Preface

Abstract

While toxicology is clearly a well defined science in its own right, it is also apparent that it is a highly eclectic science, drawing heavily on the methods and principles of other scientific disciplines. In a general and philosophical sense, toxicology is the study of poisons. However, toxicology would be of slight importance to society in general if it were not for the fact that, in an operational sense, it can also be defined as the science of chemical risk analysis. Risk analysis and its narrower subdiscipline, risk assessment, require a detailed knowledge not only of the modes of action of particular toxicants, but also of their interactions with other toxicants. Such detailed knowledge can come only from an integrative approach involving all of the events, from exposure to the expression of a toxic endpoint, carried out at all levels of biological complexity. This cascade includes such important steps as uptake at portals of entry, distribution throughout the body, detoxication and activation by phase I and phase II xenobiotic-metabolizing enzymes, further distribution of the metabolic products, and then either excretion or interaction with macromolecules (e.g., DNA, proteins) to bring about toxic endpoints. Even in its simplest summary form, this cascade consists of events that may or may not be reversible or that may or may not be rate limiting, and which may result in overall detoxication as well as the expression of one or more toxic endpoints. When two of the most important results of toxicological study, namely human health risk assessment and environmental risk assessment, are considered, it is apparent that, even in outline, they are not simple one-step processes, but complex integrative methodologies. At the same time, structural, biochemical, and molecular events at any of the various levels of biological organization—ecosystem, organism, organ, cell, subcellular organelle—can be reduced to diagrams of greater complexity. Many examples of such diagrams can be seen in standard toxicology texts (Casarett et al., 2001; Hodgson and Smart, 2001; Hodgson, 2004). To expand such a diagrammatic representation to include all of its many interactions and then integrate all of these aspects to include, under one description, a complete summary of all of the events following the exposure of an organism or ecosystem to a toxicant would result in a description so complex that only the application of the emerging discipline of systems biology could enable it to be used in a predictive manner. However, whether it be risk analysis or systems biology, it should not be forgotten that for both the initiation and the further construction of any model of toxicity, the input must be experimental data derived from actual experiments. It is in this latter regard that Current Protocols in Toxicology can play an indispensable role: first, to envision the complex of interactions involved in toxic action and second, to provide the necessary protocols for examining these interactions and for obtaining the inputs necessary to develop science-based, predictive models. The initial approach when consulting Current Protocols in Toxicology is generally to decide which chapters are likely to contain units applicable to the toxicant of interest, then to decide which of these units should be utilized first. Often of greater interest and importance is the subsequent decision, based on the initial results, as to which further units should be used to extend the findings to yield the complex model for toxicity that will, inevitably, result. Thus, it is possible to use Current Protocols in Toxicology to derive a variety of templates that can be used for the comprehensive investigation of the toxicity of a particular chemical and extrapolate the knowledge gained to an analysis of human health risk and/or environmental risk. The following are some examples of the potential utilization of Current Protocols in Toxicology, along with indications of where additional protocols are needed for more meaningful coverage. It should be emphasized that Current Protocols in Toxicology is very much a work in progress and we hope it will always be a work in progress. To further that end, suggestions concerning which units need to be revised or replaced by more contemporary methods or units that should be added are always welcome, and may be addressed to any or all of the editorial board. Toxicant 1 is an environmental contaminant of concern to public health that is readily absorbed through the skin and respiratory system and that is activated by cytochrome P450 to a metabolite that interacts with one or more neuroreceptors, with consequent behavioral effects. This toxicant is also detoxified by phase I and phase II xenobiotic-metabolizing enzymes, and its products are excreted by both biliary and renal routes. There is some indication of the involvement of oxidative stress as a secondary mode of action. Both human health and environmental health risk assessments are needed by regulatory agencies. On the basis of the abovementioned issues, recommended chapters and units of Current Protocols in Toxicology to consult would be as follows: Exposure: Exposure assessment, an integral part of human health and environmental risk analysis, will be the subject of units in a future chapter of Current Protocols in Toxicology. Uptake and distribution, redistribution of metabolites: Chapter 5, particularly UNITS Unavailable–Unavailable. Metabolism (activation and/or deactivation): Potentially any unit in Chapter 4, also Chapter 6, UNIT Unavailable. Mechanism of action: Potentially any unit in Chapters 11 and 12. Chapter 7, potentially all units. Excretion: Chapter 5, UNIT Unavailable. Risk assessment: Risk assessment, a critical step leading to risk communication and risk management, will be the subject of units in a future chapter of Current Protocols in Toxicology. Toxicant 2 is an industrial chemical, an alcohol used in the workplace that is readily absorbed through the skin and respiratory system. It is metabolized by alcohol and aldehyde dehydrogenases as well as phase II xenobiotic-metabolizing enzymes. While acute toxicity and hepatotoxicity are of primary concern, the toxicant must also be evaluated for chronic toxicity and potential carcinogenic effects in humans. The end products of metabolism are excreted by the renal route. There is also epidemiological evidence of immunotoxicity. On the basis of the abovementioned issues, recommended chapters and units of Current Protocols in Toxicology to consult would be as follows: Exposure: Exposure assessment, an integral part of human health and environmental risk analysis, will be the subject of units in a future chapter of Current Protocols in Toxicology. Uptake and distribution, redistribution of metabolites: Chapter 5, particularly UNITS Unavailable–Unavailable. Metabolism (activation and/or deactivation): Potentially any unit in Chapter 4, but particularly UNITS Unavailable & Unavailable. Also Chapter 6, UNIT Unavailable. Mechanism of action: Potentially any unit in Chapters 14 and 18. Chapter 2, UNITS Unavailable, Unavailable, & Unavailable. Excretion: Chapter 5, UNIT Unavailable. Risk Assessment: Risk assessment, a critical step leading to risk communication and risk management, will be the subject of units in a future chapter of Current Protocols in Toxicology. This publication is available in both looseleaf and online format. For looseleaf purchasers, binders are provided to accommodate the growth of the manual via the quarterly update service. This format allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. The material covered in the two versions is identical. Subjects in this manual are organized by chapters, and protocols are contained in units. Protocol units, which constitute the bulk of the book, generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method. Other units present more general information in the form of explanatory text with no protocols. Overview units contain theoretical discussions that lay the foundation for subsequent protocols. Other discussion units present more general information. Page numbering in the looseleaf version reflects the modular arrangement by unit; for example, page 1.2.3 refers to Chapter 1 (Toxicological Models), UNIT Unavailable (Statistical Methods in Toxicology), page 3 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used and recipes for common reagents are supplied in APPENDIX Unavailable. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Because this publication is first and foremost a compilation of laboratory techniques in toxicology, we have included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with special overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Chapter and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Many units in the manual contain groups of protocols, each presented with a series of steps. One or more basic protocols are presented first in each unit and generally cover the recommended or most universally applicable approaches. Alternate protocols are provided where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the basic protocol. Support protocols describe additional steps that are required to perform the basic or alternate protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the basic protocol steps. Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, while others are solutions unique to a particular protocol. Recipes for the latter solutions are provided in each unit, following the protocols (and before the Commentary), under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., electrophoresis buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are provided once in APPENDIX Unavailable. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Instructional videos are sometimes included for a unit. These videos illustrate how to perform particular steps of the protocol. For users of the looseleaf version, at the appropriate point in the text the user is directed to view the video at http://www.current protocols.com on the Current Protocols in Toxicology home page. Direct download links are included in the online version. Throughout the manual, the authors have recommended commercial suppliers of chemicals, biological materials, and equipment. In some cases, the noted brand has been found to be of superior quality or it is the only suitable product available in the marketplace. In other cases, the experience of the author of that protocol is limited to that brand. In the latter situation, recommendations are offered as an aid to the novice in obtaining the tools of the trade. Experienced investigators are therefore encouraged to experiment with substituting their own favorite brands. Addresses, phone numbers, and facsimile numbers of all suppliers mentioned in this manual are provided in the SUPPLIERS APPENDIX. Anyone carrying out these protocols may encounter the following hazardous or potentially hazardous materials: (1) radioactive substances, (2) toxic chemicals and carcinogenic or teratogenic reagents, and (3) pathogenic and infectious biological agents. Check the guidelines of your particular institution with regard to use and disposal of these hazardous materials. Although cautionary statements are included in the appropriate units, we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials must be used in strict accordance with local and national regulations. Many protocols call for use of live animals (usually rats or mice) for experiments. Prior to conducting any laboratory procedures with live subjects, the experimental approach must be submitted in writing to the appropriate Institutional Animal Care and Use Committee (IACUC) and must conform to appropriate governmental regulations regarding the care and use of laboratory animals. Written approval from the IACUC (or equivalent) is absolutely required prior to undertaking any live-animal studies. Some specific animal care and handling guidelines are provided in the protocols where live subjects are used, but check with your IACUC or governmental guidelines to obtain more extensive information. Most of the protocols included in this manual are used routinely in the authors' laboratories. These protocols work for them; to make them work for you, they have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers' observations and suggestions. Consequently, a self-mailing reader-response survey can be found at the back of the manual (and is included with each print supplement); we encourage readers to send in their comments. This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work by the Current Protocols editorial staff at John Wiley & Sons. We are extremely grateful for the critical contributions by Kathy Morgan (Series Editor), who kept the editors and the contributors on track and played a key role in bringing the entire project into existence, and by Gwen Taylor and Virginia Chanda, who provided developmental support in the early stages of the project. Other skilled members of the Current Protocols staff who contributed to the project include Joseph White, Maria Monte, Scott Holmes, and Tom Cannon. The extensive copyediting required to produce an accurate protocols manual was ably handled by Allen Ranz, Marianne Huntley, Susan Lieberman, Tom Downey, and Sylvia de Hombre, and electronic illustrations were prepared by Gae Xavier Studios and Matrix Art Services.