High–Throughput Screening Methods in Toxicity Testing

Explores the benefits and limitations of the latesthigh–throughput screening methods

With its expert coverage of high–throughput in vitroscreening methods for toxicity testing, this book makes it possiblefor researchers to accelerate and streamline the evaluation andrisk assessment of chemicals and drugs for toxicity. Moreover, itenables them to comply with the latest standards set forth by theU.S. National Research Council′s "Toxicity Testing in the 21stCentury: A Vision and Strategy" and the E.U.′s REACH legislation.Readers will discover a variety of state–of–the–science,high–throughput screening methods presented by a group of leadingauthorities in toxicology and toxicity testing.

High–Throughput Screening Methods in Toxicity Testing isdivided into five parts:

General aspects, including predicting the toxicity potential ofchemicals and drugs via high–throughput bioactivity profiling Assessing different cytotoxicity endpoints Assessing DNA damage and carcinogenesis Assessing reproductive toxicity, cardiotoxicity, andhaematotoxicity Assessing drug metabolism and receptor–related toxicity

Each chapter describes method principles and includes detailedinformation about data generation, data analysis, and applicationsin risk assessment. The authors not only enumerate the advantagesof each high–throughput method over comparable conventionalmethods, but also point out the high–throughput method′slimitations and potential pitfalls. In addition, the authorsdescribe current research efforts to make high–throughput toxicityscreening even more cost effective and streamlined.

Throughout the book, readers will find plenty of figures andillustrations to help them understand and perform the latesthigh–throughput toxicity screening methods.

This book is ideal for toxicologists and other researchers whoneed to implement high–throughput screening methods for toxicitytesting in their laboratories as well as for researchers who needto evaluate the data generated by these methods.

PREFACE ix

CONTRIBUTORS xi

PART I GENERAL ASPECTS

1 ToxCast: Predicting Toxicity Potential Through High–ThroughputBioactivity Profiling 3
Keith A. Houck, Ann M. Richard, Richard S. Judson, Matthew T.Martin, David M. Reif, and Imran Shah

2 High–Throughput Toxicity Testing in Drug Development: Aims,Strategies, and Novel Trends 33
Willem G.E.J. Schoonen, Walter M.A. Westerink, Femke M. van deWater, and Horbach G. Jean

3 Incorporating Human Dosimetry and Exposure Information withHigh–Throughput Screening Data in Chemical Toxicity Assessment77
Barbara A. Wetmore and Russell S. Thomas

4 The Use of Human Embryonic Stem Cells in High–ThroughputToxicity Assays 97
Xin Huang, Dan–yan Zhu, and Yi–jia Lou

PART II HIGH–THROUGHPUT ASSAYS TO ASSESS DIFFERENTCYTOTOXICITY ENDPOINTS

5 High–Throughput Screening Assays for the Assessment ofCytotoxicity 109
Andrew L. Niles, Richard A. Moravec, Tracy J. Worzella, NathanJ. Evans, and Terry L. Riss

6 High–Throughput Flow Cytometry Analysis of Apoptosis 129
Francesca de Giorgi and Franc¸ois Ichas

7 High Content Imaging–Based Screening for Cellular ToxicityPathways 143
Bram Herpers and Bob van de Water

8 The Keratinosens Assay: A High–Throughput Screening Assay toAssess Chemical Skin Sensitization 159
Andreas Natsch

9 High–Throughput Screening Assays to Assess ChemicalPhototoxicity 177
Satomi Onoue, Yoshiki Seto, and Shizuo Yamada

PART III HIGH–THROUGHPUT ASSAYS TO ASSESS DNA DAMAGE ANDCARCINOGENESIS

10 Ames IITM and Ames Liquid Format Mutagenicity ScreeningAssays 193
Kamala Pant

11 High–Throughput Bacterial Mutagenicity Testing: VitotoxTMAssay 213
Luc Verschaeve

12 Genotoxicity and Carcinogenicity: Regulatory and Novel TestMethods 233
Walter M.A. Westerink, Joe C.R. Stevenson, G. Jean Horbach,Femke M. van de Water, Beppy van de Waart, and Willem G.E.J.Schoonen

13 High–Throughput Genotoxicity Testing: The Greenscreen Assay271
Jorg Blumel and Nadine Krause

14 High–Throughput Assays to Quantify the Formation of DNAStrand Breaks 285
Marýa Moreno–Villanueva and Alexander Burkle

15 High–Throughput Versions of the Comet Assay 295
Irene Witte and Andre Stang

16 Automated Soft Agar Colony Formation Assay for theHigh–Throughput Screening of Malignant Cell Transformation309
Pablo Steinberg

17 High–Throughput Quantification of Morphologically TransformedFoci in Bhas 42 Cells (v–Ha–ras Transfected BALB/c 3T3) UsingSpectrophotometry 317
Kiyoshi Sasaki, Ayako Sakai, and Noriho Tanaka

PART IV HIGH–THROUGHPUT ASSAYS TO ASSESS REPRODUCTIVETOXICITY, CARDIOTOXICITY,
AND HAEMATOTOXICITY

18 ReProGlo: A New Stem–Cell–Based High–Throughput Assay toPredict the Embryotoxic Potential of Chemicals 343
Frederik Uibel and Michael Schwarz

19 Embryonic Stem Cell Test (EST): Molecular Endpoints TowardHigh–Throughput Analysis of Chemical Embryotoxic Potential357
Peter T. Theunissen, Esther de Jong, Joshua F. Robinson, andAldert H. Piersma

20 Zebrafish Development: High–Throughput Test Systems to AssessDevelopmental Toxicity 371
Stephanie Padilla

21 Single Cell Imaging Cytometry–Based High–Throughput Analysisof Drug–Induced Cardiotoxicity 385
Min Jung Kim and Joon Myong Song

22 High–Throughput Screening Assays to Evaluate the CardiotoxicPotential of Drugs 403
Carl–Fredrik Mandenius and Thomas Meyer

23 High–Throughput Screening Assays to Evaluate the HematotoxicPotential of Drugs 421
Caroline Haglund, Rolf Larsson, and Martin Hoglund

PART V HIGH–THROUGHPUT ASSAYS TO ASSESS DRUG METABOLISM ANDRECEPTOR–RELATED TOXICITY

24 High–Throughput Enzyme Biocolloid Systems for Drug Metabolismand Genotoxicity Profiling Using LC MS/MS 433
James F. Rusling and John Schenkman

25 Higher–Throughput Screening Methods to Identify CytochromeP450 Inhibitors and Inducers: Current Applications and Practice453
David M. Stresser and George Zhang

26 High–Throughput Yeast–Based Assays to Study Receptor–MediatedToxicity 479
Johanna Rajasarkka and Marko Virta

27 Evaluating the Peroxisomal Phenotype in High Content ToxicityProfiling 501
Jonathan Z. Sexton and Kevin P. Williams

28 A Panel of Quantitative Calux R Reporter Gene Assays forReliable High–Throughput Toxicity Screening of Chemicals andComplex Mixtures 519
Bart van der Burg, Sander van der Linden, Hai–yen Man, RoosWinter, Lydia Jonker, Barbara van Vugt–Lussenburg, andAbraham Brouwer

29 DR–Calux R : A High–Throughput Screening Assay for theDetection of Dioxin and Dioxin–Like Compounds in Food and Feed533
Barbara van Vugt–Lussenburg, Harrie T. Besselink, Bart van derBurg, and Abraham Brouwer

INDEX 547

PABLO STEINBERG, PhD, is Full Professor for Food Toxicology and Replacement/Complementary Methods to Animal Testing as well as Director of the Institute for Food Toxicology and Analytical Chemistry at the University of Veterinary Medicine Hannover. Prior to that, Dr. Steinberg was director of the Institute of Nutritional Science at the University of Potsdam. He has also held research positions at the University of Mainz.