Annual Review of Pharmacology and Toxicology - Volume 45, 2005

The author describes studies that led to the resolution and reconstitution of the cytochrome P450 enzyme system in microsomal membranes. The review indicates how purification and characterization of the cytochromes led to rigorous evidence for multiple isoforms of the oxygenases with distinct chemical and physical properties and different but somewhat overlapping substrate specificities. Present knowledge of the individual steps in the P450 and reductase reaction cycles is summarized, including evidence for the generation of multiple functional oxidants that may contribute to the exceptional diversity of the reactions catalyzed.

Arylamines and heterocyclic arylamines (HAAs) are of particular interest because of demonstrated carcinogenicity in animals and humans and the broad exposure to many of these compounds. The activation of these, and also some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 (P450). P450 1A2 plays a prominent role in these reactions. However, P450 1A1 and 1B1 and other P450s are also important in humans as well as experimental animals. Some arylamines (including drugs) are N-hydroxylated predominantly by P450s other than those in Family 1. Other oxygenases can also have roles. An important issue is extrapolation between species in predicting cancer risks, as shown by the low rates of HAA activation by rat P450 1A2 and low levels of P450 1A2 expression in some nonhuman primates.

This review describes the three mammalian glutathione transferase (GST) families, namely cytosolic, mitochondrial, and microsomal GST, the latter now designated MAPEG. Besides detoxifying electrophilic xenobiotics, such as chemical carcinogens, environmental pollutants, and antitumor agents, these transferases inactivate endogenous α,β-unsaturated aldehydes, quinones, epoxides, and hydroperoxides formed as secondary metabolites during oxidative stress. These enzymes are also intimately involved in the biosynthesis of leukotrienes, prostaglandins, testosterone, and progesterone, as well as the degradation of tyrosine. Among their substrates, GSTs conjugate the signaling molecules 15-deoxy-Δ^12,14-prostaglandin J2 (15d-PGJ2) and 4-hydroxynonenal with glutathione, and consequently they antagonize expression of genes trans-activated by the peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). Through metabolism of 15d-PGJ2, GST may enhance gene expression driven by nuclear factor-κB (NF-κB). Cytosolic human GST exhibit genetic polymorphisms and this variation can increase susceptibility to carcinogenesis and inflammatory disease. Polymorphisms in human MAPEG are associated with alterations in lung function and increased risk of myocardial infarction and stroke. Targeted disruption of murine genes has demonstrated that cytosolic GST isoenzymes are broadly cytoprotective, whereas MAPEG proteins have proinflammatory activities. Furthermore, knockout of mouse GSTA4 and GSTZ1 leads to overexpression of transferases in the Alpha, Mu, and Pi classes, an observation suggesting they are part of an adaptive mechanism that responds to endogenous chemical cues such as 4-hydroxynonenal and tyrosine degradation products. Consistent with this hypothesis, the promoters of cytosolic GST and MAPEG genes contain antioxidant response elements through which they are transcriptionally activated during exposure to Michael reaction acceptors and oxidative stress.

Statins are potent inhibitors of cholesterol biosynthesis. In clinical trials, statins are beneficial in the primary and secondary prevention of coronary heart disease. However, the overall benefits observed with statins appear to be greater than what might be expected from changes in lipid levels alone, suggesting effects beyond cholesterol lowering. Indeed, recent studies indicate that some of the cholesterol-independent or “pleiotropic” effects of statins involve improving endothelial function, enhancing the stability of atherosclerotic plaques, decreasing oxidative stress and inflammation, and inhibiting the thrombogenic response. Furthermore, statins have beneficial extrahepatic effects on the immune system, CNS, and bone. Many of these pleiotropic effects are mediated by inhibition of isoprenoids, which serve as lipid attachments for intracellular signaling molecules. In particular, inhibition of small GTP-binding proteins, Rho, Ras, and Rac, whose proper membrane localization and function are dependent on isoprenylation, may play an important role in mediating the pleiotropic effects of statins.

For a long time neural and endocrine messages were studied for their impact on adipocyte metabolism and control of storage/release of fatty acids. In fact, bidirectional communication exists between adipocytes and other tissues. Several molecules secreted from adipocytes are involved in fat cell signaling to other tissues. Adipocyte products could initiate antagonistic effects on target tissues. Fat cells produce peptides that can elicit insulin resistance, such as tumor necrosis factor-α and resistin, as well as hormones that can improve insulin resistance, such as leptin and adiponectin. Secretion of complement proteins, proinflammatory cytokines, procoagulant, and acute phase reactant proteins have also been observed in adipocytes. There is much to learn about how these signals function. It is unlikely that all the adipocyte's endocrine and paracrine signals have been identified. Putative pharmacological strategies aiming at modulation of afferent and efferent fat cell messages are reviewed and discussed.

Toxic degradation products are formed from a range of old and modern anesthetic agents. The common element in the formation of degradation products is the reaction of the anesthetic agent with the bases in the carbon dioxide absorbents in the anesthesia circuit. This reaction results in the conversion of trichloroethylene to dichloroacetylene, halothane to 2-bromo-2-chloro-1,1-difluoroethylene, sevoflurane to 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A), and desflurane, isoflurane, and enflurane to carbon monoxide. Dichloroacetylene, 2-bromo-2-chloro-1,1-difluoroethylene, and Compound A form glutathione S-conjugates that undergo hydrolysis to cysteine S-conjugates and bioactivation of the cysteine S-conjugates by renal cysteine conjugate β-lyase to give nephrotoxic metabolites. The elucidation of the mechanisms of formation and bioactivation of degradation products has allowed for the safe use of anesthetics that may undergo degradation in the anesthesia circuit.

The importance of reactive metabolites in the pathogenesis of drug-induced toxicity has been a focus of research interest since pioneering investigations in the 1950s revealed the link between toxic metabolites and chemical carcinogenesis. There is now a great deal of evidence that shows that reactive metabolites are formed from drugs known to cause hepatotoxicity, but how these toxic species initiate and propagate tissue damage is still poorly understood. This review summarizes the evidence for reactive metabolite formation from hepatotoxic drugs, such as acetaminophen, tamoxifen, diclofenac, and troglitazone, and the current hypotheses of how this leads to liver injury. Several hepatic proteins can be modified by reactive metabolites, but this in general equates poorly with the extent of toxicity. Much more important may be the identification of the critical proteins modified by these toxic species and how this alters their function. It is also important to note that the toxicity of reactive metabolites may be mediated by noncovalent binding mechanisms, which may also have profound effects on normal liver physiology. Technological developments in the wake of the genomic revolution now provide unprecedented power to characterize and quantify covalent modification of individual target proteins and their functional consequences; such information should dramatically improve our understanding of drug-induced hepatotoxic reactions.

Botanicals such as herbal products (HPs) and nutraceuticals (NCs) are often regarded as low risk because of their long history of human use. Anecdotal and literature reports of adverse drug events (ADEs) and clinical studies with HPs are increasing, but many of the reports are incomplete and contradictory. These reports need to identify confounding factors and explain contradictory findings if they are to help health care professionals or patients understand what risks are involved. HPs are complex botanicals, not single-active ingredient (SAI) products. Studies can be confounded by different manufacturing processes and formulations, including cosmetics and food supplements; environment; chemotypes; misidentification or adulteration; and factors associated with the patient or user population such as use, total drug load, and genetics. Future studies need to be conducted with characterized product that includes all commercially available related products. Clinical trials should be relevant to the user population and take into account the confounding factors that may influence the interpretation of the findings.

This review focuses on the current status of biomarkers and/or approaches critical to assessing novel neuroscience targets with an emphasis on new paradigms and challenges in this field of research. The importance of biomarker data integration for psychotropic drug development is illustrated with examples for clinically used medications and investigational drugs. The question remains how to verify access to the brain. Early imaging studies including micro-PET can help to overcome this. However, in case of delayed tracer development or because of no feasible application of brain imaging effects of the molecule, using CSF as a matrix could fill this gap. Proteomic research using CSF will hopefully have a major impact on the development of treatments for psychiatric disorders.

The neonicotinoids, the newest major class of insecticides, have outstanding potency and systemic action for crop protection against piercing-sucking pests, and they are highly effective for flea control on cats and dogs. Their common names are acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam. They generally have low toxicity to mammals (acute and chronic), birds, and fish. Biotransformations involve some activation reactions but largely detoxification mechanisms. In contrast to nicotine, epibatidine, and other ammonium or iminium nicotinoids, which are mostly protonated at physiological pH, the neonicotinoids are not protonated and have an electronegative nitro or cyano pharmacophore. Agonist recognition by the nicotinic receptor involves cation-π interaction for nicotinoids in mammals and possibly a cationic subsite for interaction with the nitro or cyano substituent of neonicotinoids in insects. The low affinity of neonicotinoids for vertebrate relative to insect nicotinic receptors is a major factor in their favorable toxicological profile.

Increasing evidence supports the notion that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with multiple functions, including its surprising role in apoptosis. GAPDH is overexpressed and accumulates in the nucleus during apoptosis induced by a variety of insults in diverse cell types. Knockdown of GAPDH using an antisense strategy demonstrates its involvement in the apoptotic cascade in which GAPDH nuclear translocation appears essential. Knowledge concerning the mechanisms underlying GAPDH nuclear translocation and subsequent cell death is growing. Additional evidence suggests that GAPDH may be an intracellular sensor of oxidative stress during early apoptosis. Abnormal expression, nuclear accumulation, changes in physical properties, and loss of glycolytic activity of GAPDH have been found in cellular and transgenic models as well as postmortem tissues of several neurodegenerative diseases. The interaction of GAPDH with disease-related proteins as well as drugs used to treat these diseases suggests that it is a potential molecular target for drug development.

The cytochrome P450 monooxygenases (CYPs) are the dominant enzyme system responsible for xenobiotic detoxification and drug metabolism. Several CYP isoforms exhibit non-Michaelis-Menten, or “atypical,” steady state kinetic patterns. The allosteric kinetics confound prediction of drug metabolism and drug-drug interactions, and they challenge the theoretical paradigms of allosterism. Both homotropic and heterotropic ligand effects are now widely documented. It is becoming apparent that multiple ligands can simultaneously bind within the active sites of individual CYPs, and the kinetic parameters change with ligand occupancy. In fact, the functional effect of any specific ligand as an activator or inhibitor can be substrate dependent. Divergent approaches, including kinetic modeling and X-ray crystallography, are providing new information about how multiple ligand binding yields complex CYP kinetics.

Organisms are exposed to epoxide-containing compounds from both exogenous and endogenous sources. In mammals, the hydration of these compounds by various epoxide hydrolases (EHs) can not only regulate their genotoxicity but also, for lipid-derived epoxides, their endogenous roles as chemical mediators. Recent findings suggest that the EHs as a family represent novel drug discovery targets for regulation of blood pressure, inflammation, cancer progression, and the onset of several other diseases. Knowledge of the EH mechanism provides a solid foundation for the rational design of inhibitors, and this review summarizes the current understanding of the catalytic mechanism of the EHs. Although the overall EH mechanism is now known, the molecular basis of substrate selectivity, possible allosteric regulation, and many fine details of the catalytic mechanism remain to be solved. Finally, recent development in the design of EH inhibitors and the EH biological role are discussed.

Recent discoveries of novel and potentially important biological activity have spurred interest in the chemistry and biochemistry of nitroxyl (HNO). It has become clear that, among all the nitrogen oxides, HNO is unique in its chemistry and biology. Currently, the intimate chemical details of the biological actions of HNO are not well understood. Moreover, many of the previously accepted chemical properties of HNO have been recently revised, thus requiring reevaluation of possible mechanisms of biological action. Herein, we review these developments in HNO chemistry and biology.

The clinical application of tyrosine kinase inhibitors for cancer treatment represents a therapeutic breakthrough. The rationale for developing these compounds rests on the observation that tyrosine kinase enzymes are critical components of the cellular signaling apparatus and are regularly mutated or otherwise deregulated in human malignancies. Novel tyrosine kinase inhibitors are designed to exploit the molecular differences between tumor cells and normal tissues. Herein, we will review the current state-of-the-art using agents that target as prototypes Bcr-Abl, platelet-derived growth factor receptor (PDGFR), KIT (stem cell factor receptor), and epidermal growth factor receptor (EGFR). These compounds are remarkably effective in treating diverse cancers that are highly resistant to conventional treatment, including various forms of leukemia, hypereosinophilic syndrome, mast cell disease, sarcomas, and lung cancer. It is now clear that the molecular defects underlying cancer can be targeted with designer drugs that yield striking salutary effects with minimal toxicity.

Adenosine and its receptors have been the topic of many recent reviews (1–B2, 26). These reviews provide a good summary of much of the relevant literature—including the older literature. We have, therefore, chosen to focus the present review on the insights gained from recent studies on genetically modified mice, particularly with respect to the function of adenosine receptors and their potential as therapeutic targets. The information gained from studies of drug effects is discussed in this context, and discrepancies between genetic and pharmacological results are highlighted.

Cytochrome P450–catalyzed metabolism of arachidonic acid is an important pathway for the formation of paracrine and autocrine mediators of numerous biological effects. The ω-hydroxylation of arachidonic acid generates significant levels of 20-hydroxyeicosatetraenoic acid (20-HETE) in numerous tissues, particularly the vasculature and kidney tubules. Members of the cytochrome P450 4A and 4F families are the major ω-hydroxylases, and the substrate selectivity and regulation of these enzymes has been the subject of numerous studies. Altered expression and function of arachidonic acid ω-hydroxylases in models of hypertension, diabetes, inflammation, and pregnancy suggest that 20-HETE may be involved in the pathogenesis of these diseases. Our understanding of the biological significance of 20-HETE has been greatly aided by the development and characterization of selective and potent inhibitors of the arachidonic acid ω-hydroxylases. This review discusses the substrate selectivity and expression of arachidonic acid ω-hydroxylases, regulation of these enzymes during disease, and the application of enzyme inhibitors to study 20-HETE function.

The hepatic cytochromes P450 (P450s) are monotopic endoplasmic reticulum (ER)-anchored hemoproteins engaged in the enzymatic oxidation of a wide variety of endo- and xenobiotics. In the course of these reactions, the enzymes generate reactive O2 species and/or reactive metabolic products that can attack the P450 heme and/or protein moiety and structurally and functionally damage the enzyme. The in vivo conformational unraveling of such a structurally damaged P450 signals its rapid removal via the cellular sanitation system responsible for the proteolytic disposal of structurally aberrant, abnormal, and/or otherwise malformed proteins. A key player in this process is the ubiquitin (Ub)-dependent 26S proteasome system. Accordingly, the structurally deformed P450 protein is first branded for recognition and proteolytic removal by the 26S proteasome with an enzymatically incorporated polyUb tag. P450s of the 3A subfamily such as the major human liver enzyme CYP3A4 are notorious targets for this process, and they represent excellent prototypes for the understanding of integral ER protein ubiquitination. Not all the participants in hepatic CYP3A ubiquitination and subsequent proteolytic degradation have been identified. The following discussion thus addresses the various known and plausible events and/or cellular participants involved in this multienzymatic P450 ubiquitination cascade, on the basis of our current knowledge of other eukaryotic models. In addition, because the detection of ubiquitinated P450s is technically challenging, the critical importance of appropriate methodology is also discussed.

Normal cellular functioning requires processing of proteins regulating cell cycle, growth, and apoptosis. The ubiquitin-proteasome pathway (UBP) modulates intracellular protein degradation. Specifically, the 26S proteasome is a multienzyme protease that degrades misfolded or redundant proteins; conversely, blockade of the proteasomal degradation pathways results in accumulation of unwanted proteins and cell death. Because cancer cells are more highly proliferative than normal cells, their rate of protein translation and degradation is also higher. This notion led to the development of proteasome inhibitors as therapeutics in cancer. The FDA recently approved the first proteasome inhibitor bortezomib (Velcade™), formerly known as PS-341, for the treatment of newly diagnosed and relapsed/refractory multiple myeloma (MM). Ongoing studies are examining other novel proteasome inhibitors, in addition to bortezomib, for the treatment of MM and other cancers.

CYP2C9 is a major cytochrome P450 enzyme that is involved in the metabolic clearance of a wide variety of therapeutic agents, including nonsteroidal antiinflammatories, oral anticoagulants, and oral hypoglycemics. Disruption of CYP2C9 activity by metabolic inhibition or pharmacogenetic variability underlies many of the adverse drug reactions that are associated with the enzyme. CYP2C9 is also the first human P450 to be crystallized, and the structural basis for its substrate and inhibitor selectivity is becoming increasingly clear. New, ultrapotent inhibitors of CYP2C9 have been synthesised that aid in the development of quantitative structure-activity relationship (QSAR) models to facilitate drug redesign, and extensive resequencing of the gene and studies of its regulation will undoubtedly help us understand interindividual variability in drug response and toxicity controlled by this enzyme.