RECENTLY PUBLISHED:

UNIT 1.3 Transgenic Animals in Toxicology (Warren Ladiges and Carol B. Ware, University of Washington, Seattle, Wash.). This is a clearly written guide to the design and generation of transgenic animals for use in toxicological studies. It begins with a discussion of DNA construct design and proceeds to production of transgenic animals—animal husbandry, preparation of pseudopregnant animals, production of transgenic animals by pronuclear fusion or gene targeting, production of chimeras, and colony management. This unit will be complimented by a table of trangenic animals that are useful for toxicology to be published in a supplement.

UNIT 2.2 Determination of Apoptosis and Necrosis (Boris Zhivotovsky, Afshin Samali, and Sten Orrenius, Karolinska Institute, Stockholm). This unit contains a number of assays for distinguishing apoptosis from necrosis—trypan blue, RAPI-DIFF, and Hoechst staining; analysis of size, granularity, and DNA content by flow cytometry; annexin V binding; TUNEL assays; detection of DNA fragmentation; pulsed-field gel electrophoresis; and caspase enzyme activity—to characterize the nature of cellular death arising from exposure to toxicants.

UNIT 2.5 Measurements of Intracellular Free Calcium Concentrations in Biological Systems (Karlene K. Gunter and Thomas E. Gunter, University of Rochester, Rochester, N.Y.). Intracellular calcium is used in signaling and as a second messenger in many types of cells, and calcium concentration is closely regulated. Two techniques for measuring intracellular free calcium using fluorescent ratiometric and nonratiometric probes are described in this unit—fluorescent spectroscopy to measure calcium concentrations in a cell suspension and fluorescent digital imaging microscopy (FDIM) to measure calcium concentrations in plated cells.

UNIT 3.3 Mutagenesis Assays in Mammalian Cells (Catherine B. Klein, Limor Broday, and Max Costa, New York University School of Medicine, New York). Mutagenesis assays in mammalian cells are frequently used to complement bacterial mutagenesis assays. This unit describes a mutagenesis assay using either Chinese hamster V79 cells or a V79-derivative gpt transgenic cell line to assess the effects of chemical agents on mammalian cells.

UNIT 3.4 Cell Transformation Assays (Max Costa and Jessica E. Sutherland, New York University School of Medicine). Morphological transformation of mammalian cells following exposure to a chemical agent is frequently used as a preliminary screen for the carcinogenic potential of the agent. This unit describes a protocol using Syrian hamster embryo (SHE) cells to assay transformation.

UNIT 8.6 Measurement of d-Aminolevulinate Dehydratase (ALAD) Activity (Hiroyoshi Fujita, Hokkaido University School of Medicine, Sapporo, Japan). Vertebrate ALAD is a cytosolic enzyme that catalyzes the second step of porphyrin formation; it is expressed in a number of tissues, most abundantly in liver and erythroid tissues. This unit describes a colorimetric assay for measuring ALAD activity in mammalian blood or tissues, a variation for measuring the extent of ALAD reactivation in samples in which the enzyme’s activity has been inhibited by lead poisoning, and an alternative HPLC-based assay.

UNIT 9.5 Histochemical Analysis of Heme Degradation Enzymes (James F. Ewing, ArQule, Inc., Waltham, Mass.). Histochemical analysis can be used to understand the cellular localization, expression, and regulation of enzymes responsible for catabolism of the heme molecule. This unit describes protocols for assessing the expression and regulation of specific HO-1 and HO-2 mRNAs by in situ hybridization using digoxigenin-tagged probes. Immunohistochemistry is used to visualize specific cellular sites of HO-1 and HO-2 protein expression.

UNIT 10.6 Histochemical Analysis of Nitric Oxide Synthase by NADPH Diaphorase Staining (Mirella Gonzalez-Zulueta, Valina L. Dawson, and Ted M. Dawson, Johns Hopkins University School of Medicine, Baltimore, Md.). In the early 1990s, it was observed that the presence of NOS coincides with that of NADPH diaphorase activity. For this reason, NADPH diaphorase staining is widely used to detect NOS-containing cells in neural and non-neural tissues. With the right fixation procedure, this activity is capable of detecting cells containing any of the NOS isoforms.

APPENDIX 1B Transgenic and Gene-Targeted Mouse Lines for Toxicological Studies (George Sanders, Carol Ware, and Warren Ladiges, University of Washington, Seattle). This unit contains an extensive tabular listing of transgenic and gene-targeted mice useful in toxicological studies. Each listing contains a brief description of the phenotype, relevant references, and, where applicable, commercial sources.

FORTHCOMING:

UNIT 3.5 Assays for DNA Damage (Jessica Sutherland and Max Costa, New York University School of Medicine, New York). This unit describes several assays for detecting several kinds of DNA damage--strand breaks, internal cross-linking, DNA/protein cross-links--and repair activity following exposure to genotoxic agents. The methods include single-cell electrophoresis (comet assay), filter eluting, K-SDS precipitation, and measurement of unscheduled DNA synthesis.

UNIT 8.7 Measurement of Ferrochelatase Activity (Shigeru Taketani, Kansai Medical University, Osaka, Japan). Ferrochelatase is the terminal enzyme in the heme biosynthesis pathway. Under anaerobic conditions it catalyzes the insertion of ferrous iron into the protoporphyrin IX ring to form protoheme. In the absence of iron and under aerobic conditions, the enzyme will use zinc or mercury as a substitute. The assay described in this unit uses zinc under aerobic conditions and the artificial substrate mesoporphyrin in the presence of homogenate to analyze the formation of zinc-mesoporphyrin by HPLC, with detection by absorbance at 400 nm or fluorescence intensity.

UNIT 8.8 Measurement of Erythrocyte Protoporphyrin Concentration by Double Extraction and Spectrophotometry (Patrick J. Parsons, New York State Department of Health and University of Albany, Albany, N.Y.). Quantification of the level of erythrocyte protoporphyrin can be used as means of screening for lead exposure. In this method, porphyrins and heme compounds are extracted from whole blood using ethyl acetate; porphyrins are then separated from heme by back-extraction with HCl solution, and measured by molecular fluorometric using a spectrofluorometer calibrated using protoporphyrin IX standard solutions.

UNIT 11.7 Neurobehavioral Testing in Humans (W. Kent Anger, Diane S. Rohlman, and Daniel Storzbach, Oregon Health Sciences University, Portland). The evaluation of the effects of chemical exposures on humans is a worldwide concern, and most chemicals have not been evaluated for neurotoxic effect. Human neurobehavioral research or clinical evaluations of populations exposed to chemicals must be carefully planned and structured. This unit describes the steps required to create such a study, select the appropriate measures, and evaluate the results.

UNIT 10.7 Immunocytochemical Analysis of Cyclic Nucleotides (J. deVente and H.M.W. Steinbusch, Maastricht University, Maastricht, The Netherlands). Visualization of second messenger molecules is a powerful approach for studying the role of second messengers in relation to the (sub)cellular localization and/or kinetics of the second messenger response. This unit describes the appropriate fixation procedures for cAMP and cGMP and the use of specific antibodies to localize these molecules.

UNIT 11.6 Risk Assessment and Neurotoxicology (William Slikker, Jr., National Center for Toxicological Research/Food and Drug Administration, Jefferson, Ark.). Risk assessment refers to the use of toxicological data to define the relationship between exposure to an agent and an adverse health outcome. This unit describes the factors involved in the study and evaluation of neurotoxicants to assess the risks associated with exposure.