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Utility of in vitro assays: Summary of international workshop on in vitro methods in reproductive toxicology
Reproductwe Toxwology, Vol 7, pp 171-173, 1993
0890-6238/93 $600 + 00 1993 Pergamon Press Ltd
Pnnted In the U S A All nghts reserved
• Summary of Workshop
UTILITY
OF IN VITRO
ASSAYS: SUMMARY OF INTERNATIONAL WORKSHOP O N I N V I T R O M E T H O D S IN REPRODUCTIVE TOXICOLOGY
BERNARD A . SCHWETZ National Institute of Environmental Health Sciences, Research Triangle Park, North Carohna
in in vivo studies. There was a general consensus that we do not have in vitro systems to replace those reproductive studies that we are currently using for risk assessment purposes. The next general conclusion is that the in vitro data are particularly useful for a secondary level of evaluation and the logical approach to collecting data that would be useful for risk assessment would be to incorporate m VlVO and in vitro data in the overall evaluahon. One approach for doing that would be to start out with an in vlvo screen to give you an idea of the toxicity, first of all, with a small number of animals over a range of dose levels. Does the toxicity include reproductive or developmental effects? Can It be narrowed down to some part of reproduction to help focus the reproduchve concern and guide further work9 On the basis of this preliminary in VlVO data, further studies are designed that include a combination of In vivo and in vitro studies to flesh out the rest of the data base that describes the reproductive toxicity. There was a strong feeling that there was an important place for in vitro data m the overall approach but one would not necessarily start with in vttro tests to make important decisions about what studies to do next. One of the things that became clear in these discussions was that we do not agree on what constitutes vahdation. One approach is to test the system under consideration against a long list ofpreselected agents that are either toxic or nontoxic, e.g., a list of agreed-upon reference standards The other approach to validation is that the system works for me m my laboratory. For that individual, this is a legitimate form of validation. Well, the two of them are quite different m the level of confidence that others might have m looking at that system and it
The workshop that we had on in vitro methods in reproductive toxicology was organized in four sessions. The first one dealt wtth male reproduction, the second with female reproduction and Dr. Wyrobek and Dr. Mattison chaired those sessions respectively, then a general discussion of screening methods that Dr. Palmer chaired and then the last session on developmental toxioty, which I oversaw. A couple of caveats are critical to give at the beginnmg of this review First, this was not an exhaustive review of all of the events in reproduction. There were some very important areas that we never discussed. For instance, aspects of male reproduction, other than those related to sperm, were not discussed. We did not talk about parturition. We did not talk about lactation or postnatal development so this was not an attempt to cover all the m vitro methods that relate to all the events in reproductton. Perhaps even more importantly, we dtd not attempt an exhaustive review of all of the in vitro literature. We talked about promising methods but not necessarily because they were picked as the most promising ones in the field by some process. We tried to cover a wide range of In vitro methods to stimulate discussion of concepts and general conclusions rather than to arrive at a list of specific in vitro methods that were the best. There were some overall conclusions that were independent of whether we were talking about reproduction or development, or male or female effects, and I want to summarize those before getting to more specific points. First, unfortunately, in vitro screens are not yet available to replace the in VlVO tests used to assess reproductive toxicity--reproduction m the broadest sense, including development and fertdity and the endpoints that we measure 171
172
Reproductive Toxicology
does, m my opinion, raise s o m e confusion when we say that s y s t e m s are either validated or they are not w h e n y o u h a v e such divergent opinions about what constitutes validation Regarding the use of in vitro data for risk assessment, it was also fairly clear that regulators worldwide do not yet use in vitro data as the sole basis for a regulatory decision. I must say that there was m o r e p e s s i m i s m than o p t i m i s m about the use of in vitro data for hazard identification. H o w e v e r , there was considerable support for the incorporation of these data as part of a b o d y of knowledge, c o m b i n e d with in vivo data, for making decisions about hazard identification and risk a s s e s s m e n t . Thus the best use of the in vitro data was in combination with in w v o data. A n o t h e r overall conclusion independent of reproduction or d e v e l o p m e n t was that in v~tro s y s t e m s are m o s t useful for a preselected group of chemicals for which the in vitro s y s t e m is matched to the k n o w n in VlVO toxicity of the group of chemicals This continues to be the condition under which m vitro screens are used m o s t successfully. Situations w h e r e there are n u m e r o u s chemicals that are pharmacologically, structurally, or toxicologically similar and there is an in vitro screen that identifies the toxic ones and you use ~t to c o m p a r e the relative toxicity or the p o t e n c y of others in that family, continues to be the m o s t successful use of in vitro syst e m s as o p p o s e d to screening randomly selected large n u m b e r s of chemicals that are structurally unrelated. As m all other meetings w h e r e this topic was discussed, there again was a g r e e m e n t that m vitro data are v e r y important for m e c h a m s t l c understanding, not only of the agents involved, but perhaps m o r e importantly, the biologic process that is involved. T h e mechanistic knowledge derived f r o m in vitro a p p r o a c h e s permits us not only to k n o w more about the chemicals or the other agents under study but certainly has helped us to understand the basic biology behind what we o b s e r v e as a toxicologic effect T h o s e are the overall conclusions related to the b r o a d areas we talked about. N o w some m o r e specific c o m m e n t s a b o u t male and female reproduction and d e v e l o p m e n t , starting with male reproduction. Cell culture s y s t e m s h a v e b e e n used in multiple l a b o r a t o n e s that include cultured cells of individual types or cocultures of sertoli cells, leydIg cells, or combination of these cells with germ cells. One of the difficulties of in vitro s y s t e m s is maintaining the association b e t w e e n selected cell types It is very difficult for an in vitro s y s t e m to reconstruct those
Volume7. Supplement 1. 1993 cellular associations e v e n though we can culture the cells. In vivo studies continue to confirm the Importance of the associations of the various cell types present m the testis. One big difference in the research a p p r o a c h for the male versus the female is the fact that y o u can do so m u c h ex vivo w o r k with s p e r m c o m p a r e d to the difficulty of doing such w o r k with the female. Thus, there are a lot of things that we know about male reproduction by looking at sperm, including both physiologic d a m a g e to s p e r m as well as genetic d a m a g e Most of what has b e e n done m the clinical setting, and to some extent in the experimental settmg, has b e e n to look at s p e r m counts, s p e r m morphology, s p e r m motility, and other measures of the physiology of s p e r m F r o m the standpoint of genetic damage, the methods are less well developed but there are methods to detect the effect of chemicals on the D N A of s p e r m The a p p r o a c h that was r e c o m m e n d e d for further understanding the effects of agents on s p e r m was to continue to do s e m e n evaluations in e x p o s e d men. In fact, it ~s p r o b a b l y a little easier to get h u m a n semen samples than we thought 10 years ago So we need to continue to evaluate h u m a n s e m e n and b a c k that up with s p e r m tests m animals where we can explore other questions that you cannot necessarily explore m humans, such as d o s e - r e s p o n s e curves, r e c o v e r y , and the cellular site of action The emphasis m animals would be on semen and s p e r m characteristics rather than simply fertihty trials. A n o t h e r r e c o m m e n d a t i o n was that we need studies in h u m a n s that link some of these sperm m e a s u r e s to fertility and the o u t c o m e of pregnancy, including m e a s u r e s such as the time to pregnancy and early fertility loss As it turns out, we need to do the same things in animals, not just in humans. We can m e a s u r e far m o r e things about s p e r m than we k n o w how to interpret W h e n the methods permit us to repeatedly m e a s u r e subtle changes in motility, animal models can be used to find out what those m e a n in t e r m s of reproductive success F r o m the standpoint of genetic damage to sperm, we can m e a s u r e structural and numeric changes in c h r o m o s o m e s It is m u c h m o r e difficult to m e a s u r e gene mutations. B e c a u s e of the association b e t w e e n changes in the sex c h r o m o s o m e s and phenotyplc and genotyplc changes in children, it is important to continue to monitor that association, to look at D N A changes in adults and offspring as they relate to d a m a g e to the D N A of sperm. We can do the same thing in l a b o r a t o r y animals as well as in h u m a n s R e f e r e n c e was m a d e to the upcoming use of transgenic s y s t e m s that will perhaps help us to
Summary of workshop • B A SCHWETZ understand the importance of changes in genetic structure as it relates to sperm function. Regarding comments that were specific to the female, we recognize that in order to cover the broad area of female reproductive toxicity, you would have to talk about fecundity and fertilization, in utero development, parturition, and lactation and postnatal development. We did not review all of these reproductive events, but even a limited review of In vivo data on female reproductive toxicity from humans and animals reveals problems with confounders, specificity, and limitations in statistical power. We need better blomarkers of exposure and effects to develop better data bases in humans and animals. In terms of in vitro approaches for the female we know that there are human tissue or cell culture systems that would appear to be relevant but when you take these cells out of the context of the whole animal, extrapolating back to the whole animal is very difficult. From the standpoint of cell culture systems from other mammals or nonmammals, you have the problem of both the relevancy and the difficulty of extrapolating back from an in vitro system to the whole animal. There were, however, interesting presentations on cultured human trophoblasts as well as perfused ovaries of rats. There are systems with potential for looking at parts of the female reproductive process. Regarding cell or tissue culture methods for specific female tissues, methods need to be developed for hypothalamlc cells. Methods exist for culturing pituitary cells. We do not particularly use them, and we perhaps should. In the ovary, granulosa cell cultures are being used to a limited extent From the uterus, we can culture myometrlal and endothelial cells. Perhaps these need to be used in coculture as well as isolated cell cultures in the future. In the discussions on developmental toxicity, It was clear that new knowledge IS going to come to us from molecular biology and developmental biology laboratories that will definitely increase our understanding of normal development. We will learn more about the Intricacies of the regulation of development to a much greater extent than we have ever
173
known it before. There is no doubt that we seriously underestimated how complex this process was before we began to learn about all of the factors and processes that regulate normal development. The biologists will help us understand the biology of development and will probably stimulate a new generatlon of in vitro tests that, when we are smart enough to learn how to use that information, will probably do two things for us. First, research at the molecular level will more likely identify regulatory factors and critical events that extrapolate across species. Second, this new knowledge will help clarify when mechanistic data are useful or not In risk assessment decisions. Our discussions confirmed the opinion of a number of us that whole embryos are needed to predict modulations of development of embryos caused by exogenous agents. On the other hand, in vitro data are extremely useful for us to know where to look in the whole embryo to find out what is going on, whether that whole embryo is in culture or in the uterus. In the future, new models or refinements of old models will continue to surface which we need to give serious attention. One system, for example about which we heard at this meeting and in the literature in the last few years is the chick embryo. Many of us rejected the chick embryo 20 years ago as something that we thought was not useful. Now the chick embryo is coming back as an explanted system where the control is much better. This IS an example of an old screen that we rejected that has been reevaluated under better conditions to improve the usefulness of data from the screen. We should encourage such refinements to potentially useful test systems. To summarize the overall conclusion of the workshop on in vitro methods in reproductive and developmental toxicology, the greatest benefit of in vitro data would seem to be their impact on our degree of confidence in in VlVOdata through a better understanding of the site and mechanism of action and the biologic plausibility of the toxicologic effects that we observe in vivo.
0890-6238/93 $600 + 00 1993 Pergamon Press Ltd
Pnnted In the U S A All nghts reserved
• Summary of Workshop
UTILITY
OF IN VITRO
ASSAYS: SUMMARY OF INTERNATIONAL WORKSHOP O N I N V I T R O M E T H O D S IN REPRODUCTIVE TOXICOLOGY
BERNARD A . SCHWETZ National Institute of Environmental Health Sciences, Research Triangle Park, North Carohna
in in vivo studies. There was a general consensus that we do not have in vitro systems to replace those reproductive studies that we are currently using for risk assessment purposes. The next general conclusion is that the in vitro data are particularly useful for a secondary level of evaluation and the logical approach to collecting data that would be useful for risk assessment would be to incorporate m VlVO and in vitro data in the overall evaluahon. One approach for doing that would be to start out with an in vlvo screen to give you an idea of the toxicity, first of all, with a small number of animals over a range of dose levels. Does the toxicity include reproductive or developmental effects? Can It be narrowed down to some part of reproduction to help focus the reproduchve concern and guide further work9 On the basis of this preliminary in VlVO data, further studies are designed that include a combination of In vivo and in vitro studies to flesh out the rest of the data base that describes the reproductive toxicity. There was a strong feeling that there was an important place for in vitro data m the overall approach but one would not necessarily start with in vttro tests to make important decisions about what studies to do next. One of the things that became clear in these discussions was that we do not agree on what constitutes vahdation. One approach is to test the system under consideration against a long list ofpreselected agents that are either toxic or nontoxic, e.g., a list of agreed-upon reference standards The other approach to validation is that the system works for me m my laboratory. For that individual, this is a legitimate form of validation. Well, the two of them are quite different m the level of confidence that others might have m looking at that system and it
The workshop that we had on in vitro methods in reproductive toxicology was organized in four sessions. The first one dealt wtth male reproduction, the second with female reproduction and Dr. Wyrobek and Dr. Mattison chaired those sessions respectively, then a general discussion of screening methods that Dr. Palmer chaired and then the last session on developmental toxioty, which I oversaw. A couple of caveats are critical to give at the beginnmg of this review First, this was not an exhaustive review of all of the events in reproduction. There were some very important areas that we never discussed. For instance, aspects of male reproduction, other than those related to sperm, were not discussed. We did not talk about parturition. We did not talk about lactation or postnatal development so this was not an attempt to cover all the m vitro methods that relate to all the events in reproductton. Perhaps even more importantly, we dtd not attempt an exhaustive review of all of the in vitro literature. We talked about promising methods but not necessarily because they were picked as the most promising ones in the field by some process. We tried to cover a wide range of In vitro methods to stimulate discussion of concepts and general conclusions rather than to arrive at a list of specific in vitro methods that were the best. There were some overall conclusions that were independent of whether we were talking about reproduction or development, or male or female effects, and I want to summarize those before getting to more specific points. First, unfortunately, in vitro screens are not yet available to replace the in VlVO tests used to assess reproductive toxicity--reproduction m the broadest sense, including development and fertdity and the endpoints that we measure 171
172
Reproductive Toxicology
does, m my opinion, raise s o m e confusion when we say that s y s t e m s are either validated or they are not w h e n y o u h a v e such divergent opinions about what constitutes validation Regarding the use of in vitro data for risk assessment, it was also fairly clear that regulators worldwide do not yet use in vitro data as the sole basis for a regulatory decision. I must say that there was m o r e p e s s i m i s m than o p t i m i s m about the use of in vitro data for hazard identification. H o w e v e r , there was considerable support for the incorporation of these data as part of a b o d y of knowledge, c o m b i n e d with in vivo data, for making decisions about hazard identification and risk a s s e s s m e n t . Thus the best use of the in vitro data was in combination with in w v o data. A n o t h e r overall conclusion independent of reproduction or d e v e l o p m e n t was that in v~tro s y s t e m s are m o s t useful for a preselected group of chemicals for which the in vitro s y s t e m is matched to the k n o w n in VlVO toxicity of the group of chemicals This continues to be the condition under which m vitro screens are used m o s t successfully. Situations w h e r e there are n u m e r o u s chemicals that are pharmacologically, structurally, or toxicologically similar and there is an in vitro screen that identifies the toxic ones and you use ~t to c o m p a r e the relative toxicity or the p o t e n c y of others in that family, continues to be the m o s t successful use of in vitro syst e m s as o p p o s e d to screening randomly selected large n u m b e r s of chemicals that are structurally unrelated. As m all other meetings w h e r e this topic was discussed, there again was a g r e e m e n t that m vitro data are v e r y important for m e c h a m s t l c understanding, not only of the agents involved, but perhaps m o r e importantly, the biologic process that is involved. T h e mechanistic knowledge derived f r o m in vitro a p p r o a c h e s permits us not only to k n o w more about the chemicals or the other agents under study but certainly has helped us to understand the basic biology behind what we o b s e r v e as a toxicologic effect T h o s e are the overall conclusions related to the b r o a d areas we talked about. N o w some m o r e specific c o m m e n t s a b o u t male and female reproduction and d e v e l o p m e n t , starting with male reproduction. Cell culture s y s t e m s h a v e b e e n used in multiple l a b o r a t o n e s that include cultured cells of individual types or cocultures of sertoli cells, leydIg cells, or combination of these cells with germ cells. One of the difficulties of in vitro s y s t e m s is maintaining the association b e t w e e n selected cell types It is very difficult for an in vitro s y s t e m to reconstruct those
Volume7. Supplement 1. 1993 cellular associations e v e n though we can culture the cells. In vivo studies continue to confirm the Importance of the associations of the various cell types present m the testis. One big difference in the research a p p r o a c h for the male versus the female is the fact that y o u can do so m u c h ex vivo w o r k with s p e r m c o m p a r e d to the difficulty of doing such w o r k with the female. Thus, there are a lot of things that we know about male reproduction by looking at sperm, including both physiologic d a m a g e to s p e r m as well as genetic d a m a g e Most of what has b e e n done m the clinical setting, and to some extent in the experimental settmg, has b e e n to look at s p e r m counts, s p e r m morphology, s p e r m motility, and other measures of the physiology of s p e r m F r o m the standpoint of genetic damage, the methods are less well developed but there are methods to detect the effect of chemicals on the D N A of s p e r m The a p p r o a c h that was r e c o m m e n d e d for further understanding the effects of agents on s p e r m was to continue to do s e m e n evaluations in e x p o s e d men. In fact, it ~s p r o b a b l y a little easier to get h u m a n semen samples than we thought 10 years ago So we need to continue to evaluate h u m a n s e m e n and b a c k that up with s p e r m tests m animals where we can explore other questions that you cannot necessarily explore m humans, such as d o s e - r e s p o n s e curves, r e c o v e r y , and the cellular site of action The emphasis m animals would be on semen and s p e r m characteristics rather than simply fertihty trials. A n o t h e r r e c o m m e n d a t i o n was that we need studies in h u m a n s that link some of these sperm m e a s u r e s to fertility and the o u t c o m e of pregnancy, including m e a s u r e s such as the time to pregnancy and early fertility loss As it turns out, we need to do the same things in animals, not just in humans. We can m e a s u r e far m o r e things about s p e r m than we k n o w how to interpret W h e n the methods permit us to repeatedly m e a s u r e subtle changes in motility, animal models can be used to find out what those m e a n in t e r m s of reproductive success F r o m the standpoint of genetic damage to sperm, we can m e a s u r e structural and numeric changes in c h r o m o s o m e s It is m u c h m o r e difficult to m e a s u r e gene mutations. B e c a u s e of the association b e t w e e n changes in the sex c h r o m o s o m e s and phenotyplc and genotyplc changes in children, it is important to continue to monitor that association, to look at D N A changes in adults and offspring as they relate to d a m a g e to the D N A of sperm. We can do the same thing in l a b o r a t o r y animals as well as in h u m a n s R e f e r e n c e was m a d e to the upcoming use of transgenic s y s t e m s that will perhaps help us to
Summary of workshop • B A SCHWETZ understand the importance of changes in genetic structure as it relates to sperm function. Regarding comments that were specific to the female, we recognize that in order to cover the broad area of female reproductive toxicity, you would have to talk about fecundity and fertilization, in utero development, parturition, and lactation and postnatal development. We did not review all of these reproductive events, but even a limited review of In vivo data on female reproductive toxicity from humans and animals reveals problems with confounders, specificity, and limitations in statistical power. We need better blomarkers of exposure and effects to develop better data bases in humans and animals. In terms of in vitro approaches for the female we know that there are human tissue or cell culture systems that would appear to be relevant but when you take these cells out of the context of the whole animal, extrapolating back to the whole animal is very difficult. From the standpoint of cell culture systems from other mammals or nonmammals, you have the problem of both the relevancy and the difficulty of extrapolating back from an in vitro system to the whole animal. There were, however, interesting presentations on cultured human trophoblasts as well as perfused ovaries of rats. There are systems with potential for looking at parts of the female reproductive process. Regarding cell or tissue culture methods for specific female tissues, methods need to be developed for hypothalamlc cells. Methods exist for culturing pituitary cells. We do not particularly use them, and we perhaps should. In the ovary, granulosa cell cultures are being used to a limited extent From the uterus, we can culture myometrlal and endothelial cells. Perhaps these need to be used in coculture as well as isolated cell cultures in the future. In the discussions on developmental toxicity, It was clear that new knowledge IS going to come to us from molecular biology and developmental biology laboratories that will definitely increase our understanding of normal development. We will learn more about the Intricacies of the regulation of development to a much greater extent than we have ever
173
known it before. There is no doubt that we seriously underestimated how complex this process was before we began to learn about all of the factors and processes that regulate normal development. The biologists will help us understand the biology of development and will probably stimulate a new generatlon of in vitro tests that, when we are smart enough to learn how to use that information, will probably do two things for us. First, research at the molecular level will more likely identify regulatory factors and critical events that extrapolate across species. Second, this new knowledge will help clarify when mechanistic data are useful or not In risk assessment decisions. Our discussions confirmed the opinion of a number of us that whole embryos are needed to predict modulations of development of embryos caused by exogenous agents. On the other hand, in vitro data are extremely useful for us to know where to look in the whole embryo to find out what is going on, whether that whole embryo is in culture or in the uterus. In the future, new models or refinements of old models will continue to surface which we need to give serious attention. One system, for example about which we heard at this meeting and in the literature in the last few years is the chick embryo. Many of us rejected the chick embryo 20 years ago as something that we thought was not useful. Now the chick embryo is coming back as an explanted system where the control is much better. This IS an example of an old screen that we rejected that has been reevaluated under better conditions to improve the usefulness of data from the screen. We should encourage such refinements to potentially useful test systems. To summarize the overall conclusion of the workshop on in vitro methods in reproductive and developmental toxicology, the greatest benefit of in vitro data would seem to be their impact on our degree of confidence in in VlVOdata through a better understanding of the site and mechanism of action and the biologic plausibility of the toxicologic effects that we observe in vivo.