Table Of ContentCELL CULTURE METHODS FOR IN VITRO TOXICOLOGY
CELL CULTURE METHODS
FOR IN VITRO TOXICOLOGY
Edited by
GLYNN. STACEY
National Institute of Biological Standards and Control,
Potters Bar, u.K.
ALAN DOYLE
The Wellcome Trust, London, u.K.
and
MARGHERIT A FERRO
University of Genova, Italy
SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-90-481-5936-9 ISBN 978-94-017-0996-5 (eBook)
DOI 10.1007/978-94-017-0996-5
Printed on acid-free paper
Reprinted from eell Biology and Toxicology,
VoI. 17, Issues 4 & 5, 2001.
AII Rights Reserved
© 200 1 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2001
No part of the material protected by this copyright notice may be reproduced or
utilized in any form or by any means, electronic or mechanical,
incIuding photocopying, recording, or by any information storage and
retrieval system, without written permission from the copyright owner.
Contents
List of Contributors ................................................................ vii
Preface by the Editor ................................................................. ix
Standardisation for In Vitro Toxicity Test ................................. 1
Margherita Ferro and Alan Doyle
2 Application of Cell Cultures to Toxicology .............................. 9
Ian Freshney
3 Immortalisation of Primary Cells ........................................ .27
Glyn Stacey and Caroline MacDonald
4 Safety Considerations for In Vitro Toxicology Testing .............. .43
Bruce Jones and Glyn Stacey
5 The Drug Metabolizing Enzymatic System and the
Experimental Tools Used for In Vitro Toxicology for
Metabolic Studies ..........................................................6 7
Emanuela Testai
6 Cell Culture models for Hepatotoxicology ......... '" ................. 83
Thierry Battle and Glyn Stacey
7 Intestinal Cell Culture Models: Applications of
Toxicology and Pharmacology .............. , ............................ 97
Yula Sambruy. S. Ferruzza, G. Ranaldi and I De Angelis
8 Cell Culture Models for Neurotoxicology ............................ 115
Glyn Stacey and Barabara Viviani
9 Recombinant Cell Lines for Stress Reporter Assays ................ 131
Michel Fischbach and Peter Bromley
Index ................................................................................ 149
v
List of Contributors
Thierry Battle
Computer Cell Center sa. rlle de la Marlette I.J. BI780 Sneffe. Belgium.
Peter Bromley
Gene Contorl SA .. PO Box 227. 1211 Geneve 2.J. Swit=erland
Isabella De Angelis
Istituto Na=ionale della Nutri=ione. Istituto Superiore di Sanita. Rome. Italy
Alan Doyle
The Wellcome Trust. London. UK
Margherita Ferro
University a/Genova. Italy
S. Ferruzza
Istitllto Na=ionale della Nutri=ione. Istituto Superiore di Sanita. Rome. Italy
R. Ian Freshney
CRC Department a/Medical Oncology. University a/Glasgow. Scotland. UK
Michel Fischbach
Gene Contorl SA .. PO Box 227. 1211 Geneve 2-1. Swit=erland
Bruce Jones
National Institllte 0/ Biological Standards and Control. Potters Bar. UK.
vii
Vlll
Caroline MacDonald
University of Paisley, Glasgow, Scotland, UK
G. Ranaldi
Istituto Na::ionale della Nutri::ione, r'ia Ardeatina 5'+6. 00178 Roma, Istituto Superiore di
San ita, I'ia Ie Regina Elena, 00161 Roma
Yula Sambruy
Istituto Na::ionale della Nutri::ione, /'ia Ardeatina 5'+6, 00178 Roma, Istituto Superiore di
Sanita, I'ia Ie Regina Elena, 00161 Roma
Glyn Stacey
Nationallnslitllte of Biological Standards and Control, Potters Bar, UK.
Emanuela Testai
Istituto Superiore di Sanita -Comparative Toxicology and Ecotoxicology Dept., Biochemical
Toxicology Unit, r'iale Regina Elena, 299-00165 Rome. Italy
Barbara Viviani
Institute of Toxicology. University of,Ia,4i1an. Milan, Italy
Preface
The search for methods of testing products and investigating new
compounds that avoid the use of animals has been a goal of the scientific
community for many years. The application of cell culture techniques in the
field of toxicology is now moving forward at a remarkable pace such that
alternatives to the use of animals are now emerging as a reality. This will
have a great effect on the ability of the science to make real steps towards
the complete replacement of the use animals. New approaches using our ever
expanding knowledge of cell biology and the utilisation of genetic
manipulation will help to provide in vitro models that may be much more
closely correlated with in vivo tissue responses. The development of
validation procedures for the qualification of new methods have made the
new technologies more robust and no longer limited to research laboratories.
The parallel establishment of qualified cell banks for use in specific in vitro
assays will also make a significant contribution to the reproducibility and
standardisation of these assays. This book is aimed to provide an
introduction to cell culture methods that will enable the new comer to cell
culture techniques to gain not only a general knowledge of relevant methods
from a toxicology perspective, but also to appreciate some of the challenges
that are familiar to cell culturists with hard earned experience. In particular it
is important to convey the problems that may accompany cell contamination
with microorganisms or other cell cultures. These are all too often not
evident to the novice. Two chapters deal with molecular approaches for
genetic manipulation of cells to provide new immortalised cells and
development of genetically modified cells that can provide enhanced
detection of cytotoxic responses. A whole chapter is also devoted to the
measurement of cytotoxicty and viability to give the student a grasp of the
fundamental principles and a perspective on how different assays may
perform. As a companion for students and anyone coming to cell culture for
the first time in the field of toxicology this text will provide useful advice as
well as information on the techniques themselves.
G N Stacey. PhD
ix
Chapter 1
Standardisation for In Vitro Toxicity Tests
Margherita Ferrol and Alan Doyle2
I University oiGenova, Italy
2 The WeI/come Trust, I R3 Ellston Road, London, UK
1. THE CLASSICAL "IN VIVO" TOXICITY TESTS USED IN
THE SAFETY EVALUATION OF CHEMICALS
The aim of toxicology is to study all possible harmful effects of a toxic
substance that can represent a risk for humans, In order to establish the safest
level of exposure for a certain chemical, a general and very broad screening
process is required,
Traditional toxicity tests utilize whole animal models, assuming that
small laboratory animals will react to the toxicants in a very similar way to
human subjects, There are two main arguments against this assumption: one
is that many metabolic differences do exist between animals and man, that
result in substantial changes in toxic effects, the other is that animal models
are highly standardized, whereas humans are characterized by strong inter
individual differences, Indeed it is impossible to mimic such differences by
means of animal models, unless transgenic species are produced for specific
purposes, Other limitations could be mentioned, such as the fact that they
were originally established as very empirical approaches, not subjected to
the validation procedures that now are requested for in vitro methods,
The safety evaluation of chemicals is based on the following in vivo
toxicity tests: acute, subchronic and chronic toxicity, developmental toxicity,
reproductive toxicity, ocular and skin irritancy, allergic hypersensitivity,
phototoxicity, metabolism and toxicokinetics.
G,N, Stacey et ai, (eds,), Cell Culture Methods for In Vitro Toxicology, 1-8,
© 2001 Kluwer Academic Publishers,
2 Margherita Ferro and Alan Doyle
For acute toxicity evaluation, that is the effect of a single high dose, the
LD50, proposed in 1927 by Trevan, still remains the unique standard test by
which an extremely high number of chemicals are screened today.
Subchronic and chronic toxicities are evaluated by administration of
repeated daily doses for 1-3 months and 1-2 years, respectively. After these
in vivo treatments, anatomo-pathological and biochemical parameters are
assessed, with the appearance of tumours being the most important aspect.
Developmental toxicity is based on treatment of pregnant animals
according to protocols that are specifically designed to identify all
teratogenic effects.
Reproductive toxicity evaluates reproductive activity over 2
generations.
Ocular/skin irritation and hypersensitivity have their own specific
protocols.
Metabolism and toxicokinetics are based on evaluations such as the rate
of administration, kinetics of absorption, distribution and excretion.
The experimental procedures for evaluating in vivo toxicity are codified
by regulations and directives including international recommendations for
good laboratory practice. An example of the empirical and primitive nature
of some protocols is the in vivo assessment of eye-skin irritancy. The
classic method for assessing the irritant potential of chemicals is the Draize
test proposed in 1944, in which the type and the degree of the irritation to the
rabbit eye or skin are determined visually according to a scoring procedure
(Draize et aI, 1944). Criticism arose later not least from animal welfare
groups, mainly due to the extremely subjective nature of scoring (Swanston,
1983), thus several refinements were introduced (Frazier et ai, 1987), but a
reliable alternative to this test is now more than ever a necessity.
2. IN VITRO TESTS: A MORE SCIENTIFIC AND ETHICAL
APPROACH?
The availability of new technologies, such as the in vitro culture of a
wide range of mammalian cells, including human ones, produced in the last
decade the willingness of exploring new alternative approaches to in vivo
testing, also under the pressure of animal welfare movements.
Therefore, a vast range of studies in toxicology are presently carried out
with non-animal models, mainly cultured cells, and the major parameters of
toxicity which can be evaluated are: I) cytotoxicity, 2) genotoxicity, 3)
xenobiotic biotransformation, 4) apoptotic and necrotic cell death, 5)
enzymatic induction, 6) immunotoxicity, 7) neurotoxicity, 8) intestinal
toxicity, 9) free-radical and oxidative stress-induced effects.
Standardisation for In Vitro Toxicity Tests 3
In some cases in vitro methods are in the process of pre-validation or
validation phases, as replacements of in vivo classical methods. One impor
tant example is the battery of in vitro eye/skin irritation tests that are
proposed as alternatives to the classical Draize test (Reinhardt, 1987; Wilcox
and Bruner, 1990; Balls et ai, 1995). The state-of-the-art on validation of
these and other tests have been review elsewhere (Balls and Fentem, 1997;
Frazier 1992).
The first in vitro test to be used in toxicology is the Salmonella test for
mutagenicity, proposed to the scientific community by Dr. Ames in 1971
(Ames, 1971). This test was the starting point for all genetic toxicology
testing that developed various short-term in vitro tests based on DNA
damage as an index of carcinogenicity (Bridges, 1988). Safety evaluation of
drugs, pesticides, food additives, cosmetics or other chemicals to which man
can be exposed, is undertaken with the aid of complementary genotoxicity in
vitro tests. Even if with time the predictivity of these tests has declined,
especially with adherence to routine protocols, they are still used by toxico
logists, since they remain valid scientifically and aid our understanding of
the carcinogenic process. In particular, for metabolically activated carcino
gens, the models employing human liver cells are considered a very good
system for in vitro evaluation of cancer risk.
Cytotoxicity tests focus their end-points mainly on cell death or some
measure of growth impairment, and are designed to evaluate the intrinsic
ability of a chemical to kill cells. Also when testing with animals, acute
toxicity may be assessed by evaluating extremely broad end-points, the most
common being the death of the animal. Advantages and disadvantages of
the different cytotoxicity tests are discussed in Chapter 2.
All the attempts at developing, improving, and validating both cytotox
icity and genotoxicity tests have raised many questions about their relevance
to in vivo testing, about their inter- and intra-laboratory reproducibility, and
about regulatory acceptance for human safety evaluation. However this ap
proach is also a powerful tool for mechanistic investigations. In fact, a
special area of interest for in vitro toxicologists are studies on xenobiotic
biotransformation.
Drug metabolism plays a central role in determining toxicity (see Chapter
5), and not only primary cultured hepatocytes but also many established cell
lines retain the expression of various enzyme activities involved in xeno
biotic biotransformation (Horndart and Wiebel, 1996).
In conclusion, the toxicologists of today have a variety of in vitro
systems that can be used to solve many toxicological problems, and this is
important from a scientific point of view. However, there is an urgent need
from industry for rapid, economic, more ethical and validated in vitro tests
that can replace the classical in vivo methods. This is the most frustrating
