TiPS - March 1990 [Vol. 11]
104
Alternatives to animal o×perimentation: developing in-vitro methods and changing legislation Gerhard Zbinden Despite recent changes in legislation in several countries and general reluction in the use of animals in biomedical research, the impatience of antiviwsectionists to see reductions in animal experimentation shows no signs of abating. Gerhard Z b i n d e n ana!yses the reasons for this continuing dissatisfaction, arguing that real progress has been made in biomedical research, but that the complexities of developing internationally recognized regulations constitute a barrier to rapid change in product safety testing methods. A few months ago, the respected n e w s p a p e r the Neue Z~ircher Zeitung p u b l i s h e d an editorial entitled "Wieviel Ungeduld braucht der Tierschutz?" (How much impatience do animal protectionists need?). It was a sober assessment of the recurrent requests for radical curtailment of animal experimentation. The writer, a political commentator, expressed surprise that the agitation of antivivisectionists against biomedical research with live animals is escalating at a time w h e n m a n y of the industrialized countries have passed far-reaching animal protection legislation, w h e n scientific societies and institutions have issued ethical guidelines for the care and protection of laboratory animals, and w h e n the n u m b e r of animals used in biomedical research is decreasing substantially and steadily. As a scientist interested in ethical and practical aspects of biomedical research, I often meet people o p p o s i n g animal experimentation, and I too sense their feelings of impatience very strongly. Here, I am not talking about the fruitless arguments with the violent fringe of the antivivisectionist movement. I am thinking of the people truly concerned about the fate of animals in G. Zbinden is Professor at the Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland.
research laboratories, the m e n a n d w o m e n w h o donate large sums of their own m o n e y as r e w a r d s for scientists w h o develop n e w invitro testing methods (in Europe, I k n o w of at least nine such prizes), a n d the i n d i v i d u a l s w h o are t o m b e t w e e n their rational acceptance of the crucial necessity of biomedical research, and their sense of fairness towards the millions of animals that are p r o d u c e d , used a n d discarded in the process. For m a n y of them, the d e v e l o p m e n t of in-vitro test systems seems like the ideal solution of the ethical conflict b e t w e e n the scientists' m i s s i o n to fight diseases a n d to explore the secrets of life, a n d the suffering of animals n e e d e d to achieve these i m p o r t a n t goals. The question, "Where are these
alternatives you scientists have been talking about for so long?" is asked w i t h increasing urgency. In-vitro methods in biomedical
research I too am surprised that the issue of alternative methods is b r o u g h t up now, w h e n it is e v i d e n t that the application of in-vitro m e t h o d s is increasing rapidly. I become aware of this d e v e l o p m e n t every time I sign the weekly bills of our institute and see that the expenses for tissue culture media, growth factors, antibodies, plastic culture dishes and other necessities for invitro research now exceed the cost of procurement and m a i n t e n a n c e of laboratory animals. That the socalled alternatives also p l a y an
~) 1990,Elsevier Science Publishers Ltd. (UK) 0165-6147/90/$02.00
important role in m a n y other laboratories is e v i d e n t from o u r review of the 6649 abstracts of the research c o m m u n i c a t i o n s presented at the 1989 FASEB m e e t i n g in N e w Orleans, USA (Fig. 1). In-vitro m e t h o d s - i.e. experiments w i t h tissues and b o d y fluids of untreated h u m a n s a n d animals, microorganisms, p r i m a r y ceil cultures a n d established cell lines - were u s e d in almost 50% of the research reported. Studies on vertebrate animals, most of them small rodents, accounted for r o u g h l y 40%. The r e m a i n i n g studies were performed in humans (about 8%) or were of a theoretical nature. There are interesting differences b e t w e e n the disciplines that are r e p r e s e n t e d b y FASEB: in-vitro w o r k w i t h b o d y fluids, tissues and cell cultures accounts for about 70% of the studies reported b y immunologists, whereas these techniques are u s e d in only a b o u t 20% of the research of nutritionists. The preferred research subjects of the latter are h u m a n s (30%) and, to a m u c h smaller extent, farm animals. Physiologists and p h a r macologists have traditionally w o r k e d w i t h h u m a n a n d animal tissues (Fig. 2), and the survey shows that this is still true. But they are n o w e x p a n d i n g into cell culture techniques, catching u p with the pathologists w h o rely on these m o d e l s quite extensively. Scientists w h o choose in-vitro systems do not consider them, in most instances, as 'alternatives'. The selection is b a s e d on their conviction that, for the p r o b l e m at hand, an in-vitro system is the most suitable option, and they d o not regard it as an issue to b o a s t about to outsiders. This explains w h y the extensive use of in-vitro techniques in m o d e m b i o m e d i cal research, d o c u m e n t e d b y the survey of the FASEB meeting, is not k n o w n a m o n g most of o u r critics w h o so i m p a t i e n t l y ask for the d e v e l o p m e n t of alternatives.
Regulatory agencies and toxicity testing In all controversies it is convenient to look for a scapegoat. For those w h o feel that alternatives are not as w i d e l y u s e d as they should be, the villain was found long ago. In their o p i n i o n , the b l a m e can clearly be placed with the national regulatory agencies which, in their view,
105
TiPS - March 1990 [Vol. 11]
phylogenetically highest species or material used
physiology pharmacology n
non-human prim3;e
60 - -
2070
I
ee8
pathology
nutrihon
i,:lmunology
combined
lOSS
tl;3
1497
6649
I
[]
R farm animals
220 I
dogs, cats
384 B
7_9% 1.0%
=
p
/!
I
'm
m
small laboratory animals 1939 ~
5.8%
m
reptiles, amphibians
18
0.3%
birds, eggs
7S
1.1%
fish
18
0.3%
non-vertebrates
8
0.1%
insects
4
0.1%
microorganisms tissue
mL-
42 1534
body fluid
719~._
|
primary cell culture
337
I
cell lines
sel ~
I
0.6%
m
m /
[]
23.1%
5.1% 0
tC.8~ 8.4%
I
no living material
2.6%
unclear
0 3% I
I
I
10 20 30
/
I
I
10 20 3O
i
i
i
10 :,'0 30
i
i
i
i
10 20 30
i
i
10 20 30
I
i
20 3O
Percentage of abstracts Fig. 1. Laboratory animal species or in-vitro test system used in 6649 research communications presented at the 1989 Annual Meeting of FASEB. An unequivocal distinction between the use of tissues, body fluids (e.g. peripheral blood lymphocytes) and primary cell cultures was often not possible from the inform~.tion provided. Thus, a certain degree of subjectivity was unavoidable in assigning these communications to in-vitro test categories. Whenever donor animals or human donors of tissues or body fluids were manipulated (e.g. by administering special diets, exposure to uncommon environmsntal conditions or immunization), the abstract was scored as reporting an in-vivo experimenL When preparation of a monoclonal antibody was an essential Dart of the research, the communication was scored as using 'small laboratory anima/s, even though the experimental procedures were not described. In-vitro tests using already existing or commercially available monoclonal antibodies were assigned to one of the in-vitro test categories. When more than one laboratory animal species was investigated, the abstract was assigned to the phy/ogenetical/y highest species, n, number of abstracts reviewed.
stuLbornly refuse to accept invitro test results in lieu of experiments performed with live animals. The best illustration of this development is legislation (HR 1635) introduced in the US House of Representatives in 1988 by congresswomen Barbara Boxer et al., demanding that US federal regulatory agencies be prohibited from accepting LD50 tests performed with live animals, and be compelled to propagate non-animal alternatives for this and other test procedures. There exists a widespread misconception, even among scientists, about the attitude of regulators towards in-vitro testing, and about the extent to which these individuals can control the choice of research methods. Regulatory agencies rely, to a large extent, upon the advice of scientific experts, and they have whole-
heartedly endorsed their suggestions to accept in-vitro tests for assessment of the mutagenic (not carcinogenic) hazards of chemicals, and for pyrogenicity testing of parenterally administered drugs and infusion fluids. Given properly validated scientific data, endorsed by a majority of the experts, they will undoubtedly be ready to change their requirements in other areas as well. An example is the imminent replacement of the mouse bioassay for insulin batch control by a purely chemical method of analysis 1. However, they cannot be expected to base their decision-making criteria on research conducted by individual research groups using limited numbers of test chemicals. It is also widely ignored that only a fraction of the research necessary for discovery and development of new drugs and other
chemicals is under governmental regulatory control. It is confined, essentially, to the studies for safety and a limited number of tests for quality assurance of certain biologicals. The percentage of laboratory animals used for these regulatory purposes is in the range 10-15% in industrial organizations and is close to zero in academic institutions. Nevertheless, the use of live animals for toxicological investigations has come under heavy criticism by animal rights advocates. For reasons not entirely clear, it is mainly directed against three procedures, the so-called LDs0 test and the assays for skin and eye irritancy. In these areas, animal tests serve two different purposes. First, they contribute valuable, albeit incomplete, information about the human hazard due to
TiPS - March 1990 [Vol. 11]
106
I
acute poisoning, injury caused by accidental contact of chemicals with skin and mucosal membranes and human risks related to intentional, topical use of drugs and cosmetics. For these objectives, alternative tecnniques are already widely used; these include simplified acute toxicity tests with small numbers of animals, and in-vitro models using cell cultures, chorion allantois membrane of chicken eggs, and isolated skin and eye preparations. In the hands of skilled investigators and in conjunction with clinical experience, these methods can contribute valuable information on human hazard. However, acute toxicity tests and in-vivo irritation studies are also used in a more formalized way to provide the basis for labeling of hazardous chemicals, a procedure deemed to be essential for consumer protection. In order to treat all manufacturers equally and to assure uniformity of labeling and consumer safety criteria worldwide, national regulatory agencies, under the guidance of the Organization of Economic Cooperation and Development (OECD), have adopted formalized procedures for acute oral and dermal toxicity (often referred to as LDso tests), and for skin and eye irritation testing (often referred to as Draize tests). It is clear that a replacement of these rather rigid procedures by a more flexible and scientific approach needing fewer animals, but sophisticated in-vivo monitoring and in-vitro cell and tissue culture methodology, is difficult to put into operation on a global
Fig. 2. Physiologists and pharmacologists have been experimenting on animal tissue for many years. This early example of an organ bath, dating from 1930, was devised to demonstrate humoral transference in the gut. Ringer sol-~on .~.'ck!ed slowly over a preparation of innerva~=d gut onto a test piece of gut. When the nerve-muscle preparation was stimulated an "inhibitory substance' was released into the Ringer, and caused relaxation of the test piece of gut. Keeping the air in the chamber =/arm and moist posed a major problem. This was eventually solved by heating a layer of water at the bottom of the chamber to 50-60~C.
level. Thus, despite declarations of honorable intentions by all concerned, progress in this area is and will be slow. A ray of hope is provided by the initiative of the British Toxicology Society which is validating a simplified and more humane procedure for classification of poisonous chemicals2. The society has also proposed a hierarchical scheme for dermal and mucosal irritancy testing that relies on in-vitro models for the detection of severely irritant and corrosive properties, and uses a minimal number of animals only for confirmatory investigations of chemicals and products of low degrees of irritancy3. In the other areas of industrial toxicology- testing for subchronic and chronic toxicity, reproductive toxicity and carcinogenicity - the development of in-vitro testing procedures has made considerable progress, particularly in two fields. The first is organ-directed toxicity, and encompasses organotypic cell and tissue cultures from liver, kidney, lung, testis, nervous systems and others. Apart from cytotoxicity, these systems can be used to study many specific functions, such as differentiation, contraction, synthesis and secretion of proteins and mediators, intraand intercellular signal transduction, proliferation, and malignant transformation. Their major application is in toxicological screening, i.e. the selection process of the most favorable candidate substances for further development. With these techniques toxic agents can often be recognized before formal in-vivo toxicity
studies are performed, resulting in considerable savings of laboratory animals. However, a reliable estimate of the number of animals saved by these procedures is not possible. The second area in which enormous progress has been made in recent years is the use of tissue and cell cultures for the investigation of drug metabolis:n. Here, the availability of material of human origin, particularly hepatocytes, has revolutionized the process of extrapolation of animal data to humans. As with toxicological screening, the number of laboratory animals spared by the application of these techniques is difficult to quantify. However, early detection and elimination of chemicals that are transformed into toxic metabolites by human liver enzymes will certainly lead to a considerable reduction of redundant in-vivo toxicological investigations. However, we should not forget that toxicity studies are not only performed to detect relevant adverse characteristics of the test compounds, but are also expected to demonstrate "safety', i.e. the absence of toxicity. On the basis of these findings, safe levels of human exposure are determined. Since it is impossible to prove absence of toxicity, toxicologists try to maximize the odds of detecting even the most trifling adverse effect by conducting their experiments with large numbers of animals and multiple dose levels, including one that causes overt toxicity. They also do this for their own protection, since product litigation charges have become a serious problem. New and as yet not generally accepted alternative safety tests might be as reliable as the standard in-vivo procedures from the scientific point of view, but they will not carry as much weight as the overpowering in-vivo models in the defense against unjustified claims. Thus, regulatory agencies which are blamed first, and manufacturers which are often forced to pay exorbitant sums for indemnification by compassionate but scientifically uninformed judges, are reluctant to trade time-honored safety testing procedures against the more scientific new alternatives. [] [] []
TiPS- March 1990 [Vol. 11] Biomedical scientists are using in-vitro alternative research techniques extensively and with considerable success, although their achievement in making this exciting development known to outsiders is not spectacular. In toxicology, alternative testing methods have an important place in screening for cytotoxicity and for defined specific functional and biochemical disturbances in organotypic tissue and cell culture preparations. A relatively recent development is the use of in-vitro techniques for the study of pathways and rate of metabolism of
107 foreign chemical compounds. Thanks to the development of techniques for the successful culture of human cells, extrapolation of these findings to humans now has a solid scientific basis. For the determination of product safety in-vitro methods are also being investigated, particularly in the areas of skin and eye irritation, teratogenicity and carcinogenicity. However, their widespread use and their acceptance by regulatory agencies in lieu of whole animal tests is a slow pr~,cess, since global consent on ne~ concepts for consumer
Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration Yuechueng Liu and Daniel R. Storm Neuromodulin is a neurospecific calmodulin binding protein that is implicated in neurite extension, axonal elongation and iong-term potentiation. Yuechueng Liu and Daniel Storm propose that neuromodulin binds and concentrates calmodulin on growth cone membranes and that stimulation of protein kinase C releases high local concentrations of calmodulin irJ the growth cone. Interactions between released calmodulin and cytoskeleton proteins may affect the polymerization, crosslinking and membrane attachment of cytoskeleton polymers. This local "softening" of the membrane may be an initial event in filopodia formation and extension. The Ca2+ binding protein calmodulin and its target proteins are potential drug target sites in the CNS. For example, the phenothiazines, a class of antipsychotic drug, bind to calmodulin and antagonize its interactions with calmodulin-regulated enzymes. However, the phenothiazines have a number o~ other target sites in animal cells and it is not clear to what extent the pharmacological activities of these drugs are due to antagonism of the interactions of calmodulin with its target proteins ~. For example, the K,ts for calmodulin-phenothiazine interY. Liu is a Postdoctora| Fellow and D. R. Storm is Professor at the Dep~.rtment of Pharmacology, 5]-30, University of Washington, Seattle, WA 98195, USA.
actions fall in the range 1-150 btM, which is significantly higher than the dose used in antipsychotic treatment 1. Calmodulin mediates Ca 2+ regulation of a number of enzymes and proteins that are thought to play significant neuromodulatory roles. For example, mammalian brain contains calmodulin-stimulated adenylyl cyclases, protein kinases, cyclic nucleotide phosphodiesterases and phosphoprotein phosphatases which are important components of signal transduction systems. Calmodulin-sensitive adenylyl cyclases and calmodulindependent protein kinase II have both been implicated as regulatory elements contributing to neuroplasticity. In this article, we describe the properties of neuro-
protection and product labeling is difficult to achieve. Moreover, the rapidly escalating problem of product liability claims in our litigious society is a serious impediment for the acceptance of alternative safety testing procedures.
References 1 Fisher, B. V. and Smith, D. ~. (1986) Pharmacol. Biomed. Anal. 4, 377-387 2 van den Heuvel, M. J., Daya-., A. D. and Shillaker, R. O. (1987) Hum. ToxicoL 6, 279-291 3 Fielder, R. J., Gaunt, I. F., Rhodes, C., Sullivan, F. M. and Swanston, D. W. (1987) Hum. Toxicol. 6, 269-278
modulin, a neuron-specific calmodulin binding protein that has been implicated in regulation of neuron growth and neuroplasticity. It is hypothesized that the levels of free calrnodulin present in neurons may be regulated by reversible phosphorylation of neuromodulin. Physiological functions of neuromodulin The neurobiology of axonal growth-associated proteins, including neuromodulin, has recently been reviewed 2. A variety of divergent interests have focused on neuromodulin, as a number of experimental approaches have led investigators with differing research interests to the same molecule. As a result of these efforts, several names have been assigned to the molecule, each associated with a particular branch of neurobiology (see Box). One clue to the function of neuromodulin has been found in its tissue and subcetlular distribution. The availability of substantial amounts of purified neuromodulin 3 made possible the isolation of a rabbit polyclonal antibody specific for the protein and the development of a detergent-based radioimmunoassay to quantify accurately the levels of neuromodulin in various regions of brain 4. Western blot analysis revealed the presence of neuromodulin in membrane preparations from b o v i n e cerebellum, pineal gland, internal capsule, thalamm, ~hypothalamus, striatum and hippocampus. However, the protein is absent from all bovine non-neuronal tissues examined
~ 1990,ElsevierSciencePublishersLtd. {UK) 0165- 61471901S02.00
104
Alternatives to animal o×perimentation: developing in-vitro methods and changing legislation Gerhard Zbinden Despite recent changes in legislation in several countries and general reluction in the use of animals in biomedical research, the impatience of antiviwsectionists to see reductions in animal experimentation shows no signs of abating. Gerhard Z b i n d e n ana!yses the reasons for this continuing dissatisfaction, arguing that real progress has been made in biomedical research, but that the complexities of developing internationally recognized regulations constitute a barrier to rapid change in product safety testing methods. A few months ago, the respected n e w s p a p e r the Neue Z~ircher Zeitung p u b l i s h e d an editorial entitled "Wieviel Ungeduld braucht der Tierschutz?" (How much impatience do animal protectionists need?). It was a sober assessment of the recurrent requests for radical curtailment of animal experimentation. The writer, a political commentator, expressed surprise that the agitation of antivivisectionists against biomedical research with live animals is escalating at a time w h e n m a n y of the industrialized countries have passed far-reaching animal protection legislation, w h e n scientific societies and institutions have issued ethical guidelines for the care and protection of laboratory animals, and w h e n the n u m b e r of animals used in biomedical research is decreasing substantially and steadily. As a scientist interested in ethical and practical aspects of biomedical research, I often meet people o p p o s i n g animal experimentation, and I too sense their feelings of impatience very strongly. Here, I am not talking about the fruitless arguments with the violent fringe of the antivivisectionist movement. I am thinking of the people truly concerned about the fate of animals in G. Zbinden is Professor at the Institute of Toxicology, Swiss Federal Institute of Technology and University of Zurich, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland.
research laboratories, the m e n a n d w o m e n w h o donate large sums of their own m o n e y as r e w a r d s for scientists w h o develop n e w invitro testing methods (in Europe, I k n o w of at least nine such prizes), a n d the i n d i v i d u a l s w h o are t o m b e t w e e n their rational acceptance of the crucial necessity of biomedical research, and their sense of fairness towards the millions of animals that are p r o d u c e d , used a n d discarded in the process. For m a n y of them, the d e v e l o p m e n t of in-vitro test systems seems like the ideal solution of the ethical conflict b e t w e e n the scientists' m i s s i o n to fight diseases a n d to explore the secrets of life, a n d the suffering of animals n e e d e d to achieve these i m p o r t a n t goals. The question, "Where are these
alternatives you scientists have been talking about for so long?" is asked w i t h increasing urgency. In-vitro methods in biomedical
research I too am surprised that the issue of alternative methods is b r o u g h t up now, w h e n it is e v i d e n t that the application of in-vitro m e t h o d s is increasing rapidly. I become aware of this d e v e l o p m e n t every time I sign the weekly bills of our institute and see that the expenses for tissue culture media, growth factors, antibodies, plastic culture dishes and other necessities for invitro research now exceed the cost of procurement and m a i n t e n a n c e of laboratory animals. That the socalled alternatives also p l a y an
~) 1990,Elsevier Science Publishers Ltd. (UK) 0165-6147/90/$02.00
important role in m a n y other laboratories is e v i d e n t from o u r review of the 6649 abstracts of the research c o m m u n i c a t i o n s presented at the 1989 FASEB m e e t i n g in N e w Orleans, USA (Fig. 1). In-vitro m e t h o d s - i.e. experiments w i t h tissues and b o d y fluids of untreated h u m a n s a n d animals, microorganisms, p r i m a r y ceil cultures a n d established cell lines - were u s e d in almost 50% of the research reported. Studies on vertebrate animals, most of them small rodents, accounted for r o u g h l y 40%. The r e m a i n i n g studies were performed in humans (about 8%) or were of a theoretical nature. There are interesting differences b e t w e e n the disciplines that are r e p r e s e n t e d b y FASEB: in-vitro w o r k w i t h b o d y fluids, tissues and cell cultures accounts for about 70% of the studies reported b y immunologists, whereas these techniques are u s e d in only a b o u t 20% of the research of nutritionists. The preferred research subjects of the latter are h u m a n s (30%) and, to a m u c h smaller extent, farm animals. Physiologists and p h a r macologists have traditionally w o r k e d w i t h h u m a n a n d animal tissues (Fig. 2), and the survey shows that this is still true. But they are n o w e x p a n d i n g into cell culture techniques, catching u p with the pathologists w h o rely on these m o d e l s quite extensively. Scientists w h o choose in-vitro systems do not consider them, in most instances, as 'alternatives'. The selection is b a s e d on their conviction that, for the p r o b l e m at hand, an in-vitro system is the most suitable option, and they d o not regard it as an issue to b o a s t about to outsiders. This explains w h y the extensive use of in-vitro techniques in m o d e m b i o m e d i cal research, d o c u m e n t e d b y the survey of the FASEB meeting, is not k n o w n a m o n g most of o u r critics w h o so i m p a t i e n t l y ask for the d e v e l o p m e n t of alternatives.
Regulatory agencies and toxicity testing In all controversies it is convenient to look for a scapegoat. For those w h o feel that alternatives are not as w i d e l y u s e d as they should be, the villain was found long ago. In their o p i n i o n , the b l a m e can clearly be placed with the national regulatory agencies which, in their view,
105
TiPS - March 1990 [Vol. 11]
phylogenetically highest species or material used
physiology pharmacology n
non-human prim3;e
60 - -
2070
I
ee8
pathology
nutrihon
i,:lmunology
combined
lOSS
tl;3
1497
6649
I
[]
R farm animals
220 I
dogs, cats
384 B
7_9% 1.0%
=
p
/!
I
'm
m
small laboratory animals 1939 ~
5.8%
m
reptiles, amphibians
18
0.3%
birds, eggs
7S
1.1%
fish
18
0.3%
non-vertebrates
8
0.1%
insects
4
0.1%
microorganisms tissue
mL-
42 1534
body fluid
719~._
|
primary cell culture
337
I
cell lines
sel ~
I
0.6%
m
m /
[]
23.1%
5.1% 0
tC.8~ 8.4%
I
no living material
2.6%
unclear
0 3% I
I
I
10 20 30
/
I
I
10 20 3O
i
i
i
10 :,'0 30
i
i
i
i
10 20 30
i
i
10 20 30
I
i
20 3O
Percentage of abstracts Fig. 1. Laboratory animal species or in-vitro test system used in 6649 research communications presented at the 1989 Annual Meeting of FASEB. An unequivocal distinction between the use of tissues, body fluids (e.g. peripheral blood lymphocytes) and primary cell cultures was often not possible from the inform~.tion provided. Thus, a certain degree of subjectivity was unavoidable in assigning these communications to in-vitro test categories. Whenever donor animals or human donors of tissues or body fluids were manipulated (e.g. by administering special diets, exposure to uncommon environmsntal conditions or immunization), the abstract was scored as reporting an in-vivo experimenL When preparation of a monoclonal antibody was an essential Dart of the research, the communication was scored as using 'small laboratory anima/s, even though the experimental procedures were not described. In-vitro tests using already existing or commercially available monoclonal antibodies were assigned to one of the in-vitro test categories. When more than one laboratory animal species was investigated, the abstract was assigned to the phy/ogenetical/y highest species, n, number of abstracts reviewed.
stuLbornly refuse to accept invitro test results in lieu of experiments performed with live animals. The best illustration of this development is legislation (HR 1635) introduced in the US House of Representatives in 1988 by congresswomen Barbara Boxer et al., demanding that US federal regulatory agencies be prohibited from accepting LD50 tests performed with live animals, and be compelled to propagate non-animal alternatives for this and other test procedures. There exists a widespread misconception, even among scientists, about the attitude of regulators towards in-vitro testing, and about the extent to which these individuals can control the choice of research methods. Regulatory agencies rely, to a large extent, upon the advice of scientific experts, and they have whole-
heartedly endorsed their suggestions to accept in-vitro tests for assessment of the mutagenic (not carcinogenic) hazards of chemicals, and for pyrogenicity testing of parenterally administered drugs and infusion fluids. Given properly validated scientific data, endorsed by a majority of the experts, they will undoubtedly be ready to change their requirements in other areas as well. An example is the imminent replacement of the mouse bioassay for insulin batch control by a purely chemical method of analysis 1. However, they cannot be expected to base their decision-making criteria on research conducted by individual research groups using limited numbers of test chemicals. It is also widely ignored that only a fraction of the research necessary for discovery and development of new drugs and other
chemicals is under governmental regulatory control. It is confined, essentially, to the studies for safety and a limited number of tests for quality assurance of certain biologicals. The percentage of laboratory animals used for these regulatory purposes is in the range 10-15% in industrial organizations and is close to zero in academic institutions. Nevertheless, the use of live animals for toxicological investigations has come under heavy criticism by animal rights advocates. For reasons not entirely clear, it is mainly directed against three procedures, the so-called LDs0 test and the assays for skin and eye irritancy. In these areas, animal tests serve two different purposes. First, they contribute valuable, albeit incomplete, information about the human hazard due to
TiPS - March 1990 [Vol. 11]
106
I
acute poisoning, injury caused by accidental contact of chemicals with skin and mucosal membranes and human risks related to intentional, topical use of drugs and cosmetics. For these objectives, alternative tecnniques are already widely used; these include simplified acute toxicity tests with small numbers of animals, and in-vitro models using cell cultures, chorion allantois membrane of chicken eggs, and isolated skin and eye preparations. In the hands of skilled investigators and in conjunction with clinical experience, these methods can contribute valuable information on human hazard. However, acute toxicity tests and in-vivo irritation studies are also used in a more formalized way to provide the basis for labeling of hazardous chemicals, a procedure deemed to be essential for consumer protection. In order to treat all manufacturers equally and to assure uniformity of labeling and consumer safety criteria worldwide, national regulatory agencies, under the guidance of the Organization of Economic Cooperation and Development (OECD), have adopted formalized procedures for acute oral and dermal toxicity (often referred to as LDso tests), and for skin and eye irritation testing (often referred to as Draize tests). It is clear that a replacement of these rather rigid procedures by a more flexible and scientific approach needing fewer animals, but sophisticated in-vivo monitoring and in-vitro cell and tissue culture methodology, is difficult to put into operation on a global
Fig. 2. Physiologists and pharmacologists have been experimenting on animal tissue for many years. This early example of an organ bath, dating from 1930, was devised to demonstrate humoral transference in the gut. Ringer sol-~on .~.'ck!ed slowly over a preparation of innerva~=d gut onto a test piece of gut. When the nerve-muscle preparation was stimulated an "inhibitory substance' was released into the Ringer, and caused relaxation of the test piece of gut. Keeping the air in the chamber =/arm and moist posed a major problem. This was eventually solved by heating a layer of water at the bottom of the chamber to 50-60~C.
level. Thus, despite declarations of honorable intentions by all concerned, progress in this area is and will be slow. A ray of hope is provided by the initiative of the British Toxicology Society which is validating a simplified and more humane procedure for classification of poisonous chemicals2. The society has also proposed a hierarchical scheme for dermal and mucosal irritancy testing that relies on in-vitro models for the detection of severely irritant and corrosive properties, and uses a minimal number of animals only for confirmatory investigations of chemicals and products of low degrees of irritancy3. In the other areas of industrial toxicology- testing for subchronic and chronic toxicity, reproductive toxicity and carcinogenicity - the development of in-vitro testing procedures has made considerable progress, particularly in two fields. The first is organ-directed toxicity, and encompasses organotypic cell and tissue cultures from liver, kidney, lung, testis, nervous systems and others. Apart from cytotoxicity, these systems can be used to study many specific functions, such as differentiation, contraction, synthesis and secretion of proteins and mediators, intraand intercellular signal transduction, proliferation, and malignant transformation. Their major application is in toxicological screening, i.e. the selection process of the most favorable candidate substances for further development. With these techniques toxic agents can often be recognized before formal in-vivo toxicity
studies are performed, resulting in considerable savings of laboratory animals. However, a reliable estimate of the number of animals saved by these procedures is not possible. The second area in which enormous progress has been made in recent years is the use of tissue and cell cultures for the investigation of drug metabolis:n. Here, the availability of material of human origin, particularly hepatocytes, has revolutionized the process of extrapolation of animal data to humans. As with toxicological screening, the number of laboratory animals spared by the application of these techniques is difficult to quantify. However, early detection and elimination of chemicals that are transformed into toxic metabolites by human liver enzymes will certainly lead to a considerable reduction of redundant in-vivo toxicological investigations. However, we should not forget that toxicity studies are not only performed to detect relevant adverse characteristics of the test compounds, but are also expected to demonstrate "safety', i.e. the absence of toxicity. On the basis of these findings, safe levels of human exposure are determined. Since it is impossible to prove absence of toxicity, toxicologists try to maximize the odds of detecting even the most trifling adverse effect by conducting their experiments with large numbers of animals and multiple dose levels, including one that causes overt toxicity. They also do this for their own protection, since product litigation charges have become a serious problem. New and as yet not generally accepted alternative safety tests might be as reliable as the standard in-vivo procedures from the scientific point of view, but they will not carry as much weight as the overpowering in-vivo models in the defense against unjustified claims. Thus, regulatory agencies which are blamed first, and manufacturers which are often forced to pay exorbitant sums for indemnification by compassionate but scientifically uninformed judges, are reluctant to trade time-honored safety testing procedures against the more scientific new alternatives. [] [] []
TiPS- March 1990 [Vol. 11] Biomedical scientists are using in-vitro alternative research techniques extensively and with considerable success, although their achievement in making this exciting development known to outsiders is not spectacular. In toxicology, alternative testing methods have an important place in screening for cytotoxicity and for defined specific functional and biochemical disturbances in organotypic tissue and cell culture preparations. A relatively recent development is the use of in-vitro techniques for the study of pathways and rate of metabolism of
107 foreign chemical compounds. Thanks to the development of techniques for the successful culture of human cells, extrapolation of these findings to humans now has a solid scientific basis. For the determination of product safety in-vitro methods are also being investigated, particularly in the areas of skin and eye irritation, teratogenicity and carcinogenicity. However, their widespread use and their acceptance by regulatory agencies in lieu of whole animal tests is a slow pr~,cess, since global consent on ne~ concepts for consumer
Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration Yuechueng Liu and Daniel R. Storm Neuromodulin is a neurospecific calmodulin binding protein that is implicated in neurite extension, axonal elongation and iong-term potentiation. Yuechueng Liu and Daniel Storm propose that neuromodulin binds and concentrates calmodulin on growth cone membranes and that stimulation of protein kinase C releases high local concentrations of calmodulin irJ the growth cone. Interactions between released calmodulin and cytoskeleton proteins may affect the polymerization, crosslinking and membrane attachment of cytoskeleton polymers. This local "softening" of the membrane may be an initial event in filopodia formation and extension. The Ca2+ binding protein calmodulin and its target proteins are potential drug target sites in the CNS. For example, the phenothiazines, a class of antipsychotic drug, bind to calmodulin and antagonize its interactions with calmodulin-regulated enzymes. However, the phenothiazines have a number o~ other target sites in animal cells and it is not clear to what extent the pharmacological activities of these drugs are due to antagonism of the interactions of calmodulin with its target proteins ~. For example, the K,ts for calmodulin-phenothiazine interY. Liu is a Postdoctora| Fellow and D. R. Storm is Professor at the Dep~.rtment of Pharmacology, 5]-30, University of Washington, Seattle, WA 98195, USA.
actions fall in the range 1-150 btM, which is significantly higher than the dose used in antipsychotic treatment 1. Calmodulin mediates Ca 2+ regulation of a number of enzymes and proteins that are thought to play significant neuromodulatory roles. For example, mammalian brain contains calmodulin-stimulated adenylyl cyclases, protein kinases, cyclic nucleotide phosphodiesterases and phosphoprotein phosphatases which are important components of signal transduction systems. Calmodulin-sensitive adenylyl cyclases and calmodulindependent protein kinase II have both been implicated as regulatory elements contributing to neuroplasticity. In this article, we describe the properties of neuro-
protection and product labeling is difficult to achieve. Moreover, the rapidly escalating problem of product liability claims in our litigious society is a serious impediment for the acceptance of alternative safety testing procedures.
References 1 Fisher, B. V. and Smith, D. ~. (1986) Pharmacol. Biomed. Anal. 4, 377-387 2 van den Heuvel, M. J., Daya-., A. D. and Shillaker, R. O. (1987) Hum. ToxicoL 6, 279-291 3 Fielder, R. J., Gaunt, I. F., Rhodes, C., Sullivan, F. M. and Swanston, D. W. (1987) Hum. Toxicol. 6, 269-278
modulin, a neuron-specific calmodulin binding protein that has been implicated in regulation of neuron growth and neuroplasticity. It is hypothesized that the levels of free calrnodulin present in neurons may be regulated by reversible phosphorylation of neuromodulin. Physiological functions of neuromodulin The neurobiology of axonal growth-associated proteins, including neuromodulin, has recently been reviewed 2. A variety of divergent interests have focused on neuromodulin, as a number of experimental approaches have led investigators with differing research interests to the same molecule. As a result of these efforts, several names have been assigned to the molecule, each associated with a particular branch of neurobiology (see Box). One clue to the function of neuromodulin has been found in its tissue and subcetlular distribution. The availability of substantial amounts of purified neuromodulin 3 made possible the isolation of a rabbit polyclonal antibody specific for the protein and the development of a detergent-based radioimmunoassay to quantify accurately the levels of neuromodulin in various regions of brain 4. Western blot analysis revealed the presence of neuromodulin in membrane preparations from b o v i n e cerebellum, pineal gland, internal capsule, thalamm, ~hypothalamus, striatum and hippocampus. However, the protein is absent from all bovine non-neuronal tissues examined
~ 1990,ElsevierSciencePublishersLtd. {UK) 0165- 61471901S02.00
