Annual Review of Pharmacology and Toxicology - Volume 39, 1999

Methyl conjugation is an important pathway in the biotransformation of many exogenous and endogenous compounds. Pharmacogenetic studies of methyltransferase enzymes have resulted in the identification and characterization of functionally important common genetic polymorphisms for catechol O-methyltransferase, thiopurine methyltransferase, and histamine N-methyltransferase. In recent years, characterization of these genetic polymorphisms has been extended to include the cloning of cDNAs and genes, as well as a determination of the molecular basis for the effects of inheritance on these methyltransferase enzymes. The thiopurine methyltransferase genetic polymorphism is responsible for clinically significant individual variations in the toxicity and therapeutic efficacy of thiopurine drugs such as 6-mercaptopurine. Phenotyping for the thiopurine methyltransferase genetic polymorphism represents one of the first examples in which testing for a pharmacogenetic variant has entered standard clinical practice. The full functional implications of pharmacogenetic variation in the activities of catechol O-methyltransferase and histamine N-methyltransferase remain to be determined. Finally, experimental strategies used to study methylation pharmacogenetics illustrate the rapid evolution of biochemical, pharmacologic, molecular, and genomic approaches that have been used to determine the role of inheritance in variation in drug metabolism, effect, and toxicity.

The pineal gland expresses a group of proteins essential for rhythmic melatonin production. This pineal-specific phenotype is the consequence of a temporally and spacially controlled program of gene expression. Understanding of pineal circadian biology has been greatly facilitated in recent years by a number of molecular studies, including the cloning of N-acetyltransferase, the determination of the in vivo involvement of the cAMP-inducible early repressor in the regulation of N-acetyltransferase, and the identification of a pineal transcriptional regulatory element and its interaction with the cone-rod homeobox protein. Likewise, appreciation the physiological roles of melatonin has increased dramatically with the cloning and targeted knockout of melatonin receptors. With these molecular tools in hand, we can now address more specific questions about how and why melatonin is made in the pineal at night and about how it influences the rest of the body.

Reactive oxygen intermediates are produced in all aerobic organisms during respiration and exist in the cell in a balance with biochemical antioxidants. Excess reactive oxygen resulting from exposure to environmental oxidants, toxicants, and heavy metals perturbs cellular redox balance and disrupts normal biological functions. The resulting imbalance may be detrimental to the organism and contribute to the pathogenesis of disease and aging. To counteract the oxidant effects and to restore a state of redox balance, cells must reset critical homeostatic parameters. Changes associated with oxidative damage and with restoration of cellular homeostasis often lead to activation or silencing of genes encoding regulatory transcription factors, antioxidant defense enzymes, and structural proteins. In this review, we examine the sources and generation of free radicals and oxidative stress in biological systems and the mechanisms used by reactive oxygen to modulate signal transduction cascades and redirect gene expression.

Cytochrome P4501A1 is a substrate-inducible microsomal enzyme that oxygenates polycyclic aromatic hydrocarbons, such as the carcinogen benzo(a)pyrene, as the initial step in their metabolic processing to water-soluble derivatives. Enzyme induction reflects increased transcription of the cognate CYP1A1 gene. The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin is the most potent known cytochrome P4501A1 inducer. Two regulatory proteins, the aromatic (aryl) hydrocarbon receptor (AhR) and the AhR nuclear translocator (Arnt), mediate induction. AhR and Arnt are prototypical members of the basic helix-loop-helix/Per-Arnt-Sim class of transcription factors. Mechanistic analyses of cytochrome P4501A1 induction provide insights into ligand-dependent mammalian gene expression, basic helix-loop-helix/Per-Arnt-Sim protein function, and dioxin action; such studies also impact public health issues concerned with molecular epidemiology, carcinogenesis, and risk assessment.

Ethanol and other short-chain alcohols elicit a number of cellular responses that are potentially cytotoxic and, to some extent, independent of cell type. Aberrations in phospholipid and fatty acid metabolism, changes in the cellular redox state, disruptions of the energy state, and increased production of reactive oxygen metabolites have been implicated in cellular damage resulting from acute or chronic exposure to short-chain alcohols. Resulting disruptions of intracellular signaling cascades through interference with the synthesis of phosphatidic acid, decreases in phosphorylation potential and lipid peroxidation are mechanisms by which solvent alcohols can affect the rate of cell proliferation and, consequently, cell number. Nonoxidative metabolism of short-chain alcohols, including phospholipase D–mediated synthesis of alcohol phospholipids, and the synthesis of fatty acid alcohol esters are additional mechanisms by which alcohols can affect membrane structure and compromise cell function.

Neuroglial cells of the central nervous system include the astrocytes, oligodendrocytes, and microglia. Their counterparts in the peripheral nervous system are the Schwann cells. The term neuroglia comes from an erroneous concept originally coined by Virchow (1850), in which he envisioned the neurons to be embedded in a layer of connective tissue. The term, or its shortened form—glia, has persisted as the preferred generic term for these cells. A reciprocal relationship exists between neurons and glia, and this association is vital for mutual differentiation, development, and functioning of these cell types. Therefore, perturbations in glial cell function, as well as glial metabolism of chemicals to active intermediates, can lead to neuronal dysfunction. The purpose of this review is to explore neuroglial sites of neurotoxicant actions, discuss potential mechanisms of glial-induced or glial-mediated central nervous system and peripheral nervous system damage, and review the role of glial cells in neurotoxicity development.

In recent years, there has been great interest in the study of phospholipid metabolism in intact cell systems. Such an interest arises mainly from the discovery that cellular membrane phospholipids serve not only in structural roles, but are also reservoirs of preformed second messenger molecules with key roles in cellular signaling. These second messenger molecules are generated by agonist-induced activation and secretion of intracellular and extracellular phospholipases, respectively, i.e. enzymes that cleave ester bonds within phospholipids. Prominent members of the large collection of signal-activated phospholipases are the phospholipase A2s. These enzymes hydrolyze the sn-2 ester bond of phospholipids, releasing a free fatty acid and a lysophospholipid, both of which may alter cell function. In addition to its role in cellular signaling, phospholipase A2 has recently been recognized to be involved in a wide number of pathophysiological situations, ranging from systemic and acute inflammatory conditions to cancer. A growing number of pharmacologic inhibitors will help define the role of particular phospholipase A2s in signaling cascades.

Nitric oxide (NO) regulates numerous physiological processes, including neurotransmission, smooth muscle contractility, platelet reactivity, and the cytotoxic activity of immune cells. Because of the ubiquitous nature of NO, inappropriate release of this mediator has been linked to the pathogenesis of a number of disease states. This provides the rationale for the design of therapies that modulate NO concentrations selectively. A well-characterized family of compounds are the inhibitors of NO synthase, the enzyme responsible for the generation of NO; such agents are potentially beneficial in the treatment of conditions associated with an overproduction of NO, including septic shock, neurodegenerative disorders, and inflammation. This article provides an overview of NO synthase inhibitors, focusing on agents that prevent binding of substrate l-arginine.

Transcriptional and translational regulation of glutamate receptor expression determines one of the key phenotypic features of neurons in the brain—the properties of their excitatory synaptic receptors. Up- and down-regulation of various glutamate receptor subunits occur throughout development, following ischemia, seizures, repetitive activation of afferents, or chronic administration of a variety of drugs. The promoters of the genes that encode the NR1, NR2B, NR2C, GluR1, GluR2, and KA2 subunits share several characteristics that include multiple transcriptional start sites within a CpG island, lack of TATA and CAAT boxes, and neuronal-selective expression. In most cases, the promoter regions include overlapping Sp1 and GSG motifs near the major initiation sites, and a silencer element, to guide expression in neurons. Manipulating the levels of glutamate receptors in vivo by generating transgenic and knockout mice has enhanced understanding of the role of specific glutamate receptor subunits in long-term potentiation and depression, learning, seizures, neural pattern formation, and survival. Neuron-specific glutamate receptor promoter fragments may be employed in the design of novel gene-targeting constructs to deliver future experimental transgene and therapeutic agents to selected neurons in the brain.

Reduction/oxidation (redox) reactions play a central role in the regulation of vascular cell functions. Recent studies in this laboratory have identified c-Ha-ras and osteopontin genes as critical molecular targets during oxidant-induced atherogenesis. This review focuses on the deregulation of gene transcription by redox-activated trans-acting factors after benzo(a)pyrene challenge and the modulation of extracellular matrix signaling in vascular smooth muscle cells by allylamine-induced oxidative injury. The induction of atherogenic vascular smooth muscle cell phenotypes by chemical injury exhibits remarkable parallels with those seen in other forms of atherogenesis.

Metallothioneins (MT) are low-molecular-weight, cysteine-rich, metal-binding proteins. MT genes are readily induced by various physiologic and toxicologic stimuli. Because the cysteines in MT are absolutely conserved across species, it was suspected that the cysteines are necessary for function and MT is essential for life. In attempts to determine the function(s) of MT, studies have been performed using four different experimental paradigms: (a) animals injected with chemicals known to induce MT; (b) cells adapted to survive and grow in high concentrations of MT-inducing toxicants; (c) cells transfected with the MT gene; and (d) MT-transgenic and MT-null mice. Most often, results from studies using the first three approaches have indicated multiple functions of MT in cell biology: MT (a) is a “storehouse” for zinc, (b) is a free-radical scavenger, and (c) protects against cadmium (Cd) toxicity. However, studies using MT-transgenic and null mice have not strongly supported the first two proposed functions but strongly support its function in protecting against Cd toxicity. Repeated administration of Cd to MT-null mice results in nephrotoxicity at one tenth the dose that produces nephrotoxicity in control mice. Human studies indicate that 7% of the general population have renal dysfunction from Cd exposure. Therefore, if humans did not have MT, “normal” Cd exposure would be nephrotoxic to humans. Thus, it appears that during evolution, the ability of MT to protect against Cd toxicity might have taken a more pivotal role in the maintenance of life processes, as compared with its other proposed functions (i.e. storehouse for zinc and free radical scavenger).

The eucaryotic cell cycle is regulated by the periodic synthesis and destruction of cyclins that associate with and activate cyclin-dependent kinases. Cyclin-dependent kinase inhibitors, such as p21 and p16, also play important roles in cell cycle control by coordinating internal and external signals and impeding proliferation at several key checkpoints. Understanding how these proteins interact to regulate the cell cycle has become increasingly important to researchers and clinicians with the discovery that many of the genes that encode cell cycle regulatory activities are targets for alterations that underlie the development of cancer. Several therapeutic agents, such as DNA-damaging drugs, microtubule inhibitors, antimetabolites, and topoisomerase inhibitors, take advantage of this disruption in normal cell cycle regulation to target checkpoint controls and ultimately induce growth arrest or apoptosis of neoplastic cells. Other therapeutic drugs being developed, such as UCN-01, specifically inhibit cell cycle regulatory proteins.

Dopaminergic receptors are widespread throughout the central and peripheral nervous systems, where they regulate a variety of physiological, behavioral, and endocrine functions. These receptors are also clinically important drug targets for the treatment of a number of disorders, such as Parkinson's disease, schizophrenia, and hyperprolactinemia. To date, five different dopamine receptor subtypes have been cloned and characterized. Many of these subtypes are pharmacologically similar, making it difficult to selectively stimulate or block a specific receptor subtype in vivo. Thus, the assignment of various physiological or behavioral functions to specific dopamine receptor subtypes using pharmacological tools is difficult. In view of this, a number of investigators have—in order to elucidate functional roles—begun to use highly selective genetic approaches to alter the expression of individual dopamine receptor subtypes in vivo. This review discusses recent studies involving the use of genetic approaches for the study of dopaminergic receptor function.

Cardiac β-adrenergic receptors, which respond to neuronally released and circulating catecholamines, are important regulators of cardiac function. Congestive heart failure, a common clinical condition, is associated with a number of alterations in the activation and deactivation of β-adrenergic receptor pathways. Studies with failing hearts from humans and animals indicate that such alterations include changes in the expression or function of β-adrenergic receptors, G-proteins, adenylyl cyclases, and G-protein receptor kinases. The net effect of these alterations is the substantial blunting of β-adrenergic receptor-mediated cardiac response. An important unanswered question is whether the loss of cardiac β-adrenergic receptor responsiveness is a contributing cause, or a result, of ventricular dysfunction. Even though this question remains unanswered, the concept of targeting the β-adrenergic pathway in the failing heart is becoming increasingly popular and several new therapeutic strategies are in development.

Considerable evidence has accumulated indicating that the multidrug transporter or P-glycoprotein plays a role in the development of simultaneous resistance to multiple cytotoxic drugs in cancer cells. In recent years, various approaches such as mutational analyses and biochemical and pharmacological characterization have yielded significant information about the relationship of structure and function of P-glycoprotein. However, there is still considerable controversy about the mechanism of action of this efflux pump and its function in normal cells. This review summarizes current research on the structure-function analysis of P-glycoprotein, its mechanism of action, and facts and speculations about its normal physiological role.

Either an excess or a deficiency of vitamin A and related compounds (retinoids) causes abnormal morphological development (teratogenesis). Potential retinoid sources come from dietary intake, nutritional supplements, and some therapeutic drugs. Therefore, understanding the mechanisms of retinoid teratogenesis is important. This review first gives an overview of the principles of teratology as they apply to retinoid-induced malformations. It then describes relevant aspects of the biochemical pathway and signal transduction of retinoids. The teratogenic activity of various retinoid compounds, the role of the retinoid receptors, and important toxicokinetic parameters in teratogenesis are reviewed.

As the most predominant excitatory neurotransmitter, glutamate has the potential to influence the function of most neuronal circuits in the central nervous system. To limit receptor activation during signaling and prevent the overstimulation of glutamate receptors that can trigger excitotoxic mechanisms and cell death, extracellular concentrations of excitatory amino acids are tightly controlled by transport systems on both neurons and glial cells. l-Glutamate is a potent neurotoxin, and the inadequate clearance of excitatory amino acids may contribute to the neurodegeneration seen in a variety of conditions, including epilepsy, ischemia, and amyotrophic lateral sclerosis. To establish the contributions of carrier systems to the etiology of neurological disorders, and to consider their potential utility as therapeutic targets, a detailed understanding of transporter function and pharmacology is required. This review summarizes current knowledge of the structural and functional diversity of excitatory amino acid transporters and explores how they might serve as targets for drug design.