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Predictive value of mutagenicity tests in chemical carcinogenesis
177
Mutation Research, 38 (1976) 177--190 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
PREDICTIVE VALUE OF MUTAGENICITY TESTS IN CHEMICAL CARCINOGENESIS
H. B A R T S C H International Agency for Research on Cancer, Unit o f Chemical Carcinogenesis, 150 cours Albert Thomas, 69008 Lyon, France
{Received October 10th, 1975) {Accepted November 28th, (1975)
An essential role of environmental factors in the aetiology of human cancer has been shown by the unequivocal evidence of the chemical origin of occupational cancer, as in the cases of urinary bladder tumours in workers exposed to aromatic amines [29,13,12] lung cancers in workers exposed to bis(chloromethyl) ether [30] or angiosarcomas of the liver in workers exposed to vinyl chloride [31] ; the well
Italy, Oc-
178
[50,47,3,61] ; and many mutagens, whose carcinogenicity had not yet been investigated, have now been shown to be carcinogens. This convergence of mutagens and carcinogens, which has strengthened dramatically since 1960, has resulted from the discovery that many such chemicals require metabolic activation in order to show a biological activity. This metabolic activation is often associated with binding to nucleophilic sites in nucleic acids and proteins in the organism. The growing experimental evidence linking the carcinogenic activity of numerous chemicals to their capacity to be converted into electrophilic derivatives that may also exert a mutagenic effect, had led to the suggestion that an empirical relationship between chemical carcinogenesis and mutagenesis exists [48,60,5]. However, such a correlation has so far been limited to those changes of the genotype that appear as a consequence of structural or functional alterations of nucleic acids and thus are genetically transmissible. For that reason, only mutagenicity tests that are genetically and metabolically reasonably well defined and/or that provide data shown to be valid for the prediction of the carcinogenicity of chemicals are referred to here. Other test procedures that allow the detection of the whole spectrum of genetic damage induced by chemicals are by no means disqualified by this selection. Since the genetic material of all living organisms, except for some viruses, has been found to be DNA, the major critical target molecule for chemical mutagens has been defined as DNA. A great number of chemical mutagens and carcinogens react covalently with DNA and other cellular macromolecules [10]. In contrast, for carcinogenesis, no critical target molecule has been defined, although evidence for an association between carcinogenicity and modification of DNA is provided by the appearance in patients with Xeroderma pigmentosum of a higher rate of skin cancer due to sunlight and of their cells which lack the ability to repair ultra-violet or certain chemical damage to DNA [15,18,58, 59]. Despite the diversity in chemical structures of known carcinogens and mutagens, such as alkylating agents, N-nitrosamines and N-nitrosamides, naturally occurring compounds, aromatic hydrocarbons, etc., recognition of a c o m m o n element in chemical carcinogens and mutagens has rapidly progressed since it was understood that the majority of carcinogens and many mutagens need metabolic activation in the host for transformation to their so-called ultimate reactive forms [49]. Comparison of these ultimate reactive metabolites of carcinogens has revealed that the common denominator of these substances is their electrophilicity, i.e. they are compounds whose atoms have an electron deficiency that enables them to react with electron-rich sites in cellular nucleic acids and proteins causing mutagenic effects frequently paralleled by the onset of DNA repair processes. The specificity of certain chemical compounds to produce tumours only in certain organs or in certain animal species, is primarily determined by the available concentration of ultimate reacitve metabolites which is itself a consequence of a critical balance between activation and detoxication processes. In view of the properties of ultimate reactive forms of chemical carcinogens, and, in particular, the growing evidence for an empirical relationship between the carcinogenic and the mutagenic activity of chemicals, it is n o w possible to use this relationship in short-term tests for the detection of potential carcino-
179 gens. There are many systems available at present that involve different genetic indicators and metabolic activation systems for detecting mutagenic activity; they all, however, have individual advantages and limitations. Ideally, an appropriate mutagenicity test system would include the full metabolic competency of the intact organism, that is, humans. Thus, the conclusion has been reached that a battery of test systems is needed to detect the genetic hazards caused b y chemicals [24,55,60]. In evaluating these methods, the major question is which screening tests, singly or collectively, can serve as reliable indicators or predictors of the potential carcinogenic hazard of a chemical. This can only be achieved by testing a representative number of different classes of carcinogens and non-carcinogens. A valid test should be positive for compounds with known carcinogenic activities and negative for negative compounds, within the limits of the test. Extensive studies are being carried o u t in Japan and in the United States to evaluate short-term tests [53]. Results accumulated up to the present time [1,61,67 ] using a rat-liver microsome test in vitro with Salmonella typhimurium strains developed by Bruce Ames, have shown that a b o u t 80--90% of the carcinogens tested were also mutagens, while the number of false positives and false negatives was much lower, ranging from 10 to 15%. In tests used for preliminary screening, a small proportion of false positive and false negative results may be acceptable; but, for a final test, no false negative results can be accepted. At present, mutagenicity assays, especially with sub-mammalian indicators, can be used in the following ways. (a) To trace carcinogens and/or mutagens in the complex environment of man. (b) To pre-screen compounds for their mutagenic action per se or after metabolic activation, to select high-priority compounds for long-term carcinogenicity tests. Special attention should be given to chemicals for which a substantial human exposure is known to occur and to newly-developed drugs used over a period of years in children, young adults or pregnant women. (c) The systems in vitro allow investigation of the capability of human tissues to generate electrophilic intermediates from the c o m p o u n d under test. The usual type of metabolic study, e.g. studies on distribution or excretion of the drug, are of limited value if the chemical acts by a partial conversion to a mutagenic or carcinogenic intermediate that interacts with DNA and is not excreted as such. This technique may also aid in the selection for long-term carcinogenicity tesing of those animal species most similar to man. (d) Urinary metabolites can be tested for their mutagenic action or after deconjugation by enzymes and reactivation by liver fractions. These assays, recently developed by Ames [2,16], permit the detection of mutagens in rats treated with carcinogens and can be used for monitoring the exposure of human populations. As recently shown b y Legator [42], in patients treated with the antischistosomal agent Niridazole, mutagenic activity for S. typhimurium strains was demonstrable in the urine. The validity and/or the practical application of such microbial mutagenicity tests has also been demonstrated in the IARC laboratory with vinyl chloride, which has recently been recognized as a carcinogen in animals and in man [ 3 1 ] . The validity of the microbial test system using S. typhimurium strains to predict the possible carcinogenic action of structurally related chemicals such as
180 TABLE
I
COMPARATIVE
MUTAGENICITY
OF OLEFINIC
COMPOUNDS
D a t a c o l l a t e d f r o m B a r t s e h e t al. [ 9 ] a n d u n p u b l i s h e d Compound
his + r e v e r t a n t
work. Carcinogenicity
c o l o n i e s / p m o l / h /plate a
S. l y p h i m u r i u m
TA1530 Vinyl acetate Vinyl chloride Vinylidene chloride 2-Chloro- 1,3-butadiene
TAIO0
0 b
(0) c
0 b
(0) c
_
3.7 9.5 34.5
(1.2) (0.2) (12.5)
5.6 14.6 51.2
(1.5) (0.7) (9)
+ (+) ?
a From linear dose- and time-dependent r e s p o n s e c u r v e s in t h e p r e s e n c e rant, spontaneous mutations subtracted. b In the presence of co-factors (NADP*, glucose 6-phosphate). c In the absence of co-factors.
of mouse
liver 9 000 g superna-
vinylidene chloride (1,1-dichloroethylene) and 2-chloro-l,3-butadiene was verified [8]. 2-Chloro-l,3-butadiene has been used for the production of rubber, and vinylidene chloride is used as a co-polymer with vinyl chloride in the manufacture of plastics. 2-Chloro-l,3-butadiene has been reported to induce chromosomal aberrations in lymphocytes of peripheral blood from exposed workers [38], and in the USSR it has been associated with the induction of skin and lung tumours in humans [39,40]. Table I summarizes the relative mutagenic strengths of vinyl chloride, vinylidene chloride and 2-chloro-l,3-butadiene in the presence of mouse liver fractions either in the presence or in the absence of a NADPH-generating system. Vinyl acetate is included for comparison at the top of the table. The results were calculated from linear dose- or time-dependent response curves and are expressed as the number of histidine revertant colonies per pmol of substrate per hour of exposure per plate as measured with two different S. typhimurium strains, TA1530 and TA100. 2-Chloro-l,3-butadiene exhibited the highest mutagenic effect, followed by vinylidene chloride, for which Viola and Caputo (personal communication) have recently obtained evidence for its carcinogenicity in rats. These findings indicated that epidemiological monitoring of workers exposed to vinylidene chloride and 2-chloro-l,3-butadiene may be necessary, as well as measures to prevent, or greatly reduce, exposure of humans. No mutagenic effect was detectable with vinyl acetate. This c o m p o u n d was used by Maltoni and Lefemine [45] as the control substance in long-term bioassays for vinyl chloride and was not found to be carcinogenic. Table II summarizes the relative capacity of human biopsy samples to convert halogenated hydrocarbons into mutagenic intermediates. The results are expressed as a percentage of an appropriate animal control. Samples from human individuals are represented by different characters. As previously demonstrated, in rat and mouse the major enzymic activity for conversion of vinyl chloride m o n o m e r into mutagens is located in liver and kidney, sites at which angiosarcoma or nephroblastoma are induced in animals exposed to vinyl chloride [7,44]. The enzymic activities of four different human liver biopsy sam-
181 T A B L E II M U T A G E N I C I T Y M E D I A T E D BY H U M A N A N D BY A N I M A L T I S S U E S Compound
Organ fraction
Activity % Human a
Vinyl chloride b
liver
A 170 B 70 C 64 D 46
Vinyl bromide b
liver
Z Y X
32 39 22
K Z Y X
38 18 15 10
K Z Y X
22 0 0 0
L~ M N, O P
0 0 0
Z Y X
83 37 17
Vinylidene chloride b
liver
liver 2-Chloro-l,3-butadiene b lung
1,4-Dichloro-butene-2 e
liver
Mouse
(100)
(100)
(100)
(100)
(0)
(100)
a S a m p l e s f r o m h u m a n individuals are r e p r e s e n t e d by d i f f e r e n t characters. D a t a c o l l a t e d f r o m Bartsch et al. [ 9 ] and u n p u b l i s h e d w o r k . b M u t a g e n i c i t y assay (Bartsch et al. [ 7 , 8 ] ) w i t h S. t y p h i m u r i u m T A 1 0 0 or T A 1 5 3 0 . c Plate i n c o r p o r a t i o n assay ( A m e s e t al. [ 3 ] ).
ples are expressed in relation to that of mouse liver, which is taken as 100%. Sample A was twice as active in converting vinyl chloride into mutagenic metabolites, sample B was equally active as mouse liver, and the t w o other samples, C and D, were about 50% as active. From this data, it becomes clear that rat, mouse and human liver, the target organ in which vinyl chloride causes cancer, can convert this carcinogen into electrophilic and mutagenic intermediates. These results, at least in our opinion, would have been sufficient to classify this c o m p o u n d as biologically hazardous to man. Similarly, human liver specimens were active in converting vinyl bromide, vinylidene chloride, 2-chloro-l,3-butadiene and 1,4~lichlorobutene-2 into mutagens, the activity being, in general, lower than that in mouse liver. For the latter compound, Van Duuren et al. [62] reported a local carcinogenic action in mice. Results obtained by Barbin [ 4 ] , Bartsch and Montesano [ 6 ] , Bartsch [ 7 ] , GSthe [22] and Kappus [ 3 7 ] , lend strong support to the theory that hepatic mixed-function oxidase of rat, mouse and human liver converts vinyl chloride (I) into chloroethylene oxide (II), which is known to rearrange spontaneously to 2-chloroacetaldehyde [66] (III) (Fig. 1). A further oxidation product,
182
(I)
H"
(ll)
"H
CI CH2- COOH ~ (IV)
E.R.
[H" "O" "H
.
Cl CH~CHO (1111
Fig. 1. (1) Vinyl chloride; (II) chloroethylene oxide; (III) 2-chloroacetaldehyde; (IV) monochloroacetic acid.
monochloroacetic acid (IV), is an identifiable urinary metabolite in rats and man [23,27]. The mutagenic action of these compounds was studied in S. typhimurium strains [7,44,46,56] or in Chinese hamster V79 cells, using 8-azaguanine or ouabain resistance as genetic markers [28]. The results indicated that chloroethylene oxide and 2-chloroacetaldehyde were the most mutagenic compounds of this series in both systems (see also ref. [68]). Chloroethylene oxide is a primary reactive metabolite formed from vinyl chloride by mouse liver microsomes, oxygen and a NADPH generating system and is a rapidly reacting alkylating agent [4]. This evidence was obtained by comparison of the absorption spectra of the adducts obtained after reaction of 4-(4-nitrobenzyl)pyridine with chloroethylene oxide with the spectrum of a volatile vinyl chloride metabolite, which turned out to be identical with the one obtained with chloroethylene oxide. In the absence of the NADPH-generating system no such product was formed. Since chloroethylene oxide rearranges spontaneously to chloroacetaldehyde, those compounds are strong mutagens in several systems, and both now appear to be carcinogenic metabolites of vinyl chloride [6]. A number of N-nitrosamines, some examples of which are listed in Table III, are known to be p o t e n t carcinogens to various animal species, and man is exposed to some of them. (For a review, see Magee [43] .) Although no epidemiological investigations or case reports have so far given evidence that they have actually induced cancer in man, there is a strong possibility that they are hazardous to man [51]. N-Nitroso-morpholine, for example, was the most active of a series of cyclic nitrosamines in inducing point mutations in S. typhimurium TA1530 strain after metabolic conversion by rat liver enzymes [9]. The possible biological hazard to man of this c o m p o u n d becomes evident since liver samples, A, B, C and D from four humans efficiently converted N-nitrosomorpholine into mutagenic intermediates (Table III); the average enzymic activity was closest to that of rat liver, in which N-nitrosomorpholine is a p o t e n t carcinogen [20]. The differences in the enzymic activities of samples A, B, C and D indicate a promising means of obtaining valuable information on the variations within human populations, if a large number of human individuals is analysed. From the genetic background or the induced state of certain enzymes in those individuals one might obtain an idea of their susceptibility towards certain carcinogens. We
183 T A B L E III M U T A G E N I C I T Y M E D I A T E D BY H U M A N A N D BY A N I M A L T I S S U E S Compound
Organ fraction
Activity % Human a
N-Nitrosomorpholine b
liver
A B C D
90 50 40 30
Mouse
(100)
lung
E,F 0 G,H
N-Nitrosopyrrolidine
liver
Z 115 Y 90 X 5O
(100)
N-Nitrosopiperidine
liver
Z 215 Y 185 X 85
(100)
N-Nitroso-N'-methyl
liver
Z 3200 Y 1800 X 400
(I00)
piperazine
(0)
a S a m p l e s f r o m h u m a n individuals are r e p r e s e n t e d by d i f f e r e n t characters. D a t a c o l l a t e d f r o m Bartsch et al. [ 9 ] and u n p u b l i s h e d w o r k . b M u t a g e n i e i t y assay ( A m e s e t al. [ 3 ] ) w i t h S. t y p h i m u r i u m T A 1 5 3 0 .
are pursuing such a study in the IARC laboratory; some results are shown in Fig. 2. The relative enzymic activities of the liver samples described in Table III were the same when, instead of N-nitrosomorpholine, vinyl chloride was used as substrate. Sample A converted vinyl chloride and N-nitrosomorpholine into mutagenic intermediates the most efficiently while the other samples, B, C and D, were less active. As can be seen, there was an almost linear correlation between these t w o substrates which are chemically so diverse in structure. An absolute correlation, when such a plot is used, between the rates of metabolism of a chemical carcinogen in tissues obtained from different individuals would suggest that the two carcinogens here, vinyl chloride and N-nitrosomorpholine, are metabolized by a single enzymic system or by enzymic systems that are under a similar regulatory control [ 1 7 ] . The same almost linear relationship is observed by plotting the activities of three samples, X, Y and Z, withN-nitrosopiperidine versusN-nitrosopyrrolidine. If N-nitrosopiperidine metabolism is plotted versus that of N'-methyl-N-nitrosopiperazine, the correlation is not as good. However, it was remarkable that the three human biopsy samples were three to 30 times more active than rat liver in converting N'-methyl-N-nitrosopiperazine into mutagens. These data, although preliminary, already allow one important conclusion to be drawn: individuals A and Z appeared to be liable to a higher risk owing to their higher enzymic activity to convert not only one carcinogen into reactive
184 NO-PIPERAZINE
150
I
- N':'M e
i
'
NO-PIPERIDINE
150
I
®r
lOO
®z
:
Z O i -<
100
RAT
0 f-
50 Z m
/plate >
I 0
,
50
I
~
150
I
I
250
350
0
VINYL
NO-PIPERAZlNE-N:-Me
150
i
'
i
i
.............
300
I
50 ~..A
100
150
CHLORIDE ..........
~.. . . . . . . . . . . . . . .
J.......
I "1
RAT
R...A.T.........................................OZ 100
50
200
®X
I 0
50
100
I
I 150
,
I 250
c
i
L
350
0
100
I
200
,
I
t
3o0 350
F i g . 2. L i v e r - e n z y m e - m e d i a t e d m u t a g e n i e i t y ( h i s + r e v e r t a n t c o l o n i e s o f S. t y p h i m u r i u m 1530/plate) of BD-VI rat tissue and individual human biopsy samples, represented by different characters. N-nitrosamine w e r e a s s a y e d a c c o r d i n g t o A m e s e t al. [ 3 ] , a n d v i n y l c h l o r i d e a s d e s c r i b e d b y B a r t s c h e t al. [ 7 ] . T h e c o n c e n t r a t i o n o f t h e c a r c i n o g e n s u s e d w a s w i t h i n a l i n e a r r a n g e o f t h e d o s e - r e s p o n s e c u r v e s o b t a i n e d i n a n assay system containin~ 9 000 ~ supernatant equivalent to 39 mg liver/plate. Number of spontaneous his + revertant colonies from appropriate controls (no co-factors or no substrate) was subtracted. Data from B a r t s c h e t al. [ 9 ] a n d u n p u b l i s h e d w o r k .
metabolites, but, as measured for individual Z, several carcinogens, which even belong to different chemical classes. This has been shown for five different compounds (Fig. 2). The identification of people who may be at exceptionally high risk for cancer because of a genetically determined enzyme profile may in future be of some help in establishing regulatory measures to limit the levels of carcinogens in the environment. Such levels should be established by taking into consideration the possible risk for the most susceptible individuals. The last part of this discussion concerns the usefulness of these mutagenicity tests in predicting the possible carcinogenic effects of chemicals in man. Despite the converging tendency of chemicals to be both carcinogenic and mutagenic, it is not known at present whether in future all carcinogens will be found to be mutagens and all mutagens carcinogenic. For the strong mutagens, such as base analogues, nitrous acid and hydroxylamine, no carcinogenic effect in animals has been reported so far [48]. These chemicals do not act via electrophilic intermediates. On the other hand, synthetic steroidal sex hormones, which are carcinogenic in animals [31], have not yet been reported to be mutagenic, and we have also been unable in our own laboratory to detect a mutagenic effect in tissue-mediated assays in vitro using the human and animal carcinogen a,a'-di-
185
ethylstilboestrol as substrate with the T A 1 0 0 strain of S. typhimurium (Bartsch, unpublished). These findings would suggest that for this class of compounds, a different cancer-inducing mechanism may be implied. There are various limitations of the mutagenicity test systems, since some of the factors that determine the processes of cancer development in vivo cannot be duplicated in, for example, mutagenicity systems in vitro. Some of the most important of these determining factors are the concentration of ultimate reactive metabolites available for reaction in organs and animal species with cellular macromolecules, which is a consequence of a critical balance between metabolic activation and detoxication processes; an organ-specific release of proximate or ultimate carcinogens by enzymic deconjugation; the biological half-life of metabolites; and organ-specific D N A repair or frequency of D N A replication in target or non-target organs and immuno-surveillance. The use of mutagenicity tests in the assessment of the carcinogenic risk due to chemicals justifies their availability, since, although an enormous number of new chemicals is entering the environment, the world capacity for testing for carcinogenicity is only about 500 compounds per year, and it is not possible to test all of them in long-term carcinogenicity tests [ 3 2 , 3 3 , 3 4 , 3 5 , 3 6 ] . It is necessary, therefore, to pre-screen in order to establish priorities for long-term testing. In evaluating these tests, four points should be emphasized: (1) positive results from mutagenicity tests given at level C {Table IV) cannot automatically be taken to imply a definite carcinogenic effect in man {level A); (2) positive or negative results from these tests cannot substitute for long-term carcinogenicity
TABLE
IV
FRAMEWORK
OF CARCINOGENICITY
Valid data o n
TEST PROCEDURES
Test s y s t e m
N o data o n
Carcinogenic in m a n
T h r e s h o l d dose; individual
T_ Target organ in man; high risk groups
Species and organ specificity d o s e response in animals
Mechanism o f m e t a b o l i c activation in animals and man; t y p e o f genetic damage
;
risk
A
Epidemiologicalstudies
l
Level
Positive
]
Predictive value for extrap o l a t i o n (at present limited); target organ; threshold d o s e
J
Careinogenieity test in animals I Positive
Mutagenicity tests Microbial, m a m m a l i a n , h u m a n cells/activation in vivo and in vitro Chemicals
Level
~ --
B
Species a n d / o r organ specificity correlation b e t w e e n mutagenic and carcinogenic p o t e n c y Level
C
;
186 testing in animals (level B); (3) the present methodology, in particular tissuemediated mutagenicity assays, is effective in predicting the carcinogenic potential of chemicals with a high probability, ranging at present between 80 and 90%, but gives no indication of target organs or species specificity of the carcinogenic action of the chemical; and, (4) the relative potency of a chemical as a mutagen cannot at present be correlated with its p o t e n c y as a carcinogen in experimental animals or man. Taking into account present reproducibility, cost, number of chemicals that can be examined in a short time and the scientific basis of the test, tissue-mediated mutagenicity procedures using well-characterized genetic indicators and a metabolically defined activation system in vitro (apart from the Drosophila test [63] ) appear at present to be the most promising short-term tests for the detection of adverse biological effects produced by chemicals. The present methodology still incurs the chance of false negative results, depending on the system used, either due to mutagen specificity, lack of appropriate cofactors for activation or to extreme reactivity and volatility of the c o m p o u n d or its metabolites [9]. However, the number of false negative results obtained in the tissue-mediated assay in vitro is very small in comparison with that obtained with other mutagenicity test procedures. As has often been mentioned, theoretical limitations are implied in the short-term tests currently used, because the development of cancer in vivo is determined by factors that cannot yet be duplicated in many of the mutagenicity test systems. Although there are still many problems involved in the interpretation of results of mutagenicity tests in terms of evaluating the carcinogenicity of chemicals, short-term tests can already be used in detecting possible cancer-causing agents with a sensitivity which did n o t exist ten years ago. They could thus be a powerful tool, when used in combination with epidemiological studies, in environmental control. This possibility, therefore, raises two important issues: (1) the validity of the short-term tests must be based on reliable corroboration by data from long-term tests in experimental animals; so, first of all the long-term data must be critically analysed and accepted to provide solid and definite background information; and, (2) the scientific community, as well as health authorities, must reach a concensus on the usefulness of results from these test systems in predicting possible carcinogenic effects of c o m p o u n d s in man. Otherwise, and if no public health measures are taken, for instance, to remove the agent from the environment or greatly to reduce exposure to it, cancer in man can only be treated and not prevented.
Acknowledgements
The author's experimental work was partially supported by the National Cancer Institute of the USA, Contract No. 1CP-55630. For valuable discussion and/or experimental assistance, the author is indebted to L. Tomatis, R. Montesano, C. Malaveille, A. Barbin, A. Camus, G. Planche, G. Brun and H. Br~sil. The help of Miss P. Stafford Smith and Mrs E. Ward in preparing the manuscript is gratefully acknowledged.
187
Note added in proof Loprieno et al. [68] have observed a pronounced mutagenic effect of vinyl chloride in Schizosaccharomyces pombe and Saccharomyces cerevisiae, only when mouse-liver microsomes were added to the assays in vitro. Vinyl chloride was also active in the host-mediated assay in mice. References 1 A m e s , B.N. a n d J. M c C a n n , C a r c i n o g e n s are m u t a g e n s : a s i m p l e t e s t s y s t e m , in R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s (eds.) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , L y o n ( I A R C S c i e n t i f i c P u b lications No. 12), 1976. 2 A m e s , B.N., A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s in u r i n e : s t u d i e s w i t h t h e c a r c i n o g e n 2a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. (Wash.), 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 3 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d D . F . 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5 5 R a m e l , C., e d , R e p o r t of a s y m p o s i u m h e l d a t S k o k l o s t e r , S w e d e n , 1 9 7 2 , A m b i o S p e c i a l R e p o r t , No. 3, 1 9 7 3 . 5 6 R a n n u g , U., A. J o h a n s s o n , C. R a m e l a n d C . A . W a c h m e i s t e r , T h e m u t a g e n i c i t y o f v i n y l c h l o r i d e a f t e r metabolic activation, Ambio, 3 (1974) 194--197. 5 7 De S e r r e s , F . J . a n d W. S h e r i d a n , (eds.) T h e E v a l u a t i o n o f C h e m i c a l M u t a g e n i c i t y D a t a in R e l a t i o n t o P o p u l a t i o n R i s k , E n v i r o n m e n t a l H e a l t h P e r s p e c t i v e s , No. 6 , 1 9 7 3 . 5 8 S t i c h , H . F . , R . H . C . S a n , J . A . Miller a n d E.C. Miller, V a r i o u s levels of D N A r e p a i r s y n t h e s i s i n X e r o d e r m a p i g m e n t o s i u m cells e x p o s e d t o t h e c a r c i n o g e n s N - h y d r o x y a n d N - a c e t o x y - 2 - a c e t y l a m i n o f l u o r e n e , N a t u r e N e w Biol., 2 3 8 ( 1 9 7 2 ) 9 - - 1 0 . 5 9 S t i e h , H . F . , R . H . C . S a n a n d Y. K a w a z o e , I n c r e a s e d s e n s i t i v i t y of X e r o d e r m a p i g m e n t o s u m cells t o s o m e c h e m i c a l c a r c i n o g e n s a n d m u t a g e n s , M u t a t i o n Res., 1 7 ( 1 9 7 3 ) 1 2 7 - - 1 3 7 . 6 0 S t o l t z , D . R . , L . A . Poirier, C.C. Irving, H . F . S t i c h , J . H . W e i s b u r g e r a n d H.C. Grice, E v a l u a t i o n o f s h o r t t e r m t e s t s f o r c a r c i n o g e n i c i t y , T o x i c o l . A p p l . P h a r m a c o l . , 29 ( 1 9 7 4 ) 1 5 7 - - 1 8 0 . 6 1 S u g i m u r a , T., T. Y a h a g i , M. N a g a o , M. T a k e u c h i , T. K a w a c h i , K. H a r a , E. Y a m a s a k i , T. M a t s u s h i m a , Y. H a s h i m o t o a n d M. O k a d a , V a l i d i t y o f m u t a g e n i c i t y t e s t s u s i n g m i c r o b e s as a r a p i d s c r e e n i n g m e t h o d f o r e n v i r o n m e n t a l c h e m i c a l s . In: R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s (eds.) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , L y o n , I A R C S c i e n t i f i c P u b l i c a t i o n s No. 1 2 ) 1 9 7 6 . 6 2 V a n D u u r e n , B . L . , B.M. G o l d s c h m i d t a n d I. S e i d m a n , C a r c i n o g e n i c a c t i v i t y o f di- a n d t r i f u n c t i o n a l ~c h l o r o e t h e r s a n d o f 1 , 4 - d i c h l o r o b u t e n e - 2 in I C R / H A Swiss m i c e , C a n c e r Res., 3 5 ( 1 9 7 5 ) 2 5 5 3 - - 2 5 5 7 . 6 3 V o g e l , E., T h e r e l a t i o n b e t w e e n m u t a t i o n a l p a t t e r n a n d c o n c e n t r a t i o n b y c h e m i c a l m u t a g e n s in D r o s o p h i l a , in R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s ( e d s . ) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , Lyon, IARC Scientific Publications No. 12), 1976. 64 WHO, Evaluation and Testing of Drugs for Mutagenicity: Principles and Problems, WHO Techn. Rep. Set., N o . 4 8 2 , 1 9 7 1 . 6 5 W H O , A s s e s s m e n t o f t h e C a r c i n o g e n i c i t y a n d M u t a g e n i e i t y of C h e m i c a l s , W H O T e e h n . R e p . Set., No. 546, 1974. 6 6 Zief, M. a n d C.H. S c h r a m m , C h l o r o e t h y l e n e o x i d e , C h e m . I n d . , ( 1 9 6 4 ) 6 6 0 - - - 6 6 1 . 6 7 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B. A m e s , D e t e c t i o n o f c a r c i n o g e n s as m u t a g e n s in t h e Salmonella/microsome t e s t : A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. (Wash.) 7 2 ( 1 9 7 5 ) 5 1 3 5 - - 5 1 3 9 . 6 8 L o p r i e n o , N., R. B a r a l e , S. B a r o n c e l l i , C. B a u e r , G. B r o n z e t t i . A. C a m m e l l i n i , G. C e r c i g n a n i , C. Corsi, G. Gervasi, C. L e p o r i n i , R. Nieri, A . M . Rossi, G. S t r e t t i a n d G. T u r c h i , E v a l u a t i o n o f t h e g e n e t i c eff e c t s i n d u c e d b y v i n y l c h l o r i d e m o n o m e r ( V C M ) u n d e r m a m m a l i a n m e t a b o l i c a c t i v a t i o n : s t u d i e s in v i t r o a n d in vivo, M u t a t i o n Res., 4 0 ( 1 9 7 6 ) 85---96.
Mutation Research, 38 (1976) 177--190 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
PREDICTIVE VALUE OF MUTAGENICITY TESTS IN CHEMICAL CARCINOGENESIS
H. B A R T S C H International Agency for Research on Cancer, Unit o f Chemical Carcinogenesis, 150 cours Albert Thomas, 69008 Lyon, France
{Received October 10th, 1975) {Accepted November 28th, (1975)
An essential role of environmental factors in the aetiology of human cancer has been shown by the unequivocal evidence of the chemical origin of occupational cancer, as in the cases of urinary bladder tumours in workers exposed to aromatic amines [29,13,12] lung cancers in workers exposed to bis(chloromethyl) ether [30] or angiosarcomas of the liver in workers exposed to vinyl chloride [31] ; the well
Italy, Oc-
178
[50,47,3,61] ; and many mutagens, whose carcinogenicity had not yet been investigated, have now been shown to be carcinogens. This convergence of mutagens and carcinogens, which has strengthened dramatically since 1960, has resulted from the discovery that many such chemicals require metabolic activation in order to show a biological activity. This metabolic activation is often associated with binding to nucleophilic sites in nucleic acids and proteins in the organism. The growing experimental evidence linking the carcinogenic activity of numerous chemicals to their capacity to be converted into electrophilic derivatives that may also exert a mutagenic effect, had led to the suggestion that an empirical relationship between chemical carcinogenesis and mutagenesis exists [48,60,5]. However, such a correlation has so far been limited to those changes of the genotype that appear as a consequence of structural or functional alterations of nucleic acids and thus are genetically transmissible. For that reason, only mutagenicity tests that are genetically and metabolically reasonably well defined and/or that provide data shown to be valid for the prediction of the carcinogenicity of chemicals are referred to here. Other test procedures that allow the detection of the whole spectrum of genetic damage induced by chemicals are by no means disqualified by this selection. Since the genetic material of all living organisms, except for some viruses, has been found to be DNA, the major critical target molecule for chemical mutagens has been defined as DNA. A great number of chemical mutagens and carcinogens react covalently with DNA and other cellular macromolecules [10]. In contrast, for carcinogenesis, no critical target molecule has been defined, although evidence for an association between carcinogenicity and modification of DNA is provided by the appearance in patients with Xeroderma pigmentosum of a higher rate of skin cancer due to sunlight and of their cells which lack the ability to repair ultra-violet or certain chemical damage to DNA [15,18,58, 59]. Despite the diversity in chemical structures of known carcinogens and mutagens, such as alkylating agents, N-nitrosamines and N-nitrosamides, naturally occurring compounds, aromatic hydrocarbons, etc., recognition of a c o m m o n element in chemical carcinogens and mutagens has rapidly progressed since it was understood that the majority of carcinogens and many mutagens need metabolic activation in the host for transformation to their so-called ultimate reactive forms [49]. Comparison of these ultimate reactive metabolites of carcinogens has revealed that the common denominator of these substances is their electrophilicity, i.e. they are compounds whose atoms have an electron deficiency that enables them to react with electron-rich sites in cellular nucleic acids and proteins causing mutagenic effects frequently paralleled by the onset of DNA repair processes. The specificity of certain chemical compounds to produce tumours only in certain organs or in certain animal species, is primarily determined by the available concentration of ultimate reacitve metabolites which is itself a consequence of a critical balance between activation and detoxication processes. In view of the properties of ultimate reactive forms of chemical carcinogens, and, in particular, the growing evidence for an empirical relationship between the carcinogenic and the mutagenic activity of chemicals, it is n o w possible to use this relationship in short-term tests for the detection of potential carcino-
179 gens. There are many systems available at present that involve different genetic indicators and metabolic activation systems for detecting mutagenic activity; they all, however, have individual advantages and limitations. Ideally, an appropriate mutagenicity test system would include the full metabolic competency of the intact organism, that is, humans. Thus, the conclusion has been reached that a battery of test systems is needed to detect the genetic hazards caused b y chemicals [24,55,60]. In evaluating these methods, the major question is which screening tests, singly or collectively, can serve as reliable indicators or predictors of the potential carcinogenic hazard of a chemical. This can only be achieved by testing a representative number of different classes of carcinogens and non-carcinogens. A valid test should be positive for compounds with known carcinogenic activities and negative for negative compounds, within the limits of the test. Extensive studies are being carried o u t in Japan and in the United States to evaluate short-term tests [53]. Results accumulated up to the present time [1,61,67 ] using a rat-liver microsome test in vitro with Salmonella typhimurium strains developed by Bruce Ames, have shown that a b o u t 80--90% of the carcinogens tested were also mutagens, while the number of false positives and false negatives was much lower, ranging from 10 to 15%. In tests used for preliminary screening, a small proportion of false positive and false negative results may be acceptable; but, for a final test, no false negative results can be accepted. At present, mutagenicity assays, especially with sub-mammalian indicators, can be used in the following ways. (a) To trace carcinogens and/or mutagens in the complex environment of man. (b) To pre-screen compounds for their mutagenic action per se or after metabolic activation, to select high-priority compounds for long-term carcinogenicity tests. Special attention should be given to chemicals for which a substantial human exposure is known to occur and to newly-developed drugs used over a period of years in children, young adults or pregnant women. (c) The systems in vitro allow investigation of the capability of human tissues to generate electrophilic intermediates from the c o m p o u n d under test. The usual type of metabolic study, e.g. studies on distribution or excretion of the drug, are of limited value if the chemical acts by a partial conversion to a mutagenic or carcinogenic intermediate that interacts with DNA and is not excreted as such. This technique may also aid in the selection for long-term carcinogenicity tesing of those animal species most similar to man. (d) Urinary metabolites can be tested for their mutagenic action or after deconjugation by enzymes and reactivation by liver fractions. These assays, recently developed by Ames [2,16], permit the detection of mutagens in rats treated with carcinogens and can be used for monitoring the exposure of human populations. As recently shown b y Legator [42], in patients treated with the antischistosomal agent Niridazole, mutagenic activity for S. typhimurium strains was demonstrable in the urine. The validity and/or the practical application of such microbial mutagenicity tests has also been demonstrated in the IARC laboratory with vinyl chloride, which has recently been recognized as a carcinogen in animals and in man [ 3 1 ] . The validity of the microbial test system using S. typhimurium strains to predict the possible carcinogenic action of structurally related chemicals such as
180 TABLE
I
COMPARATIVE
MUTAGENICITY
OF OLEFINIC
COMPOUNDS
D a t a c o l l a t e d f r o m B a r t s e h e t al. [ 9 ] a n d u n p u b l i s h e d Compound
his + r e v e r t a n t
work. Carcinogenicity
c o l o n i e s / p m o l / h /plate a
S. l y p h i m u r i u m
TA1530 Vinyl acetate Vinyl chloride Vinylidene chloride 2-Chloro- 1,3-butadiene
TAIO0
0 b
(0) c
0 b
(0) c
_
3.7 9.5 34.5
(1.2) (0.2) (12.5)
5.6 14.6 51.2
(1.5) (0.7) (9)
+ (+) ?
a From linear dose- and time-dependent r e s p o n s e c u r v e s in t h e p r e s e n c e rant, spontaneous mutations subtracted. b In the presence of co-factors (NADP*, glucose 6-phosphate). c In the absence of co-factors.
of mouse
liver 9 000 g superna-
vinylidene chloride (1,1-dichloroethylene) and 2-chloro-l,3-butadiene was verified [8]. 2-Chloro-l,3-butadiene has been used for the production of rubber, and vinylidene chloride is used as a co-polymer with vinyl chloride in the manufacture of plastics. 2-Chloro-l,3-butadiene has been reported to induce chromosomal aberrations in lymphocytes of peripheral blood from exposed workers [38], and in the USSR it has been associated with the induction of skin and lung tumours in humans [39,40]. Table I summarizes the relative mutagenic strengths of vinyl chloride, vinylidene chloride and 2-chloro-l,3-butadiene in the presence of mouse liver fractions either in the presence or in the absence of a NADPH-generating system. Vinyl acetate is included for comparison at the top of the table. The results were calculated from linear dose- or time-dependent response curves and are expressed as the number of histidine revertant colonies per pmol of substrate per hour of exposure per plate as measured with two different S. typhimurium strains, TA1530 and TA100. 2-Chloro-l,3-butadiene exhibited the highest mutagenic effect, followed by vinylidene chloride, for which Viola and Caputo (personal communication) have recently obtained evidence for its carcinogenicity in rats. These findings indicated that epidemiological monitoring of workers exposed to vinylidene chloride and 2-chloro-l,3-butadiene may be necessary, as well as measures to prevent, or greatly reduce, exposure of humans. No mutagenic effect was detectable with vinyl acetate. This c o m p o u n d was used by Maltoni and Lefemine [45] as the control substance in long-term bioassays for vinyl chloride and was not found to be carcinogenic. Table II summarizes the relative capacity of human biopsy samples to convert halogenated hydrocarbons into mutagenic intermediates. The results are expressed as a percentage of an appropriate animal control. Samples from human individuals are represented by different characters. As previously demonstrated, in rat and mouse the major enzymic activity for conversion of vinyl chloride m o n o m e r into mutagens is located in liver and kidney, sites at which angiosarcoma or nephroblastoma are induced in animals exposed to vinyl chloride [7,44]. The enzymic activities of four different human liver biopsy sam-
181 T A B L E II M U T A G E N I C I T Y M E D I A T E D BY H U M A N A N D BY A N I M A L T I S S U E S Compound
Organ fraction
Activity % Human a
Vinyl chloride b
liver
A 170 B 70 C 64 D 46
Vinyl bromide b
liver
Z Y X
32 39 22
K Z Y X
38 18 15 10
K Z Y X
22 0 0 0
L~ M N, O P
0 0 0
Z Y X
83 37 17
Vinylidene chloride b
liver
liver 2-Chloro-l,3-butadiene b lung
1,4-Dichloro-butene-2 e
liver
Mouse
(100)
(100)
(100)
(100)
(0)
(100)
a S a m p l e s f r o m h u m a n individuals are r e p r e s e n t e d by d i f f e r e n t characters. D a t a c o l l a t e d f r o m Bartsch et al. [ 9 ] and u n p u b l i s h e d w o r k . b M u t a g e n i c i t y assay (Bartsch et al. [ 7 , 8 ] ) w i t h S. t y p h i m u r i u m T A 1 0 0 or T A 1 5 3 0 . c Plate i n c o r p o r a t i o n assay ( A m e s e t al. [ 3 ] ).
ples are expressed in relation to that of mouse liver, which is taken as 100%. Sample A was twice as active in converting vinyl chloride into mutagenic metabolites, sample B was equally active as mouse liver, and the t w o other samples, C and D, were about 50% as active. From this data, it becomes clear that rat, mouse and human liver, the target organ in which vinyl chloride causes cancer, can convert this carcinogen into electrophilic and mutagenic intermediates. These results, at least in our opinion, would have been sufficient to classify this c o m p o u n d as biologically hazardous to man. Similarly, human liver specimens were active in converting vinyl bromide, vinylidene chloride, 2-chloro-l,3-butadiene and 1,4~lichlorobutene-2 into mutagens, the activity being, in general, lower than that in mouse liver. For the latter compound, Van Duuren et al. [62] reported a local carcinogenic action in mice. Results obtained by Barbin [ 4 ] , Bartsch and Montesano [ 6 ] , Bartsch [ 7 ] , GSthe [22] and Kappus [ 3 7 ] , lend strong support to the theory that hepatic mixed-function oxidase of rat, mouse and human liver converts vinyl chloride (I) into chloroethylene oxide (II), which is known to rearrange spontaneously to 2-chloroacetaldehyde [66] (III) (Fig. 1). A further oxidation product,
182
(I)
H"
(ll)
"H
CI CH2- COOH ~ (IV)
E.R.
[H" "O" "H
.
Cl CH~CHO (1111
Fig. 1. (1) Vinyl chloride; (II) chloroethylene oxide; (III) 2-chloroacetaldehyde; (IV) monochloroacetic acid.
monochloroacetic acid (IV), is an identifiable urinary metabolite in rats and man [23,27]. The mutagenic action of these compounds was studied in S. typhimurium strains [7,44,46,56] or in Chinese hamster V79 cells, using 8-azaguanine or ouabain resistance as genetic markers [28]. The results indicated that chloroethylene oxide and 2-chloroacetaldehyde were the most mutagenic compounds of this series in both systems (see also ref. [68]). Chloroethylene oxide is a primary reactive metabolite formed from vinyl chloride by mouse liver microsomes, oxygen and a NADPH generating system and is a rapidly reacting alkylating agent [4]. This evidence was obtained by comparison of the absorption spectra of the adducts obtained after reaction of 4-(4-nitrobenzyl)pyridine with chloroethylene oxide with the spectrum of a volatile vinyl chloride metabolite, which turned out to be identical with the one obtained with chloroethylene oxide. In the absence of the NADPH-generating system no such product was formed. Since chloroethylene oxide rearranges spontaneously to chloroacetaldehyde, those compounds are strong mutagens in several systems, and both now appear to be carcinogenic metabolites of vinyl chloride [6]. A number of N-nitrosamines, some examples of which are listed in Table III, are known to be p o t e n t carcinogens to various animal species, and man is exposed to some of them. (For a review, see Magee [43] .) Although no epidemiological investigations or case reports have so far given evidence that they have actually induced cancer in man, there is a strong possibility that they are hazardous to man [51]. N-Nitroso-morpholine, for example, was the most active of a series of cyclic nitrosamines in inducing point mutations in S. typhimurium TA1530 strain after metabolic conversion by rat liver enzymes [9]. The possible biological hazard to man of this c o m p o u n d becomes evident since liver samples, A, B, C and D from four humans efficiently converted N-nitrosomorpholine into mutagenic intermediates (Table III); the average enzymic activity was closest to that of rat liver, in which N-nitrosomorpholine is a p o t e n t carcinogen [20]. The differences in the enzymic activities of samples A, B, C and D indicate a promising means of obtaining valuable information on the variations within human populations, if a large number of human individuals is analysed. From the genetic background or the induced state of certain enzymes in those individuals one might obtain an idea of their susceptibility towards certain carcinogens. We
183 T A B L E III M U T A G E N I C I T Y M E D I A T E D BY H U M A N A N D BY A N I M A L T I S S U E S Compound
Organ fraction
Activity % Human a
N-Nitrosomorpholine b
liver
A B C D
90 50 40 30
Mouse
(100)
lung
E,F 0 G,H
N-Nitrosopyrrolidine
liver
Z 115 Y 90 X 5O
(100)
N-Nitrosopiperidine
liver
Z 215 Y 185 X 85
(100)
N-Nitroso-N'-methyl
liver
Z 3200 Y 1800 X 400
(I00)
piperazine
(0)
a S a m p l e s f r o m h u m a n individuals are r e p r e s e n t e d by d i f f e r e n t characters. D a t a c o l l a t e d f r o m Bartsch et al. [ 9 ] and u n p u b l i s h e d w o r k . b M u t a g e n i e i t y assay ( A m e s e t al. [ 3 ] ) w i t h S. t y p h i m u r i u m T A 1 5 3 0 .
are pursuing such a study in the IARC laboratory; some results are shown in Fig. 2. The relative enzymic activities of the liver samples described in Table III were the same when, instead of N-nitrosomorpholine, vinyl chloride was used as substrate. Sample A converted vinyl chloride and N-nitrosomorpholine into mutagenic intermediates the most efficiently while the other samples, B, C and D, were less active. As can be seen, there was an almost linear correlation between these t w o substrates which are chemically so diverse in structure. An absolute correlation, when such a plot is used, between the rates of metabolism of a chemical carcinogen in tissues obtained from different individuals would suggest that the two carcinogens here, vinyl chloride and N-nitrosomorpholine, are metabolized by a single enzymic system or by enzymic systems that are under a similar regulatory control [ 1 7 ] . The same almost linear relationship is observed by plotting the activities of three samples, X, Y and Z, withN-nitrosopiperidine versusN-nitrosopyrrolidine. If N-nitrosopiperidine metabolism is plotted versus that of N'-methyl-N-nitrosopiperazine, the correlation is not as good. However, it was remarkable that the three human biopsy samples were three to 30 times more active than rat liver in converting N'-methyl-N-nitrosopiperazine into mutagens. These data, although preliminary, already allow one important conclusion to be drawn: individuals A and Z appeared to be liable to a higher risk owing to their higher enzymic activity to convert not only one carcinogen into reactive
184 NO-PIPERAZINE
150
I
- N':'M e
i
'
NO-PIPERIDINE
150
I
®r
lOO
®z
:
Z O i -<
100
RAT
0 f-
50 Z m
/plate >
I 0
,
50
I
~
150
I
I
250
350
0
VINYL
NO-PIPERAZlNE-N:-Me
150
i
'
i
i
.............
300
I
50 ~..A
100
150
CHLORIDE ..........
~.. . . . . . . . . . . . . . .
J.......
I "1
RAT
R...A.T.........................................OZ 100
50
200
®X
I 0
50
100
I
I 150
,
I 250
c
i
L
350
0
100
I
200
,
I
t
3o0 350
F i g . 2. L i v e r - e n z y m e - m e d i a t e d m u t a g e n i e i t y ( h i s + r e v e r t a n t c o l o n i e s o f S. t y p h i m u r i u m 1530/plate) of BD-VI rat tissue and individual human biopsy samples, represented by different characters. N-nitrosamine w e r e a s s a y e d a c c o r d i n g t o A m e s e t al. [ 3 ] , a n d v i n y l c h l o r i d e a s d e s c r i b e d b y B a r t s c h e t al. [ 7 ] . T h e c o n c e n t r a t i o n o f t h e c a r c i n o g e n s u s e d w a s w i t h i n a l i n e a r r a n g e o f t h e d o s e - r e s p o n s e c u r v e s o b t a i n e d i n a n assay system containin~ 9 000 ~ supernatant equivalent to 39 mg liver/plate. Number of spontaneous his + revertant colonies from appropriate controls (no co-factors or no substrate) was subtracted. Data from B a r t s c h e t al. [ 9 ] a n d u n p u b l i s h e d w o r k .
metabolites, but, as measured for individual Z, several carcinogens, which even belong to different chemical classes. This has been shown for five different compounds (Fig. 2). The identification of people who may be at exceptionally high risk for cancer because of a genetically determined enzyme profile may in future be of some help in establishing regulatory measures to limit the levels of carcinogens in the environment. Such levels should be established by taking into consideration the possible risk for the most susceptible individuals. The last part of this discussion concerns the usefulness of these mutagenicity tests in predicting the possible carcinogenic effects of chemicals in man. Despite the converging tendency of chemicals to be both carcinogenic and mutagenic, it is not known at present whether in future all carcinogens will be found to be mutagens and all mutagens carcinogenic. For the strong mutagens, such as base analogues, nitrous acid and hydroxylamine, no carcinogenic effect in animals has been reported so far [48]. These chemicals do not act via electrophilic intermediates. On the other hand, synthetic steroidal sex hormones, which are carcinogenic in animals [31], have not yet been reported to be mutagenic, and we have also been unable in our own laboratory to detect a mutagenic effect in tissue-mediated assays in vitro using the human and animal carcinogen a,a'-di-
185
ethylstilboestrol as substrate with the T A 1 0 0 strain of S. typhimurium (Bartsch, unpublished). These findings would suggest that for this class of compounds, a different cancer-inducing mechanism may be implied. There are various limitations of the mutagenicity test systems, since some of the factors that determine the processes of cancer development in vivo cannot be duplicated in, for example, mutagenicity systems in vitro. Some of the most important of these determining factors are the concentration of ultimate reactive metabolites available for reaction in organs and animal species with cellular macromolecules, which is a consequence of a critical balance between metabolic activation and detoxication processes; an organ-specific release of proximate or ultimate carcinogens by enzymic deconjugation; the biological half-life of metabolites; and organ-specific D N A repair or frequency of D N A replication in target or non-target organs and immuno-surveillance. The use of mutagenicity tests in the assessment of the carcinogenic risk due to chemicals justifies their availability, since, although an enormous number of new chemicals is entering the environment, the world capacity for testing for carcinogenicity is only about 500 compounds per year, and it is not possible to test all of them in long-term carcinogenicity tests [ 3 2 , 3 3 , 3 4 , 3 5 , 3 6 ] . It is necessary, therefore, to pre-screen in order to establish priorities for long-term testing. In evaluating these tests, four points should be emphasized: (1) positive results from mutagenicity tests given at level C {Table IV) cannot automatically be taken to imply a definite carcinogenic effect in man {level A); (2) positive or negative results from these tests cannot substitute for long-term carcinogenicity
TABLE
IV
FRAMEWORK
OF CARCINOGENICITY
Valid data o n
TEST PROCEDURES
Test s y s t e m
N o data o n
Carcinogenic in m a n
T h r e s h o l d dose; individual
T_ Target organ in man; high risk groups
Species and organ specificity d o s e response in animals
Mechanism o f m e t a b o l i c activation in animals and man; t y p e o f genetic damage
;
risk
A
Epidemiologicalstudies
l
Level
Positive
]
Predictive value for extrap o l a t i o n (at present limited); target organ; threshold d o s e
J
Careinogenieity test in animals I Positive
Mutagenicity tests Microbial, m a m m a l i a n , h u m a n cells/activation in vivo and in vitro Chemicals
Level
~ --
B
Species a n d / o r organ specificity correlation b e t w e e n mutagenic and carcinogenic p o t e n c y Level
C
;
186 testing in animals (level B); (3) the present methodology, in particular tissuemediated mutagenicity assays, is effective in predicting the carcinogenic potential of chemicals with a high probability, ranging at present between 80 and 90%, but gives no indication of target organs or species specificity of the carcinogenic action of the chemical; and, (4) the relative potency of a chemical as a mutagen cannot at present be correlated with its p o t e n c y as a carcinogen in experimental animals or man. Taking into account present reproducibility, cost, number of chemicals that can be examined in a short time and the scientific basis of the test, tissue-mediated mutagenicity procedures using well-characterized genetic indicators and a metabolically defined activation system in vitro (apart from the Drosophila test [63] ) appear at present to be the most promising short-term tests for the detection of adverse biological effects produced by chemicals. The present methodology still incurs the chance of false negative results, depending on the system used, either due to mutagen specificity, lack of appropriate cofactors for activation or to extreme reactivity and volatility of the c o m p o u n d or its metabolites [9]. However, the number of false negative results obtained in the tissue-mediated assay in vitro is very small in comparison with that obtained with other mutagenicity test procedures. As has often been mentioned, theoretical limitations are implied in the short-term tests currently used, because the development of cancer in vivo is determined by factors that cannot yet be duplicated in many of the mutagenicity test systems. Although there are still many problems involved in the interpretation of results of mutagenicity tests in terms of evaluating the carcinogenicity of chemicals, short-term tests can already be used in detecting possible cancer-causing agents with a sensitivity which did n o t exist ten years ago. They could thus be a powerful tool, when used in combination with epidemiological studies, in environmental control. This possibility, therefore, raises two important issues: (1) the validity of the short-term tests must be based on reliable corroboration by data from long-term tests in experimental animals; so, first of all the long-term data must be critically analysed and accepted to provide solid and definite background information; and, (2) the scientific community, as well as health authorities, must reach a concensus on the usefulness of results from these test systems in predicting possible carcinogenic effects of c o m p o u n d s in man. Otherwise, and if no public health measures are taken, for instance, to remove the agent from the environment or greatly to reduce exposure to it, cancer in man can only be treated and not prevented.
Acknowledgements
The author's experimental work was partially supported by the National Cancer Institute of the USA, Contract No. 1CP-55630. For valuable discussion and/or experimental assistance, the author is indebted to L. Tomatis, R. Montesano, C. Malaveille, A. Barbin, A. Camus, G. Planche, G. Brun and H. Br~sil. The help of Miss P. Stafford Smith and Mrs E. Ward in preparing the manuscript is gratefully acknowledged.
187
Note added in proof Loprieno et al. [68] have observed a pronounced mutagenic effect of vinyl chloride in Schizosaccharomyces pombe and Saccharomyces cerevisiae, only when mouse-liver microsomes were added to the assays in vitro. Vinyl chloride was also active in the host-mediated assay in mice. References 1 A m e s , B.N. a n d J. M c C a n n , C a r c i n o g e n s are m u t a g e n s : a s i m p l e t e s t s y s t e m , in R. M o n t e s a n o , H. B a r t s c h a n d L. T o m a t i s (eds.) S c r e e n i n g Tests in C h e m i c a l C a r c i n o g e n e s i s , L y o n ( I A R C S c i e n t i f i c P u b lications No. 12), 1976. 2 A m e s , B.N., A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s in u r i n e : s t u d i e s w i t h t h e c a r c i n o g e n 2a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. (Wash.), 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 3 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d D . F . 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