway. P450 enzymes may also convert the drug to a chemically reactive metabolite, which, if not detoxified, may lead to various forms of hepatic and extrahepatic toxicity, including cellular necrosis, hypersensitivity, teratogenicity, and carcinogenicity, depending on the site of formation and the relative stability of the metabolite, and the cellular macromolecule with which it reacts. Variation in the regulation and expression of the drug metabolising enzymes may play a key role in both interindividual variation in sensitivity to drug toxicity and tissue-specific damage. Avoidance of toxicity may be possible in rare instances by prediction of individual susceptibility or by designing new chemical entities that are metabolised by a range of enzymes (both cytochromes P450 and others) and do not undergo bioactivation.

Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease

Liver biology and function, drug-induced liver injury (DILI) and liver diseases are difficult to study using current in vitro models such as primary human hepatocyte (PHH) monolayer cultures, as their rapid de-differentiation restricts their usefulness substantially. Thus, we have developed and extensively characterized an easily scalable 3D PHH spheroid system in chemically-defined, serum-free conditions. Using whole proteome analyses, we found that PHH spheroids cultured this way were similar to the liver in vivo and even retained their inter-individual variability. Furthermore, PHH spheroids remained phenotypically stable and retained morphology, viability, and hepatocyte-specific functions for culture periods of at least 5 weeks. We show that under chronic exposure, the sensitivity of the hepatocytes drastically increased and toxicity of a set of hepatotoxins was detected at clinically relevant concentrations. An interesting example was the chronic toxicity of fialuridine for which hepatotoxicity was mimicked after repeated-dosing in the PHH spheroid model, not possible to detect using previous in vitro systems. Additionally, we provide proof-of-principle that PHH spheroids can reflect liver pathologies such as cholestasis, steatosis and viral hepatitis. Combined, our results demonstrate that the PHH spheroid system presented here constitutes a versatile and promising in vitro system to study liver function, liver diseases, drug targets and long-term DILI.

Physical and functional interaction of sequestosome 1 with Keap1 regulates the Keap1-Nrf2 cell defense pathway.

Nrf2 regulates the expression of numerous cytoprotective genes in mammalian cells. The activity of Nrf2 is regulated by the Cul3 adaptor Keap1, yet little is known regarding mechanisms of regulation of Keap1 itself. Here, we have used immunopurification of Keap1 and mass spectrometry, in addition to immunoblotting, to identify sequestosome 1 (SQSTM1) as a cellular binding partner of Keap1. SQSTM1 serves as a scaffold in various signaling pathways and shuttles polyubiquitinated proteins to the proteasomal and lysosomal degradation machineries. Ectopic expression of SQSTM1 led to a decrease in the basal protein level of Keap1 in a panel of cells. Furthermore, RNA interference (RNAi) depletion of SQSTM1 resulted in an increase in the protein level of Keap1 and a concomitant decrease in the protein level of Nrf2 in the absence of changes in Keap1 or Nrf2 mRNA levels. The increased protein level of Keap1 in cells depleted of SQSTM1 by RNAi was linked to a decrease in its rate of degradation; the half-life of Keap1 was almost doubled by RNAi depletion of SQSTM1. The decreased level of Nrf2 in cells depleted of SQSTM1 by RNAi was associated with decreases in the mRNA levels, protein levels, and function of several Nrf2-regulated cell defense genes. SQSTM1 was dispensable for the induction of the Keap1-Nrf2 pathway, as Nrf2 activation by tert-butylhydroquinone or iodoacetamide was not affected by RNAi depletion of SQSTM1. These findings demonstrate a physical and functional interaction between Keap1 and SQSTM1 and reveal an additional layer of regulation in the Keap1-Nrf2 pathway.

Advances in molecular toxicology–towards understanding idiosyncratic drug toxicity

Idiosyncratic drug toxicity is a major complication of drug therapy and drug development. Such adverse drug reactions (ADRs) include anaphylaxis, blood dyscrasias, hepatotoxicity and severe cutaneous reactions. They are usually serious and can be fatal. At present, prediction of idiosyncratic ADRs at the preclinical stage of drug development is not possible because there are no suitable animal models and we do not understand the basic mechanisms involved in the toxicity when it does occur in man. Many idiosyncratic reactions appear to have an immunological aetiology. For example, there is increasing evidence for the role of T lymphocytes in severe skin reactions. Nevertheless, the sequence of events by which a simple chemical can elicit severe tissue damage remains poorly understood and alternative novel mechanisms of toxicity must also be explored. The purpose of this article will be to review the currently accepted mechanisms of idiosyncratic drug toxicity at the chemical and the molecular levels. In particular, we will consider how recent advances in cellular immunology and molecular biology can improve our understanding of both the chemical and clinical aspects of drug hypersensitivity. Recent advances in the role of both inter- and intra-cellular signalling in the regulation of the immune response to drugs and their metabolites will be discussed. The long-term aim of such research is to provide test systems for the evaluation of drug safety and patient susceptibility to idiosyncratic drug toxicity.

Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity

Bioactivation of sulphamethoxazole (SMX) to chemically‐reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro. Flow cytometry revealed binding of N‐hydroxy (SMX‐NHOH) and nitroso (SMX‐NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX‐NO was reduced by exogenous glutathione (GSH). SMX‐NHOH and SMX‐NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism. Fluorescence HPLC showed that SMX‐NHOH and SMX‐NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma. Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX‐NHOH and SMX‐NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX‐NO than neutrophils. Partitioning of SMX‐NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone. Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.

Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

BackgroundNrf2 is a key transcriptional regulator of a battery of genes that facilitate phase II/III drug metabolism and defence against oxidative stress. Nrf2 is largely regulated by Keap1, which directs Nrf2 for proteasomal degradation. The Nrf2/Keap1 system is dysregulated in lung, head and neck, and breast cancers and this affects cellular proliferation and response to therapy. Here, we have investigated the integrity of the Nrf2/Keap1 system in pancreatic cancer.ResultsKeap1, Nrf2 and the Nrf2 target genes AKR1c1 and GCLC were detected in a panel of five pancreatic cancer cell lines. Mutation analysis of NRF2 exon 2 and KEAP1 exons 2-6 in these cell lines identified no mutations in NRF2 and only synonomous mutations in KEAP1. RNAi depletion of Nrf2 caused a decrease in the proliferation of Suit-2, MiaPaca-2 and FAMPAC cells and enhanced sensitivity to gemcitabine (Suit-2), 5-flurouracil (FAMPAC), cisplatin (Suit-2 and FAMPAC) and gamma radiation (Suit-2). The expression of Nrf2 and Keap1 was also analysed in pancreatic ductal adenocarcinomas (n = 66 and 57, respectively) and matching normal benign epithelium (n = 21 cases). Whilst no significant correlation was seen between the expression levels of Keap1 and Nrf2 in the tumors, interestingly, Nrf2 staining was significantly greater in the cytoplasm of tumors compared to benign ducts (P < 0.001).ConclusionsExpression of Nrf2 is up-regulated in pancreatic cancer cell lines and ductal adenocarcinomas. This may reflect a greater intrinsic capacity of these cells to respond to stress signals and resist chemotherapeutic interventions. Nrf2 also appears to support proliferation in certain pancreatic adenocarinomas. Therefore, strategies to pharmacologically manipulate the levels and/or activity of Nrf2 may have the potential to reduce pancreatic tumor growth, and increase sensitivity to therapeutics.