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The toxicological evaluation of mutagenic events
Mutation Research, 25 (1974) 145-157 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - P r i n t e d in T h e N e t h e r l a n d s
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T H E TOXICOLOGICAL E V A L U A T I O N OF MUTAGENIC EVENTS
DIETHER
NEUBERT Pharmakologisches Institut der Freien Universitiit Berlin, Embryonal-Pharmahologie, x Berlin 33, Thielallee 69/73 (Germany)
(Received April 4th, 1974) (Revision received J u l y 8th, 1974)
SUMMARY
At present no mammalian test system which meets the toxicological requirements is available for routine testing of mutagenicity. Therefore, emphasis should be laid primarily on basic research in this area and not on large-scale screening of possible mutagens with methods known to be inadequate in m a n y respects, if mutagenicity is a maior hazard to man, a view certainly not shared by all toxicologists. Furthermore, if carcinogenicity is based on a mutagenic event occurring in somatic cells, the well established tests for carcinogenicity would provide a better way for evaluating irreversible somatic mutations than the tests now suggested for mutagenicity testing. In the present situation a drastic reduction of the noxes men are exposed to would be the most reliable means of preventing a toxicological disaster. We are still in the situation of continuously performing "mass human experiments" and detecting hazards only after considerable harm has been done. Consequently, the goal must be neither to expose a considerable proportion of our population to environmental hazards nor to give drugs to thousands or even millions of healthy people for any reasons whatsoever, unless test systems are available which would allow effective prevention of disaster.
INTRODUCTION
The evaluation of mutagenic events taking place in a mammalian organism is basically a toxicological problem. This is not only so because pharmacology and toxicology are the medical fields concerned with the interactions of drugs and a mammalian organism*, but more so because mutagenicity in mammals is governed by basic pharmacological parameters (Fig. I). However, toxicological considerations connected with mutagenicity are complicated by a number of additional factors as they are in a number of other special toxic events such as carcinogenicity or teratogenicity. As an applied science, pharmacology and toxicology are basically anthro* A d r u g is defined as a n y foreign c o m p o u n d able to affect t h e m a m m a l i a n cell, or a physiological s u b s t a n c e u s e d in u n p h y s i o l o g i c a l l y h i g h c o n c e n t r a t i o n s .
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pocentric. Therefore I should like to concentrate on problems primarily connected with the detection of possible mutagenic health hazards to man. There is no doubt that, owing to our limited knowledge in this comparatively new field, conclusions concerning the prediction and detection of such health hazards will be rather pessimistic. Since the toxicological point of view has so far not been adequately considered by many investigators I should like to survey some of the basic toxicological principles that govern mutagenic eventsin mammals. Many of these principles are well established and there is no need to "rediscover" them. Two aspects are especially important from the toxicological point of view: (z) problems of extrapolating experimental findings in laboratory animals to man; and, especially, (2) the dose-response relationship to be expected in mutagenicity. In addition to these, some special problems connected with mutagenic-toxicological evaluations will be discussed. PROBLEMS OF EXTRAPOLATING EXPERIMENTAL FINDINGS IN LABORATORY ANIMALS TO MAN
The pharmacological effect of a drug is governed by a number of principles. These often have to be evaluated and investigated separately since the ideal conditions that would allow a direct extrapolation to man cannot be obtained. These pharmacological parameters (Fig. I) include pharmacokinetics, drug metabolism, species differences, and the problem of measuring drug concentrations at the target (or "receptor"). Principal difficulties arising during a toxicological evaluation are discussed elsewhere and pertinent references are given 5.
Parameters that govern the mutagenic-toxic effect Fig. I gives the basic parameters that influence the effect of a mutagenic drug in mammals. (a) Pharmacokineties. The serum concentration of the drug, and thereby tissue concentrations, critically depend on parameters that include rates of absorption,
/ PHARMACOKINETICS Resorption Distribution Elimination
A
DRUG META.BOL!SM _
outside of target cell
B
PERMEABILITY
C
DRUG METABOLISM within target cell
I MOLECULAR] MUTAGENIC EVENT
D
CONCENTRATION at target
E
I REPA,R etc. Fig. i. Factors that govern the outcome of a mutagenic-toxic event in mammals.
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d i s t r i b u t i o n , m e t a b o l i s m a n d e l i m i n a t i o n ; factors t h a t m a y be described as p h a r m a c o kinetics. Unless a s u b s t a n c e is i n t r o d u c e d into a m a m m a l i a n o r g a n i s m c o n t i n u o u s l y the effective c o n c e n t r a t i o n of the active d r u g species changes w i t h time in a t y p i c a l w a y (Fig. 2). The fact t h a t high concentrations of the d r u g to be t e s t e d m a y be present for only a s h o r t t i m e has i m p o r t a n t consequences in tests such as the " h o s t - m e d i a t e d assay". Since considerable species differences in t h e p h a r m a c o k i n e t i c s of a d r u g are a n t i c i p a t e d , the e x a c t p h a r m a c o k i n e t i c s m u s t be known for the e x p e r i m e n t a l species used as well as for h u m a n s if e x t r a p o l a t i o n of toxicological d a t a to the conditions possibly existing in m a n is the goal. F u r t h e r m o r e , the c o n c e n t r a t i o n reached b y the d r u g in the m a m m a l i a n organism m a y be highly d e p e n d e n t on the m o d e ot application. Because the rates of a b s o r p t i o n v a r y , a drug has to be a d m i n i s t e r e d b y the r o u t e most likely to be used in humans. F o r e x a m p l e it seems unjustified to test a herbicide exclusively as i.v. or s.c. injections. (b) D r u g metabolism. D r u g m e t a b o l i s m is a n o t h e r i m p o r t a n t factor for the m a n i f e s t a t i o n of a possible m u t a g e n i c effect. Since a d r u g can be i n a c t i v a t e d a n d a c t i v a t e d b y m a m m a l i a n m e t a b o l i s m this factor could be " n e g a t i v e " or " p o s i t i v e " for t h e m u t a g e n i c event. Some of the aspects of drug m e t a b o l i s m have been s u r v e y e d b y Dr. Uhleke d u r i n g this conference. There are n u m e r o u s factors r e l e v a n t to the p r o b l e m of t e s t i n g for m u t a g e n i c i t y in a m a m m a l i a n system. A r a t h e r c o m p l e x situation m a y arise if only a few of these factors are changed b y the e x p e r i m e n t a l conditions. A n e x a m p l e is given in Fig. 3 for an a l k y l a t i n g agent (cyclophosphamide) well k n o w n for being " a c t i v a t e d " in the m a m m a l i a n organism. W h e n drug m e t a b o l i s m is modified b y the s i m u l t a n e o u s a d m i n i s t r a t i o n of an i n h i b i t o r (CFT I 2 o I - - d i e t h y l a
b
serum concentration 14-
experimental period c d • f ~ t
¢
f
t
g
t
I _h_
A54o/mlserum (16-
121080.3642-
/ jj time
Fig. 2. Schematic example of a typical change in the serum concentration of a drug and a metabolite to which the drug is converted in vivo. On the top of the figure examples of experimental periods are given that might be chosen to evaluate a mutagenic-toxic effect. (a), Almost no drug effect is measured; (b)-(c), predominantly evaluates the effect produced by the original drug while (d)-(h), the effect of the metabolite predominates. The effect of the original drug plus that possibly produced by the metabolite could only be evaluated if the experimental period is extended over (b)-(h) ! Fig. 3- Serum concentration in rats after a single dose of Endoxan. (Data according to BAss et al. z and ENGELS2.)Rats were injected with i oo mg/kg Endoxan i.p. at o-time. The concentration of the activated form of Endoxan was measured with the NBP-test (data given for 7 ml of the organic solvent). Inhibition of the drug metabolizing enzyme system in liver microsomes by CFT 12Ol drastically changes the pharmacokinetics of the activated form of Endoxan and produced a less intense but longer lasting effect. CFT 12Ol was given orally at 75 mg/kg i h before Endoxan and 5° mg/kg I, 3 and 5 h after the alkylating drug.
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aminoethanol ester of phenyldiallyl acetic acid) which is known to interfere with the drug-oxidizing enzyme system in liver microsomes% the peak concentration of the activated drug species is decreased in the serum--as m a y be expected. But the pharmacokinetics are changed drastically and the activated form oi Endoxan can be detected in the serum for a considerably longer time interval in the presence of the inhibitor of drug metabolism. This result m a y be explained because the non-activated form of the drug is available to the organism for a longer period. A similar situation m a y be expected to occur in a species of experimental animals that are able to "activate" Endoxan only at a slow rate. So a high rate of "activation" in the liver is not necessarily correlated with a high concentration of activated compound reached in another cell type of the organism for a long period but m a y lead to the opposite situation. Therefore, it is helpful to differentiate between drug metabolism in cells other than the target cell (Fig. IB) and drug metabolism within a target cell (Fig. ID) because these two processes are often not identical. The serum concentration of the active drug species, and thereby the concentration of the mutagen offered to the target cell, will be a function of the predominant metabolizing cell type. Very otten the hepatocyte is believed to represent the cell type most important in drug metabolism, and within that cell the microsomal drugoxidizing system, characterized especially by BRODIE and his coworkersL is of primary significance. But this view is certainly an over-simplification since m a n y other cell types in a mammalian organism metabolize drugs, and within a cell m a n y other enzymatic systems exist that are capable of metabolizing drugs apart from the microsomal "drug-oxidizing system". For instance in "inactivating" reactions several esterases are not localized at the microsomes and e.g. dechlorinating reactions take place in the eytosoP. On the other hand, most of the antimetabolites (purine or pyrimidine antagonists)--some of which are well known as mutagens (6-mercaptopurine, bromodeoxyuridine etc.)--are converted to the biologically "active" form outside the microsome. Furthermore, it is well known that m a n y enzyme systems have different intracellular locations in different animal species. (c) Species differences. Drug metabolism m a y vary drastically for a given drug in different species. This and other factors can result in considerable species differences in the sensitivity towards drugs. An example of such a species difference and the consequences for extrapolation of data from experimental animals to man is given in Table I. Even from the very few data compiled in this table two basic facts are obvious: (z) species differences can easily amount to one or two orders of magnitude; and (2) no correlation exists in the comparative sensitivity for different drugs in a given animal species. As a consequence of such results, which are well established in pharmacology and toxicology, toxicological evaluations have to be performed with at least two different mammalian species. An exception is only justified if it is known that drug metabolism is identical in the experimental animals chosen and in man. This is seldom true. Therefore, tests for possible mutagenic hazards, even in mammals, are of limited value if only one species is used. (d) Permeability. Since m a n y drugs or their metabolites do not readily penetrate membrane systems the serum concentration m a y not give a true picture of the concentration of the active drug species at the target. A complex situation arises if the drug itself or some of its metabolites are partially excluded from entering a target cell. Conclusions based on the drug metabolism occurring within a hepatocyte or in one
T H E T O X I C O L O G I C A L E V A L U A T I O N OF M U T A G E N I C E V E N T S
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I
DIFFERENCES IN THE SENSITIVITY OF SOME SPECIES TO CYTOSTATICS
LDlo (mg/m ~) A methopterin F UdR Mouse Hamster Rat Dog Monkey
Io lO3 3 2 35
Simplified from Freireich
500 17o 47 ° 760 690
et at., 1966.
compartment of a cell are therefore not representative for the different cell types of an organism. (e) Drug metabolism within the target cell. In principle two different possibilities can be visualized: (z) a drug or its active metabolite is available to the target cell and penetrates the cell membrane at a rate sufficient to create an effective drug concentration at the target within the cell; or (2) the active drug species has to be generated within the target cell. The latter m a y be so because the active metabolite cannot penetrate the cell membrane or because the active metabolite has such a short half-life that only the formation within a target cell can lead to an effective concentration. Examples of the latter possibility are given in Dr. l~hleke's paper at this conference. It is noteworthy that most antimetabolites are converted to the active drug species within the target cell (cf. ref. 7). The drug-oxidizing system in microsomes is only occasionally involved in such conversions. (f) Effective drug concentration within the target cell. The effective drug concentration within the target cell therefore depends on a considerable number of variables some of which have already been mentioned. Additional factors certainly exist. The mutagen could be rapidly inactivated within the target cell or "unspecifically" bound to cell components other than the target. Since the extent of the molecular mutagenic event critically depends on the concentration of the mutagen at the target, all these parameters must be known and considered in order to make a valid analysis. Even then the final result m a y be altered by subsequent events such as repair processes (Fig. I F).
Toxicological significance of different mutagenicity test systems With the principal parameters depicted in Fig. I in mind, we can try to evaluate some of the mutagenicity test systems presently used. (a) Tests with microorganisms. All test systems using the genome of microorganisms as an indicator for a drug-induced mutagenicity suffer from the disadvantage that only part E in Fig. I can be evaluated. No evidence can be obtained that the active drug species possibly present at the target of a mammalian organism reaches the indicator system in the microorganism. Furthermore, considerable differences exist between a bacterial genome and animal chromosomes as well as in the enzymes involved in replication and cell division. Supplementing a test system based on the genome of microorganisms with enzyme systems isolated from certain mammalian cells again can only be of limited value because the given drug would have to be metabolized iust by this enzyme system. It has been pointed out before that drug metabolism is not confined to one
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enzyme system. From this point of view it is hard to see why a microsomal system is used to supplement the test with microorganisms instead of homogenates--or even intact animal cells. Caution is necessary to avoid overestimating the value of such a combined system since most of the drugs studied so far were known to be converted and activated by this microsomal enzymatic system so that it is possible to predict the results without actually doing the experiments. It is hard to imagine the value of such a system in a situation where the metabolism of a given drug is unknown. (b) "Host-mediated" assay. In the "host-mediated assay" the genome of a microorganism is used as an indicator for mutagenic events and a mammalian species provides the environment for the drug action. In a number of variations of this test the microorganisms are either injected into the peritoneal cavity, injected into a special organ (cf. Dr. Fahrig's lecture at this conference*), or injected intravenously. Good examples have been presented that the concentration in the peritoneal fluid of tile investigated drug and its metabolites might not be sufficient to guarantee a successful evaluation of the results. Surprisingly, only few people have bothered to measure the drug concentrations reached in the peritoneal fluid! As shown by Dr. Malling during this conference, better results are obtained when the microorganisms are injected intravenously. But according to the criteria mentioned before, this approach also cannot be satisfactory for two principal reasons. (~) If the microorganisms remain in the blood stream, essentially the effect of the drug concentration in the serum is tested. Since, in general, the period during which the microorganisms are exposed to the drug and its metabolites within the serum is comparatively short, only a small section of the pharmacokinetic profile can be checked (cf. Fig. 2). This disadvantage can only be overcome if the time of exposure of the microorganisms can be lengthened to an experimental period which covers most of the pharmacokinetic events. So far, this has not been accomplished because the microorganisms leave the blood stream rapidly after the injection. (2) Another objection results from tile assumption that most of the microorganisms, if they are introduced into a cell of the host, might be expected to be incorporated into cells of the reticuloendothelial system. But again these are not the cells in which typical drug metabolism can be expected nor the cells most interesting for mutagenicity testing. On the contrary, it m a y be assumed that the concentration of metabolites in these cells is much lower when compared with that present in hepatocytes or other cells actively engaged in drug metabolism. Again basic research has not been performed which--for example with electron microscopic techniques--would unambiguously show the exact location and the fate ot the microorganisms injected into the host. Furthermore, the "host-mediated" assay basically is a test with the genome of a microorganism and cannot be considered a truly mammalian test system. Very often a "host-mediated" assay is performed under conditions which cast serious doubt on the significance of tile results obtained. Not only is the mammalian organism infected with a number of microorganisms able to cause a lethal sepsis but also doses of the drug to be tested are used that would kill the animal over a longer period. Can metabolism be considered "normal" under such conditions ? An incubation of microorganisms with sera from different experimental animals treated with the compounds to be studied would--although again insufficient when used as the only * N o t published in this issue of Mutation Research, cf. Vol. 26 (1974) 29-36.
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test--provide similar advantages and also provide the possibility of accounting for the drug metabolism occurring in different animal species. Such a test could also conveniently be performed with the sera of patients treated with the drug to be tested for therapeutic reasons. But the "host-mediated" assay basically suffers from the disadvantage that it only covers the reactions A, B and in part E of our scheme (Fig. I). (c) Non-mammalian animal systems. After the recognition that, owing to a lack of specificity, prokaryotic test systems are of limited value for a toxicological evaluation of possible mutagenic hazards in man, the oldest animal system used in studies on mutagenicity--Drosophila--is apparently having a renaissance. Although nobody would believe the metabolism of a fly to be identical with that of a mammalian organism, a certain degree of metabolic similarity and a fairly high degree of sensitivity could suggest Drosophila as a useful prescreening system before one proceeds to a more specific but certainly much less sensitive mammalian test for mutagenicity. The impressive state of knowledge on the theoretical background of the mutagenic events occurring in this organism--as depicted, for example, by Dr. Wtirgler during this conference--greatly facilitates the interpretation of experimental findings after drug administration. This is in striking contrast with a mammalian dominant lethal test which Dr. Auerbach during this conference referred to as a "mutagenic black box". (d) Mammalian systems in vivo. Mammalian systems in vivo should offer the experimental approach from which the greatest information regarding a toxicological analysis m a y be expected. This would especially be true if a suitable test could be performed in humans exposed, for various reasons, to the drugs to be studied. But apparently no test system has been established up to now which allows an adequate monitoring of all the mutagenic hazards to which a human organism m a y be exposed. Therefore animal systems have to be used instead. Although the dominant lethal test is the one most widely used in this respect it also suffers from a number ot disadvantages. Often a positive result is reached only if the drug to be tested is administered in exceedingly high doses. Furthermore, the mutagenic events tested are apparently predominantly chromosomal aberrations and there is no indication that for example point mutations m a y be detected. On the other hand, the multiple loci test can scarcely be used on a routine basis for the screening of drugs because of the huge number of animals necessary for one test. For the purpose of detecting somatic mutations--chromosomal aberrations-the micronucleus test proposed by SCHMID5 m a y be especially suitable since it makes use of the drug metabolism in the intact animal, and rapidly proliferating cell types of bone marrow are used. (e) Mammalian cells in vitro. Extensive and rather informative data on mammalian test systems were presented during this conterence (see papers of Drs. J. Simons and D. Wild). It seems that, in the majority of systems investigated, concentrations of the drug have to be used which induce a variety of toxic effects during mutagenicity tests including cell deaths in a significant percentage. Although these systems have the advantage of using a mammalian genome as an indicator, the methods again m a y suffer from the inability of the given cell type to activate a number of drugs in a way similar to that occurring in a whole mammalian organism. Therefore, at present it seems only feasible to combine a number of tests in order to get the required information. It also appears necessary to combine the
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"genetic" studies with purely pharmacological studies on the pharmacokinetics, and especially on the problem of what concentrations of the active drug species in question are reached at the target. The studies by Dr. Generoso and his coworkers, as presented at this meeting, in which data on the mutagenic effect were combined with information on the degree of alkylation reached in the target cell at different dose levels, certainly point in this direction and should be performed much more extensively. D O S E - - R E S P O N S E R E L A T I O N S H I P S TO BE E X P E C T E D
IN M U T A G E N I C I T Y
For an evaluation of the possible hazards to be expected for man in ~11 toxicological studies, knowledge of the dose-response relationship is of utmost importance. Mutagenicity is certainly not an exception in this respect! Several investigators have attempted to extrapolate a linear dose-response relationship to doses several orders of magnitude lower, arguing that mutagenicity is a "single-hit event". With the information now available such an attempt is not justified for a number of reasons, as discussed below.
Different mutagenic events A common event leading to a mutagenic effect does not exist. But several mechanisms can give rise to a change of the genetic material in a mutagenic-toxic effect. In principle two different interferences with the genetic material m a y be differentiated with consequences also on the dose-response relationship to be expected: (a) a direct attack of a drug on the DNA. This would lead to a sequence alteration or to a point mutation or comparable mutagenic defects. (b) An indirect interference with the genetic material. This m a y occur by an alteration of replicating enzymes, specific proteins involved in cell division or reactions involved in protein synthesis or transcription not originating from an altered template. Furthermore, the final outcome of the mutagenic effect m a y be greatly modified by subsequent repair processes or similar events. (a) A direct alteration of the genome by the mutagen. The mutagenic events leading to a direct alteration of the DNA (point mutations) might be expected to proceed linearly over an extended dose range in certain circumstances. But again, the purely pharmacological parameters governing the concentration of the active drug species at the target are not likely to represent linear processes in an extremely low dose range. Absorption, elimination, and "unspecific" adsorption might occur at a different rate with very low doses of a drug. Only few pertinent pharmacological data exist since it has seldom been necessary to follow a pharmacological or toxic effect over a dose range of several orders of magnitude. Therefore much more experimental data are needed. But as general prediction one might assume that it is unlikely that the concentration at a target is linear with the dose applied if minute amounts of a drug are given to a mammalian organism (cf. discussion on "threshold" in carcinogenicity~). Since qualitative statements are useless in toxicology, it is certainly up to the scientist involved in mutagenic-toxicological research to provide pertinent quantitative data for the dose ranges of interest if an evaluation of the possible hazards for man is anticipated. (b) An indirect alteration of the gene expression by the mutagen. A different situation might arise if a drug acts indirectly on the genome. It is noteworthy that several
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substances reported to cause mutagenic effects, and especially chromosomal aberrations, belong to the class of antimetabolites or cytostatic agents that interfere with the formation of nucleic acid precursors or with the replicating process and cell division. If one accepts interference with intermediary metabolism as the sole mechanism of action of these drugs then the mutagenie events produced by these agents must be caused by the inhibition of enzymes or a stoichiometric combination with certain cell components, such as spindle proteins. If this holds true, a different dose-response relationship m a y be expected for mutagenic events triggered by such agents as compared with dlugs directly interfering with the genetic material, for example through alkylations. The well-established criteria of enzyme inhibitions would govern such toxic drug effects. But this would exclude dose-response relationships of the final mutagenic effect, linear over several orders of magnitude of the drug concentrations, since the inhibition of an enzymatic reaction would be without biological interest in the majority of the enzymatic reactions if the drug concentration was to drop below I/IO of the K~. No deleterious toxic effects could be expected from such drug actions at extremely low doses. This discussion shows that much more basic research is necessary in mammalian systems to allow a clear-cut evaluation of experimental data on the hazards to be expected for man exposed to certain concentrations of potentially mutagenic substances.
"Threshold" doses of mutagenic drugs Although the term "threshold dose" or "zero effect dose" is frequently used, such a dose limit does not actually exist. People therefore put these terms in parentheses and define the limits for which these terms should stand. Therefore it seems to be more appropriate to replace these terms by expressions like "predictable risk" or "acceptable dose" which mean basically the same without implying that a drug is ineffective beyond a certain dose limit. One would then have clearly to define "acceptable", "predictable" and "risk". Without proceeding too far with this discussion I personally feel that an agreement should have been reached on these terms before an elaborate screening program was begun. Otherwise the situation will arise--or has already arisen-- in which nobody really knows how to interpret the accumulating data.
Problems connected with the administration of extremely high doses of a potential mutagen The evaluation of the dose response relationship is often difficult, especially when mammalian test systems are used, because extremely high doses of the drugs have to be administered to detect a mutagenic effect. We have seen that cyclophosphamide is necessary at about 50 mg/kg to produce a significant mutagenic effect in the dominant lethal test. Such a dose results in severe changes of cell metabolism in m a n y tissues of a mammalian organism, and clearly detectable effects of this drug can be demonstrated with about i/IO this dose in tumor cells and embryonic tissues as well as in bone marrow and the gonads. Replication, transcription and translation are drastically changed in m a n y mammalian cells after the administration of cyclophosphamide at 50 mg/kg, and one is no longer dealing with toxic effects which are predominantly mutagenic. A similar situation exists with m a n y other potentially mutagenic drugs. It is a pharmacological fact that with increasing doses more effects of a drug
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manifest themselves. This problem is especially pertinent in a test for mutagenesis because, in the dose range which is sublethal for the cells to be tested, one is dealing with severely altered cells whose metabolism m a y be completely upset. This might influence the outcome of a possible mutagenic effect either in a positive or in a negative way but certainly would not allow an extrapolation to a lower dose range. Furthermore, if it is true that interference with the replication processes, protein synthesis or cell division m a y lead to chromosomal aberrations, then such an effect m a y occur in an "unspecific" way under all conditions that damage cell metabolism extensively, but subsequently allow cells to survive. Similar effects of "unspecific" toxic lesions are also well known in teratology. Since m a n y of such "unspecific" toxic effects m a y be based on enzyme inhibitions they m a y also be without significance at a lower dose level. But we are not basically interested in a mutagenic evaluation of toxic effects which nearly kill a mammalian organism or kill a significant proportion of its cells. For a valid toxicological evaluation, therefore, mammalian test systems with a higher sensitivity are badly needed. SOME S P E C I A L P R O B L E M S C O N N E C T E D W I T H M U T A G E N I C - T O X I C O L O G I C A L E V A L U A T I O N S
In addition to the problems mentioned, a number of additional questions arise which have to be answered to allow conclusions regarding the possible hazard for man to be based on solid scientific ground. Point mutations and the size of the "active" genome For a quantitative toxicological evaluation of mutagenic events occurring at a mammalian genome, some judgement of the degree of alteration present in the genetic material is inevitable. Here again, basic research is almost completely lacking. In contrast with a prokaryotic genome, the chromosomal material of a mammalian cell does not consist, even in the haploid stage, of unique DNA copies exclusively. The two following points are of special significance in this respect. (z) The discussion during this symposium cleally indicated that, for manlmalian cells, experimental evidence is lacking so far on whether the chromosomal material is attacked randomly, for example by an alkylating agent, or whether certain regions, for example "euchromatin", are primarily altered. This unsettled question is closely connected with another ploblem. (2) W h a t is the percentage of "sensitive" regions within a mammalian genome whose alteration is critically connected with the mutagenic hazard for the organism ? Studies performed in our laboratory with embryonic tissues indicated that, with cyclophosphamide, alkylation rates of up to i mole of alkylating agent per lO 5 to lO 6 moles DNA nucleotides, or an average alkylating rate of lO s to lO 4 alkyiations per cell, permit normal embryonic development if the alkylating agent is applied at the stage of organogenesis l& Therefore it can be assumed either that the maiority of the alkylations, under such experimental conditions, do not affect the gene expression in a way incompatible with normal cell functions and the ability of the cells to differentiate in a typical way, or that extensive repair processes or similar events are also operative in a mammalian cell. Our ignorance of the composition, and the regulation of the gene expiession, of a mammalian nuclear genome is extensive and m a y remain so for years.
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Extrachromosomal mutations Mutagenicity research so far has concentrated exclusively on possible hazards resulting from interference with the nuclear genome. But the existence of extlanuclear genetic material is now well established although its function during normal and abnormal cell development is still poorly understood 1. But if one believes in a mutagenic origin of certain pathological conditions, including carcinogenesis, there is no sensible reason to ignore this extrachromosomal genetic material as a target of mutagenic events. If a mutation within mitochondrial DNA were of significance in mutagenicity research the possible hazards involved m a y be much greater than with an average mutational event hitting nuclear DNA. These possible consequences have been discussed extensively elsewhere 1 and a mathematical evaluation of a mutagenic event linked to mitochondrial DNA is given. None of the systems used tor mutagenicity testing today would detect such extrachromosomal mutations which might turn out to be more significant in toxicology than mutations occurring in the nuclear material. Drug combi,~ations If mutations within a genome become irreversibly fixed in the genetic material, mutagenic events hitting the same cell m a y add up even over very long times. If this is so, in a toxicological evaluation the possible hazard of the combination of several mutagenic drugs has to be considered since, owing to environmental conditions, an individual is very rarely exposed to one drug only. The effect of a substance repeatedly acting on an organism must be investigated in detail. Examples of such possibilities are well known in toxicology, e.g. the effects of some carcinogens. But drug combinations m a y not only be of importance if both drugs represent mutagens, since it is also well known that cytostatic agents especially can potentiate the actions of each other. Since m a n y drugs are known to interfere with the metabolism of other drugs--either enhancing or inhibiting drug metabolism or elimination--the evaluation of such combinations has also to be taken into account during mutagenicity testing. So far almost nothing has been done in this respect. At the beginning of this discussion I mentioned that the present prospects tor evaluating the possible hazards of a mutagen for man are rather pessimistic. In this respect mutagenicity testing is still in the worst situation of all toxicological evaluations. Although there are several problems left in connection with the ability to detect carcinogenic changes, and we are surely not yet in a situation to exclude all teratogenic-toxicological hazards, good progress has been made in both these fields in recent years. It is m y feeling that, in the field of detecting possible mutagenic hazards, m a n y of the basic toxicological problems have not even been recognized or taken into account by the majority ot the investigators. Therefore, this confeIence m a y have been helpful in clearing up the present situation and enhancing recognition of the problems that have to be faced. CONCLUDING REMARKS
(I) Mutagenic-toxic events acting upon a mammalian organism are governed by the same pharmacokinetic parameters as are all toxic actions. It depends on these parameters whether sufficient active drug to induce a mutagenic effect accumulates at the target.
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(2) For an evaluation of the dose-response relationship typical for a given mutagen, and especially an extrapolation to much lower doses than those used in the experimental set-up, as well as a prediction of the possible risk for man, the mode of action of the mutagen has to be known. A quite different dose-response relationship is expected for a mutagen interfering with the enzymatic reactions involved in replication or cell division, compared with those mutagens directly interfering with the genetic material. (3) Up to now all test systems (for mutagenic events) using the mammalian genome as an indicator, often respond only to extremely high doses of the mutagen which simultaneously cause a variety of other toxic events at the mammalian cells. This may not be too critical in the testing of drugs to be used for therapeutic reasons but certainly is a major drawback for the evaluation of compounds present in the environment in trace amounts and to which man is exposed for prolonged periods. Therefore the present approaches are highly unsatisfactory and greatly limit the applicability of mammalian mutagenic test systems for evaluation of mutagenic hazards possibly endangering man. (4) No reliable and sensitive mammalian screening system is available for detecting point mutations or similar events, and for evaluating the dose-response relationships typical of such events. Because of the extremely large number of animals needed to detect only a few mutations the "specific locus test" also cannot fill this gap. (5) Bacterial test systems are only of limited value for toxicological evaluations of potentially mutagenic drugs because only some mutagenic effects can be tested, and furthermore the special mammalian metabolism is not taken into account. For obvious toxicological reasons the supplementation of the bacterial test system with liver microsomes adds only little to the solution of the problem of specificity in a routine assay of substances of unknown metabolism. (6) Results obtained with some non-mammalian animal test systems, such as those using Drosophila, may often show a remarkable degree of similarity to results obtained with mammalian systems. Tests using Drosophila may be useful for prescreening. Of course, no direct conclusions can be drawn regarding to potential hazards for man from such data only. (7) Mutations occurring in extrachromosomal DNA would not be detected with any of the methods used. REFERENCES I BASS, R., F. BECK, K.ENGELS, H . - J . MERKER, D. NEUBERT AND B. RANDHAHN (Eds,), Metabolic Pathways in Mammalian Embryos during Organogenesis and its Modification by Drugs, Free U n i v e r s i t y Press, Berlin, 197 ° . 2 ENGELS, K., U n t e r s u c h u n g e n tiber m i t o e h o n d r i a l e D N A - P o l y m e r a s e u n d fiber W i r k u n g e n v o n C y c l o p h o s p h a m i d , Inaugural-Dissertation, Fachbereich Chemie, Freie Universittit Berlin, 1972. 3 GILLETTE, J. I~., M e t a b o l i s m of d r u g s a n d o t h e r foreign c o m p o u n d s b y e n z y m a t i c m e c h a n i s m s , Fortschr. Arzneimittel-Forsch., 6 (1963) 13-73. 4 MANTEL,N., T h e c o n c e p t of t h r e s h o l d in carcinogenesis, Clin. Pharmacol. Therap., 4 (1963) lO4. 5 NEUBERT, D., Die Toxihologischen Voraussetzungen fi2r die Klinische Anwendung einer Neuen Substanz, S y m p o s i u m B u n d e s g e s u n d h e i t s a m t der B R D , 19746 NEUBERT, D., AND H. HERKEN, W i r k u n g s s t e i g e r u n g v o n S c h l a f m i t t e l n d u r c h d e n P h e n y l diallylessigsAureester des Di~kthylaminoi~thanols, Arch. Exptl. Pathol. Pharmakol., 225 (I955) 453-462. 7 NEUBERT, D., Wirkungsweise yon Antimetaboliten, " P a r a c e l s u s - B e i h e f t e " , Vortritge des 2o. fi,rztetreffens, 1968, pp. 13-3o.
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8 NEUBERT, D., R. BASS AND H.-J. MERKER, Occurrence and possible functions of mitochondrial DNA in animal development, in R. WEBER (Ed.), The Biochemistry of Animal Development, Vol. 3, Academic Press, New York, 1974. 9 PORTIG, J., P. KRAUS, S. SODOMANNAND G. •OACK, Biodegradation of alpha-hexachlorocyclohexane, I. Glutathione-dependent conversion to a hydrophilic metabolite by rat liver cytosol, Arch. Pharmacol., 279 (1973) 185-198. io YON LEDEBUR, M., AND W. SCHMID, The micronucleus test. Methodological aspects, Mutation Res., 19 (1973) IO9-II7.
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T H E TOXICOLOGICAL E V A L U A T I O N OF MUTAGENIC EVENTS
DIETHER
NEUBERT Pharmakologisches Institut der Freien Universitiit Berlin, Embryonal-Pharmahologie, x Berlin 33, Thielallee 69/73 (Germany)
(Received April 4th, 1974) (Revision received J u l y 8th, 1974)
SUMMARY
At present no mammalian test system which meets the toxicological requirements is available for routine testing of mutagenicity. Therefore, emphasis should be laid primarily on basic research in this area and not on large-scale screening of possible mutagens with methods known to be inadequate in m a n y respects, if mutagenicity is a maior hazard to man, a view certainly not shared by all toxicologists. Furthermore, if carcinogenicity is based on a mutagenic event occurring in somatic cells, the well established tests for carcinogenicity would provide a better way for evaluating irreversible somatic mutations than the tests now suggested for mutagenicity testing. In the present situation a drastic reduction of the noxes men are exposed to would be the most reliable means of preventing a toxicological disaster. We are still in the situation of continuously performing "mass human experiments" and detecting hazards only after considerable harm has been done. Consequently, the goal must be neither to expose a considerable proportion of our population to environmental hazards nor to give drugs to thousands or even millions of healthy people for any reasons whatsoever, unless test systems are available which would allow effective prevention of disaster.
INTRODUCTION
The evaluation of mutagenic events taking place in a mammalian organism is basically a toxicological problem. This is not only so because pharmacology and toxicology are the medical fields concerned with the interactions of drugs and a mammalian organism*, but more so because mutagenicity in mammals is governed by basic pharmacological parameters (Fig. I). However, toxicological considerations connected with mutagenicity are complicated by a number of additional factors as they are in a number of other special toxic events such as carcinogenicity or teratogenicity. As an applied science, pharmacology and toxicology are basically anthro* A d r u g is defined as a n y foreign c o m p o u n d able to affect t h e m a m m a l i a n cell, or a physiological s u b s t a n c e u s e d in u n p h y s i o l o g i c a l l y h i g h c o n c e n t r a t i o n s .
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pocentric. Therefore I should like to concentrate on problems primarily connected with the detection of possible mutagenic health hazards to man. There is no doubt that, owing to our limited knowledge in this comparatively new field, conclusions concerning the prediction and detection of such health hazards will be rather pessimistic. Since the toxicological point of view has so far not been adequately considered by many investigators I should like to survey some of the basic toxicological principles that govern mutagenic eventsin mammals. Many of these principles are well established and there is no need to "rediscover" them. Two aspects are especially important from the toxicological point of view: (z) problems of extrapolating experimental findings in laboratory animals to man; and, especially, (2) the dose-response relationship to be expected in mutagenicity. In addition to these, some special problems connected with mutagenic-toxicological evaluations will be discussed. PROBLEMS OF EXTRAPOLATING EXPERIMENTAL FINDINGS IN LABORATORY ANIMALS TO MAN
The pharmacological effect of a drug is governed by a number of principles. These often have to be evaluated and investigated separately since the ideal conditions that would allow a direct extrapolation to man cannot be obtained. These pharmacological parameters (Fig. I) include pharmacokinetics, drug metabolism, species differences, and the problem of measuring drug concentrations at the target (or "receptor"). Principal difficulties arising during a toxicological evaluation are discussed elsewhere and pertinent references are given 5.
Parameters that govern the mutagenic-toxic effect Fig. I gives the basic parameters that influence the effect of a mutagenic drug in mammals. (a) Pharmacokineties. The serum concentration of the drug, and thereby tissue concentrations, critically depend on parameters that include rates of absorption,
/ PHARMACOKINETICS Resorption Distribution Elimination
A
DRUG META.BOL!SM _
outside of target cell
B
PERMEABILITY
C
DRUG METABOLISM within target cell
I MOLECULAR] MUTAGENIC EVENT
D
CONCENTRATION at target
E
I REPA,R etc. Fig. i. Factors that govern the outcome of a mutagenic-toxic event in mammals.
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d i s t r i b u t i o n , m e t a b o l i s m a n d e l i m i n a t i o n ; factors t h a t m a y be described as p h a r m a c o kinetics. Unless a s u b s t a n c e is i n t r o d u c e d into a m a m m a l i a n o r g a n i s m c o n t i n u o u s l y the effective c o n c e n t r a t i o n of the active d r u g species changes w i t h time in a t y p i c a l w a y (Fig. 2). The fact t h a t high concentrations of the d r u g to be t e s t e d m a y be present for only a s h o r t t i m e has i m p o r t a n t consequences in tests such as the " h o s t - m e d i a t e d assay". Since considerable species differences in t h e p h a r m a c o k i n e t i c s of a d r u g are a n t i c i p a t e d , the e x a c t p h a r m a c o k i n e t i c s m u s t be known for the e x p e r i m e n t a l species used as well as for h u m a n s if e x t r a p o l a t i o n of toxicological d a t a to the conditions possibly existing in m a n is the goal. F u r t h e r m o r e , the c o n c e n t r a t i o n reached b y the d r u g in the m a m m a l i a n organism m a y be highly d e p e n d e n t on the m o d e ot application. Because the rates of a b s o r p t i o n v a r y , a drug has to be a d m i n i s t e r e d b y the r o u t e most likely to be used in humans. F o r e x a m p l e it seems unjustified to test a herbicide exclusively as i.v. or s.c. injections. (b) D r u g metabolism. D r u g m e t a b o l i s m is a n o t h e r i m p o r t a n t factor for the m a n i f e s t a t i o n of a possible m u t a g e n i c effect. Since a d r u g can be i n a c t i v a t e d a n d a c t i v a t e d b y m a m m a l i a n m e t a b o l i s m this factor could be " n e g a t i v e " or " p o s i t i v e " for t h e m u t a g e n i c event. Some of the aspects of drug m e t a b o l i s m have been s u r v e y e d b y Dr. Uhleke d u r i n g this conference. There are n u m e r o u s factors r e l e v a n t to the p r o b l e m of t e s t i n g for m u t a g e n i c i t y in a m a m m a l i a n system. A r a t h e r c o m p l e x situation m a y arise if only a few of these factors are changed b y the e x p e r i m e n t a l conditions. A n e x a m p l e is given in Fig. 3 for an a l k y l a t i n g agent (cyclophosphamide) well k n o w n for being " a c t i v a t e d " in the m a m m a l i a n organism. W h e n drug m e t a b o l i s m is modified b y the s i m u l t a n e o u s a d m i n i s t r a t i o n of an i n h i b i t o r (CFT I 2 o I - - d i e t h y l a
b
serum concentration 14-
experimental period c d • f ~ t
¢
f
t
g
t
I _h_
A54o/mlserum (16-
121080.3642-
/ jj time
Fig. 2. Schematic example of a typical change in the serum concentration of a drug and a metabolite to which the drug is converted in vivo. On the top of the figure examples of experimental periods are given that might be chosen to evaluate a mutagenic-toxic effect. (a), Almost no drug effect is measured; (b)-(c), predominantly evaluates the effect produced by the original drug while (d)-(h), the effect of the metabolite predominates. The effect of the original drug plus that possibly produced by the metabolite could only be evaluated if the experimental period is extended over (b)-(h) ! Fig. 3- Serum concentration in rats after a single dose of Endoxan. (Data according to BAss et al. z and ENGELS2.)Rats were injected with i oo mg/kg Endoxan i.p. at o-time. The concentration of the activated form of Endoxan was measured with the NBP-test (data given for 7 ml of the organic solvent). Inhibition of the drug metabolizing enzyme system in liver microsomes by CFT 12Ol drastically changes the pharmacokinetics of the activated form of Endoxan and produced a less intense but longer lasting effect. CFT 12Ol was given orally at 75 mg/kg i h before Endoxan and 5° mg/kg I, 3 and 5 h after the alkylating drug.
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aminoethanol ester of phenyldiallyl acetic acid) which is known to interfere with the drug-oxidizing enzyme system in liver microsomes% the peak concentration of the activated drug species is decreased in the serum--as m a y be expected. But the pharmacokinetics are changed drastically and the activated form oi Endoxan can be detected in the serum for a considerably longer time interval in the presence of the inhibitor of drug metabolism. This result m a y be explained because the non-activated form of the drug is available to the organism for a longer period. A similar situation m a y be expected to occur in a species of experimental animals that are able to "activate" Endoxan only at a slow rate. So a high rate of "activation" in the liver is not necessarily correlated with a high concentration of activated compound reached in another cell type of the organism for a long period but m a y lead to the opposite situation. Therefore, it is helpful to differentiate between drug metabolism in cells other than the target cell (Fig. IB) and drug metabolism within a target cell (Fig. ID) because these two processes are often not identical. The serum concentration of the active drug species, and thereby the concentration of the mutagen offered to the target cell, will be a function of the predominant metabolizing cell type. Very otten the hepatocyte is believed to represent the cell type most important in drug metabolism, and within that cell the microsomal drugoxidizing system, characterized especially by BRODIE and his coworkersL is of primary significance. But this view is certainly an over-simplification since m a n y other cell types in a mammalian organism metabolize drugs, and within a cell m a n y other enzymatic systems exist that are capable of metabolizing drugs apart from the microsomal "drug-oxidizing system". For instance in "inactivating" reactions several esterases are not localized at the microsomes and e.g. dechlorinating reactions take place in the eytosoP. On the other hand, most of the antimetabolites (purine or pyrimidine antagonists)--some of which are well known as mutagens (6-mercaptopurine, bromodeoxyuridine etc.)--are converted to the biologically "active" form outside the microsome. Furthermore, it is well known that m a n y enzyme systems have different intracellular locations in different animal species. (c) Species differences. Drug metabolism m a y vary drastically for a given drug in different species. This and other factors can result in considerable species differences in the sensitivity towards drugs. An example of such a species difference and the consequences for extrapolation of data from experimental animals to man is given in Table I. Even from the very few data compiled in this table two basic facts are obvious: (z) species differences can easily amount to one or two orders of magnitude; and (2) no correlation exists in the comparative sensitivity for different drugs in a given animal species. As a consequence of such results, which are well established in pharmacology and toxicology, toxicological evaluations have to be performed with at least two different mammalian species. An exception is only justified if it is known that drug metabolism is identical in the experimental animals chosen and in man. This is seldom true. Therefore, tests for possible mutagenic hazards, even in mammals, are of limited value if only one species is used. (d) Permeability. Since m a n y drugs or their metabolites do not readily penetrate membrane systems the serum concentration m a y not give a true picture of the concentration of the active drug species at the target. A complex situation arises if the drug itself or some of its metabolites are partially excluded from entering a target cell. Conclusions based on the drug metabolism occurring within a hepatocyte or in one
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I
DIFFERENCES IN THE SENSITIVITY OF SOME SPECIES TO CYTOSTATICS
LDlo (mg/m ~) A methopterin F UdR Mouse Hamster Rat Dog Monkey
Io lO3 3 2 35
Simplified from Freireich
500 17o 47 ° 760 690
et at., 1966.
compartment of a cell are therefore not representative for the different cell types of an organism. (e) Drug metabolism within the target cell. In principle two different possibilities can be visualized: (z) a drug or its active metabolite is available to the target cell and penetrates the cell membrane at a rate sufficient to create an effective drug concentration at the target within the cell; or (2) the active drug species has to be generated within the target cell. The latter m a y be so because the active metabolite cannot penetrate the cell membrane or because the active metabolite has such a short half-life that only the formation within a target cell can lead to an effective concentration. Examples of the latter possibility are given in Dr. l~hleke's paper at this conference. It is noteworthy that most antimetabolites are converted to the active drug species within the target cell (cf. ref. 7). The drug-oxidizing system in microsomes is only occasionally involved in such conversions. (f) Effective drug concentration within the target cell. The effective drug concentration within the target cell therefore depends on a considerable number of variables some of which have already been mentioned. Additional factors certainly exist. The mutagen could be rapidly inactivated within the target cell or "unspecifically" bound to cell components other than the target. Since the extent of the molecular mutagenic event critically depends on the concentration of the mutagen at the target, all these parameters must be known and considered in order to make a valid analysis. Even then the final result m a y be altered by subsequent events such as repair processes (Fig. I F).
Toxicological significance of different mutagenicity test systems With the principal parameters depicted in Fig. I in mind, we can try to evaluate some of the mutagenicity test systems presently used. (a) Tests with microorganisms. All test systems using the genome of microorganisms as an indicator for a drug-induced mutagenicity suffer from the disadvantage that only part E in Fig. I can be evaluated. No evidence can be obtained that the active drug species possibly present at the target of a mammalian organism reaches the indicator system in the microorganism. Furthermore, considerable differences exist between a bacterial genome and animal chromosomes as well as in the enzymes involved in replication and cell division. Supplementing a test system based on the genome of microorganisms with enzyme systems isolated from certain mammalian cells again can only be of limited value because the given drug would have to be metabolized iust by this enzyme system. It has been pointed out before that drug metabolism is not confined to one
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enzyme system. From this point of view it is hard to see why a microsomal system is used to supplement the test with microorganisms instead of homogenates--or even intact animal cells. Caution is necessary to avoid overestimating the value of such a combined system since most of the drugs studied so far were known to be converted and activated by this microsomal enzymatic system so that it is possible to predict the results without actually doing the experiments. It is hard to imagine the value of such a system in a situation where the metabolism of a given drug is unknown. (b) "Host-mediated" assay. In the "host-mediated assay" the genome of a microorganism is used as an indicator for mutagenic events and a mammalian species provides the environment for the drug action. In a number of variations of this test the microorganisms are either injected into the peritoneal cavity, injected into a special organ (cf. Dr. Fahrig's lecture at this conference*), or injected intravenously. Good examples have been presented that the concentration in the peritoneal fluid of tile investigated drug and its metabolites might not be sufficient to guarantee a successful evaluation of the results. Surprisingly, only few people have bothered to measure the drug concentrations reached in the peritoneal fluid! As shown by Dr. Malling during this conference, better results are obtained when the microorganisms are injected intravenously. But according to the criteria mentioned before, this approach also cannot be satisfactory for two principal reasons. (~) If the microorganisms remain in the blood stream, essentially the effect of the drug concentration in the serum is tested. Since, in general, the period during which the microorganisms are exposed to the drug and its metabolites within the serum is comparatively short, only a small section of the pharmacokinetic profile can be checked (cf. Fig. 2). This disadvantage can only be overcome if the time of exposure of the microorganisms can be lengthened to an experimental period which covers most of the pharmacokinetic events. So far, this has not been accomplished because the microorganisms leave the blood stream rapidly after the injection. (2) Another objection results from tile assumption that most of the microorganisms, if they are introduced into a cell of the host, might be expected to be incorporated into cells of the reticuloendothelial system. But again these are not the cells in which typical drug metabolism can be expected nor the cells most interesting for mutagenicity testing. On the contrary, it m a y be assumed that the concentration of metabolites in these cells is much lower when compared with that present in hepatocytes or other cells actively engaged in drug metabolism. Again basic research has not been performed which--for example with electron microscopic techniques--would unambiguously show the exact location and the fate ot the microorganisms injected into the host. Furthermore, the "host-mediated" assay basically is a test with the genome of a microorganism and cannot be considered a truly mammalian test system. Very often a "host-mediated" assay is performed under conditions which cast serious doubt on the significance of tile results obtained. Not only is the mammalian organism infected with a number of microorganisms able to cause a lethal sepsis but also doses of the drug to be tested are used that would kill the animal over a longer period. Can metabolism be considered "normal" under such conditions ? An incubation of microorganisms with sera from different experimental animals treated with the compounds to be studied would--although again insufficient when used as the only * N o t published in this issue of Mutation Research, cf. Vol. 26 (1974) 29-36.
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test--provide similar advantages and also provide the possibility of accounting for the drug metabolism occurring in different animal species. Such a test could also conveniently be performed with the sera of patients treated with the drug to be tested for therapeutic reasons. But the "host-mediated" assay basically suffers from the disadvantage that it only covers the reactions A, B and in part E of our scheme (Fig. I). (c) Non-mammalian animal systems. After the recognition that, owing to a lack of specificity, prokaryotic test systems are of limited value for a toxicological evaluation of possible mutagenic hazards in man, the oldest animal system used in studies on mutagenicity--Drosophila--is apparently having a renaissance. Although nobody would believe the metabolism of a fly to be identical with that of a mammalian organism, a certain degree of metabolic similarity and a fairly high degree of sensitivity could suggest Drosophila as a useful prescreening system before one proceeds to a more specific but certainly much less sensitive mammalian test for mutagenicity. The impressive state of knowledge on the theoretical background of the mutagenic events occurring in this organism--as depicted, for example, by Dr. Wtirgler during this conference--greatly facilitates the interpretation of experimental findings after drug administration. This is in striking contrast with a mammalian dominant lethal test which Dr. Auerbach during this conference referred to as a "mutagenic black box". (d) Mammalian systems in vivo. Mammalian systems in vivo should offer the experimental approach from which the greatest information regarding a toxicological analysis m a y be expected. This would especially be true if a suitable test could be performed in humans exposed, for various reasons, to the drugs to be studied. But apparently no test system has been established up to now which allows an adequate monitoring of all the mutagenic hazards to which a human organism m a y be exposed. Therefore animal systems have to be used instead. Although the dominant lethal test is the one most widely used in this respect it also suffers from a number ot disadvantages. Often a positive result is reached only if the drug to be tested is administered in exceedingly high doses. Furthermore, the mutagenic events tested are apparently predominantly chromosomal aberrations and there is no indication that for example point mutations m a y be detected. On the other hand, the multiple loci test can scarcely be used on a routine basis for the screening of drugs because of the huge number of animals necessary for one test. For the purpose of detecting somatic mutations--chromosomal aberrations-the micronucleus test proposed by SCHMID5 m a y be especially suitable since it makes use of the drug metabolism in the intact animal, and rapidly proliferating cell types of bone marrow are used. (e) Mammalian cells in vitro. Extensive and rather informative data on mammalian test systems were presented during this conterence (see papers of Drs. J. Simons and D. Wild). It seems that, in the majority of systems investigated, concentrations of the drug have to be used which induce a variety of toxic effects during mutagenicity tests including cell deaths in a significant percentage. Although these systems have the advantage of using a mammalian genome as an indicator, the methods again m a y suffer from the inability of the given cell type to activate a number of drugs in a way similar to that occurring in a whole mammalian organism. Therefore, at present it seems only feasible to combine a number of tests in order to get the required information. It also appears necessary to combine the
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"genetic" studies with purely pharmacological studies on the pharmacokinetics, and especially on the problem of what concentrations of the active drug species in question are reached at the target. The studies by Dr. Generoso and his coworkers, as presented at this meeting, in which data on the mutagenic effect were combined with information on the degree of alkylation reached in the target cell at different dose levels, certainly point in this direction and should be performed much more extensively. D O S E - - R E S P O N S E R E L A T I O N S H I P S TO BE E X P E C T E D
IN M U T A G E N I C I T Y
For an evaluation of the possible hazards to be expected for man in ~11 toxicological studies, knowledge of the dose-response relationship is of utmost importance. Mutagenicity is certainly not an exception in this respect! Several investigators have attempted to extrapolate a linear dose-response relationship to doses several orders of magnitude lower, arguing that mutagenicity is a "single-hit event". With the information now available such an attempt is not justified for a number of reasons, as discussed below.
Different mutagenic events A common event leading to a mutagenic effect does not exist. But several mechanisms can give rise to a change of the genetic material in a mutagenic-toxic effect. In principle two different interferences with the genetic material m a y be differentiated with consequences also on the dose-response relationship to be expected: (a) a direct attack of a drug on the DNA. This would lead to a sequence alteration or to a point mutation or comparable mutagenic defects. (b) An indirect interference with the genetic material. This m a y occur by an alteration of replicating enzymes, specific proteins involved in cell division or reactions involved in protein synthesis or transcription not originating from an altered template. Furthermore, the final outcome of the mutagenic effect m a y be greatly modified by subsequent repair processes or similar events. (a) A direct alteration of the genome by the mutagen. The mutagenic events leading to a direct alteration of the DNA (point mutations) might be expected to proceed linearly over an extended dose range in certain circumstances. But again, the purely pharmacological parameters governing the concentration of the active drug species at the target are not likely to represent linear processes in an extremely low dose range. Absorption, elimination, and "unspecific" adsorption might occur at a different rate with very low doses of a drug. Only few pertinent pharmacological data exist since it has seldom been necessary to follow a pharmacological or toxic effect over a dose range of several orders of magnitude. Therefore much more experimental data are needed. But as general prediction one might assume that it is unlikely that the concentration at a target is linear with the dose applied if minute amounts of a drug are given to a mammalian organism (cf. discussion on "threshold" in carcinogenicity~). Since qualitative statements are useless in toxicology, it is certainly up to the scientist involved in mutagenic-toxicological research to provide pertinent quantitative data for the dose ranges of interest if an evaluation of the possible hazards for man is anticipated. (b) An indirect alteration of the gene expression by the mutagen. A different situation might arise if a drug acts indirectly on the genome. It is noteworthy that several
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substances reported to cause mutagenic effects, and especially chromosomal aberrations, belong to the class of antimetabolites or cytostatic agents that interfere with the formation of nucleic acid precursors or with the replicating process and cell division. If one accepts interference with intermediary metabolism as the sole mechanism of action of these drugs then the mutagenie events produced by these agents must be caused by the inhibition of enzymes or a stoichiometric combination with certain cell components, such as spindle proteins. If this holds true, a different dose-response relationship m a y be expected for mutagenic events triggered by such agents as compared with dlugs directly interfering with the genetic material, for example through alkylations. The well-established criteria of enzyme inhibitions would govern such toxic drug effects. But this would exclude dose-response relationships of the final mutagenic effect, linear over several orders of magnitude of the drug concentrations, since the inhibition of an enzymatic reaction would be without biological interest in the majority of the enzymatic reactions if the drug concentration was to drop below I/IO of the K~. No deleterious toxic effects could be expected from such drug actions at extremely low doses. This discussion shows that much more basic research is necessary in mammalian systems to allow a clear-cut evaluation of experimental data on the hazards to be expected for man exposed to certain concentrations of potentially mutagenic substances.
"Threshold" doses of mutagenic drugs Although the term "threshold dose" or "zero effect dose" is frequently used, such a dose limit does not actually exist. People therefore put these terms in parentheses and define the limits for which these terms should stand. Therefore it seems to be more appropriate to replace these terms by expressions like "predictable risk" or "acceptable dose" which mean basically the same without implying that a drug is ineffective beyond a certain dose limit. One would then have clearly to define "acceptable", "predictable" and "risk". Without proceeding too far with this discussion I personally feel that an agreement should have been reached on these terms before an elaborate screening program was begun. Otherwise the situation will arise--or has already arisen-- in which nobody really knows how to interpret the accumulating data.
Problems connected with the administration of extremely high doses of a potential mutagen The evaluation of the dose response relationship is often difficult, especially when mammalian test systems are used, because extremely high doses of the drugs have to be administered to detect a mutagenic effect. We have seen that cyclophosphamide is necessary at about 50 mg/kg to produce a significant mutagenic effect in the dominant lethal test. Such a dose results in severe changes of cell metabolism in m a n y tissues of a mammalian organism, and clearly detectable effects of this drug can be demonstrated with about i/IO this dose in tumor cells and embryonic tissues as well as in bone marrow and the gonads. Replication, transcription and translation are drastically changed in m a n y mammalian cells after the administration of cyclophosphamide at 50 mg/kg, and one is no longer dealing with toxic effects which are predominantly mutagenic. A similar situation exists with m a n y other potentially mutagenic drugs. It is a pharmacological fact that with increasing doses more effects of a drug
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manifest themselves. This problem is especially pertinent in a test for mutagenesis because, in the dose range which is sublethal for the cells to be tested, one is dealing with severely altered cells whose metabolism m a y be completely upset. This might influence the outcome of a possible mutagenic effect either in a positive or in a negative way but certainly would not allow an extrapolation to a lower dose range. Furthermore, if it is true that interference with the replication processes, protein synthesis or cell division m a y lead to chromosomal aberrations, then such an effect m a y occur in an "unspecific" way under all conditions that damage cell metabolism extensively, but subsequently allow cells to survive. Similar effects of "unspecific" toxic lesions are also well known in teratology. Since m a n y of such "unspecific" toxic effects m a y be based on enzyme inhibitions they m a y also be without significance at a lower dose level. But we are not basically interested in a mutagenic evaluation of toxic effects which nearly kill a mammalian organism or kill a significant proportion of its cells. For a valid toxicological evaluation, therefore, mammalian test systems with a higher sensitivity are badly needed. SOME S P E C I A L P R O B L E M S C O N N E C T E D W I T H M U T A G E N I C - T O X I C O L O G I C A L E V A L U A T I O N S
In addition to the problems mentioned, a number of additional questions arise which have to be answered to allow conclusions regarding the possible hazard for man to be based on solid scientific ground. Point mutations and the size of the "active" genome For a quantitative toxicological evaluation of mutagenic events occurring at a mammalian genome, some judgement of the degree of alteration present in the genetic material is inevitable. Here again, basic research is almost completely lacking. In contrast with a prokaryotic genome, the chromosomal material of a mammalian cell does not consist, even in the haploid stage, of unique DNA copies exclusively. The two following points are of special significance in this respect. (z) The discussion during this symposium cleally indicated that, for manlmalian cells, experimental evidence is lacking so far on whether the chromosomal material is attacked randomly, for example by an alkylating agent, or whether certain regions, for example "euchromatin", are primarily altered. This unsettled question is closely connected with another ploblem. (2) W h a t is the percentage of "sensitive" regions within a mammalian genome whose alteration is critically connected with the mutagenic hazard for the organism ? Studies performed in our laboratory with embryonic tissues indicated that, with cyclophosphamide, alkylation rates of up to i mole of alkylating agent per lO 5 to lO 6 moles DNA nucleotides, or an average alkylating rate of lO s to lO 4 alkyiations per cell, permit normal embryonic development if the alkylating agent is applied at the stage of organogenesis l& Therefore it can be assumed either that the maiority of the alkylations, under such experimental conditions, do not affect the gene expression in a way incompatible with normal cell functions and the ability of the cells to differentiate in a typical way, or that extensive repair processes or similar events are also operative in a mammalian cell. Our ignorance of the composition, and the regulation of the gene expiession, of a mammalian nuclear genome is extensive and m a y remain so for years.
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Extrachromosomal mutations Mutagenicity research so far has concentrated exclusively on possible hazards resulting from interference with the nuclear genome. But the existence of extlanuclear genetic material is now well established although its function during normal and abnormal cell development is still poorly understood 1. But if one believes in a mutagenic origin of certain pathological conditions, including carcinogenesis, there is no sensible reason to ignore this extrachromosomal genetic material as a target of mutagenic events. If a mutation within mitochondrial DNA were of significance in mutagenicity research the possible hazards involved m a y be much greater than with an average mutational event hitting nuclear DNA. These possible consequences have been discussed extensively elsewhere 1 and a mathematical evaluation of a mutagenic event linked to mitochondrial DNA is given. None of the systems used tor mutagenicity testing today would detect such extrachromosomal mutations which might turn out to be more significant in toxicology than mutations occurring in the nuclear material. Drug combi,~ations If mutations within a genome become irreversibly fixed in the genetic material, mutagenic events hitting the same cell m a y add up even over very long times. If this is so, in a toxicological evaluation the possible hazard of the combination of several mutagenic drugs has to be considered since, owing to environmental conditions, an individual is very rarely exposed to one drug only. The effect of a substance repeatedly acting on an organism must be investigated in detail. Examples of such possibilities are well known in toxicology, e.g. the effects of some carcinogens. But drug combinations m a y not only be of importance if both drugs represent mutagens, since it is also well known that cytostatic agents especially can potentiate the actions of each other. Since m a n y drugs are known to interfere with the metabolism of other drugs--either enhancing or inhibiting drug metabolism or elimination--the evaluation of such combinations has also to be taken into account during mutagenicity testing. So far almost nothing has been done in this respect. At the beginning of this discussion I mentioned that the present prospects tor evaluating the possible hazards of a mutagen for man are rather pessimistic. In this respect mutagenicity testing is still in the worst situation of all toxicological evaluations. Although there are several problems left in connection with the ability to detect carcinogenic changes, and we are surely not yet in a situation to exclude all teratogenic-toxicological hazards, good progress has been made in both these fields in recent years. It is m y feeling that, in the field of detecting possible mutagenic hazards, m a n y of the basic toxicological problems have not even been recognized or taken into account by the majority ot the investigators. Therefore, this confeIence m a y have been helpful in clearing up the present situation and enhancing recognition of the problems that have to be faced. CONCLUDING REMARKS
(I) Mutagenic-toxic events acting upon a mammalian organism are governed by the same pharmacokinetic parameters as are all toxic actions. It depends on these parameters whether sufficient active drug to induce a mutagenic effect accumulates at the target.
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(2) For an evaluation of the dose-response relationship typical for a given mutagen, and especially an extrapolation to much lower doses than those used in the experimental set-up, as well as a prediction of the possible risk for man, the mode of action of the mutagen has to be known. A quite different dose-response relationship is expected for a mutagen interfering with the enzymatic reactions involved in replication or cell division, compared with those mutagens directly interfering with the genetic material. (3) Up to now all test systems (for mutagenic events) using the mammalian genome as an indicator, often respond only to extremely high doses of the mutagen which simultaneously cause a variety of other toxic events at the mammalian cells. This may not be too critical in the testing of drugs to be used for therapeutic reasons but certainly is a major drawback for the evaluation of compounds present in the environment in trace amounts and to which man is exposed for prolonged periods. Therefore the present approaches are highly unsatisfactory and greatly limit the applicability of mammalian mutagenic test systems for evaluation of mutagenic hazards possibly endangering man. (4) No reliable and sensitive mammalian screening system is available for detecting point mutations or similar events, and for evaluating the dose-response relationships typical of such events. Because of the extremely large number of animals needed to detect only a few mutations the "specific locus test" also cannot fill this gap. (5) Bacterial test systems are only of limited value for toxicological evaluations of potentially mutagenic drugs because only some mutagenic effects can be tested, and furthermore the special mammalian metabolism is not taken into account. For obvious toxicological reasons the supplementation of the bacterial test system with liver microsomes adds only little to the solution of the problem of specificity in a routine assay of substances of unknown metabolism. (6) Results obtained with some non-mammalian animal test systems, such as those using Drosophila, may often show a remarkable degree of similarity to results obtained with mammalian systems. Tests using Drosophila may be useful for prescreening. Of course, no direct conclusions can be drawn regarding to potential hazards for man from such data only. (7) Mutations occurring in extrachromosomal DNA would not be detected with any of the methods used. REFERENCES I BASS, R., F. BECK, K.ENGELS, H . - J . MERKER, D. NEUBERT AND B. RANDHAHN (Eds,), Metabolic Pathways in Mammalian Embryos during Organogenesis and its Modification by Drugs, Free U n i v e r s i t y Press, Berlin, 197 ° . 2 ENGELS, K., U n t e r s u c h u n g e n tiber m i t o e h o n d r i a l e D N A - P o l y m e r a s e u n d fiber W i r k u n g e n v o n C y c l o p h o s p h a m i d , Inaugural-Dissertation, Fachbereich Chemie, Freie Universittit Berlin, 1972. 3 GILLETTE, J. I~., M e t a b o l i s m of d r u g s a n d o t h e r foreign c o m p o u n d s b y e n z y m a t i c m e c h a n i s m s , Fortschr. Arzneimittel-Forsch., 6 (1963) 13-73. 4 MANTEL,N., T h e c o n c e p t of t h r e s h o l d in carcinogenesis, Clin. Pharmacol. Therap., 4 (1963) lO4. 5 NEUBERT, D., Die Toxihologischen Voraussetzungen fi2r die Klinische Anwendung einer Neuen Substanz, S y m p o s i u m B u n d e s g e s u n d h e i t s a m t der B R D , 19746 NEUBERT, D., AND H. HERKEN, W i r k u n g s s t e i g e r u n g v o n S c h l a f m i t t e l n d u r c h d e n P h e n y l diallylessigsAureester des Di~kthylaminoi~thanols, Arch. Exptl. Pathol. Pharmakol., 225 (I955) 453-462. 7 NEUBERT, D., Wirkungsweise yon Antimetaboliten, " P a r a c e l s u s - B e i h e f t e " , Vortritge des 2o. fi,rztetreffens, 1968, pp. 13-3o.
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8 NEUBERT, D., R. BASS AND H.-J. MERKER, Occurrence and possible functions of mitochondrial DNA in animal development, in R. WEBER (Ed.), The Biochemistry of Animal Development, Vol. 3, Academic Press, New York, 1974. 9 PORTIG, J., P. KRAUS, S. SODOMANNAND G. •OACK, Biodegradation of alpha-hexachlorocyclohexane, I. Glutathione-dependent conversion to a hydrophilic metabolite by rat liver cytosol, Arch. Pharmacol., 279 (1973) 185-198. io YON LEDEBUR, M., AND W. SCHMID, The micronucleus test. Methodological aspects, Mutation Res., 19 (1973) IO9-II7.