Mutation Research, 250 (1991) 25-33 © 1991 Elsevier Science Publishers B.V. All rights reserved 0027-5107/91/$03.50 ADONIS 0027510791001618
25
MUT 02530
Implications of newly recognized relationships between mutagenicity, genotoxicity and carcinogenicity of molecules Herbert S. Rosenkranz a, Ying Ping Zhang and Gilles Klopman b a
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 and b Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Accepted 16 April 1991)
Keywords: Relationships, between molecules; CASE structure-activity relational method
Summary The CASE structure-activity relational method was used to predict the mutagenicity, cytogenotoxicity, carcinogenicity, sensory irritation, male rat-specific a2/z-nephrotoxicity and maximum tolerated dose of a population of molecules (N >_ 1300). These chemicals were then sorted out by their predicted responses to specific tests and sub-populations of molecules with different prevalence with respect to described endpoints were constructed, i.e. 0-100% prevalences of mutagens, rodent carcinogens and SCE inducers. The predicted properties of these populations were analyzed and the overlap among tests was determined. The method also permits the determination of the dependence among assays and the level of false-positive and false-negative predictions.
The recent realization that a number of shortterm tests used to predict potential carcinogenicity may not be highly predictive has raised some questions regarding the selections of combinations of tests (batteries) for predicting carcinogenicity, the mechanisms of carcinogenicity itself and the significance of a finding of mutagenicity and/or genotoxicity in the absence of knowledge regarding carcinogenicity. Recently we have developed a method for addressing these problems. We have taken advantage of the ability of the CASE structure-activity
Correspondence: Prof. Herbert S. Rosenkranz, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.).
relational system (Klopman et al., 1990; Rosenkranz and Klopman, 1989) to predict the carcinogenic, mutagenic and genotoxic properties of molecules to simulate the properties of populations of molecules containing different proportions (prevalences) of chemicals with specific properties. Moreover, by comparing experimental with simulated results we have established the validity of the procedure. In a preliminary analysis we showed (Klopman and Rosenkranz, 1991) that, in simulated populations of molecules in which the prevalence of carcinogens ranged from 0% to 100%, mutagenicity ranged from 14% to 44% suggesting (a) that mutagenicity in Salmonella when used as a surrogate for carcinogenicity, may yield 14% false-positive predictions and (b) that 44% of carcinogens may be 'nongenotoxic' (see also Table 1, Number 10). How-
26 ever, that analysis did not address the usual situa-
primarily the relationships between the projected
t i o n , i.e. t h e s i g n i f i c a n c e o f t h e r e s u l t s o f s h o r t -
r e s u l t s o f a g r o u p o f in v i t r o a n d c a n c e r b i o a s s a y s ,
t e r m t e s t s in t h e a b s e n c e o f r o d e n t c a n c e r b i o a s -
on the one hand, and the results of the Salmonella
say d a t a .
m u t a g e n i c i t y assay, p r o b a b l y t h e m o s t w i d e l y u s e d
In t h e
present
analysis we
consider
TABLE 1 SUMMARY OF THE OVERLAP OF POSITIVE RESPONSES b No.
1 2 3 4 5
6
7 8 9
10 11 12 13
14 15 16 17 18
Biological system
Extent of overlaps I With Salmonella
II With carcinogenicity in rodents
(1%
10(1
0%
100 %
Slope
Structural alerts Carcinogenicity: rodents Carcinogenicity: mice
12.5 22.5
65.6 54.9
0.531 0.324
9.2
26.6
0.174
Carcinogenicity: rats Non-genotoxic Rodent Carcinogens MTD: Mice
15.4
30.6
0.152
9.2
7.8
- 11.1114
6.4
9.5
(I.(131
MTD: Rats Sister-chromatid exchanges (SCE) Chromosomal aberrations (Cvt)
6.8 35.0
11.0 69.9
0.043 0.349
36.8
64.9
0.281
22.8
48.3
11.255
2(1. I
47.7
0.276
12.6
39.9
0.273
7.2
8.1
11.11tl9
3.5
3.2
- 0.003
1.2
11.6
- 0.006
Salmonella (Sty) Sensory irritation a2gNephrotoxicity Formation of genotoxic diazonium ion Sty and Cvt Sty and SCE SCE and Cvt SCE, Cvt and Sty Sty, SCE and Cvt, All Negative
Slope
III With structural alerts
IV With SCE
11%
(1%
100
Slope
9.9
52.3
0.424
7.7
5.5 8.1
23.5 33.8
0.181 ;' 11.257~'
15.1 4.1
40.5 21.9
0.254 " 0.178
7.9
29.8
11.219
I(XI
9.0
Slope
I).1113
Abbreviations: Sty, Salmonella; SCE: sister-chromatid exchanges; (5,'t, chromosomal aberrations. The slopes expected if the tests were independent is 11.08 for each pair of tests. The significance of the differences between the actual and expected results is p < 0.(XI01. , Only prevalences of 0 and 100% are shown for illustrative purposes. Complete distributions are shown in Figs. 1-6.
27 short-term test, on the other. Such analyses may lead to a better understanding of the significance of these assays with respect to carcinogenic mechanisms as well as provide knowledge of the relationships of surrogates to one another (e.g. dependence, partial dependence, independence) which is necessary for the rational selection of predictive batteries of short-term tests (Rosenkranz and Ennever, 1988; Pet-Edwards et al., 1989).
Methodology The CASE method has been described on a number of occasions (Klopman et al., 1990; Rosenkranz and Klopman, 1989, 1990a,b). CASE selects its own descriptors from a learning set composed of active as well as inactive molecules. These descriptors are readily recognized as continuous structural fragments embedded in the molecule. The descriptors consist of either activating (biophore) or deactivating (biophobe) fragments. Each biophobe and biophore is characterized by its distribution among active and inactive molecules and the associated p value. Once biophores and biophobes have been identified, unknown molecules may be analyzed. Upon submission of such a molecule, the CASE program will generate all possible fragments ranging from 2 to 10 'heavy' atoms accompanied by their hydrogens and these will be compared to the previously identified biophores and biophobes. On the basis of the presence a n d / o r absence of these descriptors, CASE predicts activity or lack thereof.
Ddta bases A number of the data bases used by CASE as 'learning sets' were generated under the aegis of the U.S. National Toxicology Program: Mutagenicity in Salmonella typhimurium, induction of sister-chromatid exchanges and chromosomal aberrations in cultured CHO cells carcinogenicity in rodents (Ashby et al., 1989; Ashby and Tennant, 1988, 1991; Galloway et al., 1985, 1987; Gulati et al., 1989; Loveday et al., 1989; Tennant and Ashby, 1991; Tennant et al., 1987; Zeiger, 1987). Data on the maximum tolerated doses of
the chemicals used in the NTP cancer bioassays were kindly provided by Dr. R.W. Tennant, National Institute of Environmental Health Sciences. A data base related to binding to a2/~ in male rats was made available through the generosity of Dr. Lois D. Lehman-McKeeman, The Procter and Gamble Company. For the generation of a data base on non-genotoxic carcinogens, the results of the NTP cancer bioassays and Salmonella tests were used: A chemical was considered positive if it was carcinogenic in either the rat or the mouse or both and not mutagenic in Salmonella. Non-carcinogens or carcinogens which were also Salmonella mutagens were designated as negative. A data base of sensory irritants (Alarie, 1973; Alarie and Luo, 1986) was kindly assembled by Dr. Michelle Schaper of this Department. Some of the biophores and biophobes associated with individual endpoints have been described previously: Carcinogenicity in rodents (combined rats and mice), separately rats and mice (Rosenkranz and Klopman, 1990a,c,d), mutagenicity in Salmonella (Rosenkranz and Klopman, 1990b), induction of chromosomal aberrations and sister chromatid exchanges in cultured cells (Rosenkranz et al., 1990a), genotoxic diazonium ions following nitrosation of phenols (Rosenkranz et al., 1990b).
Simulation studies In order to construct a population of molecules which is representative of the universe of chemicals, chemicals were selected randomly from the Merck Index (10th edn.), the Handbook of Biochemistry (CRC) and various other publications and data bases. Our final population was composed of approximately 19% therapeutic agents, 29% natural products, 6% physiological chemicals, 9, 4, 17% respectively nitro-, amino- and halogen-containing substances. The potential toxicological properties of these molecules were determined using the above described data bases and the CASE program. Populations of varying prevalences with respect to specific properties were constructed by selecting active and inactive molecules at random.
28 100 ¸
Results and discussion
i
i---t-
Mutagenicity in Salmonella and structural alerts for genotoxicity The basis of the mutagenicity of chemicals is derived from their ability to interact with a n d / o r modify cellular DNA. These interactions include intercalation between DNA base pairs, (e.g. proflavin, ethidium bromide) and oxidative damage (e.g. peroxides). However, by far the largest group of chemicals derive their DNA-modifying ability from their actual or potential electrophilicity (e.g. alkylating agents, nitrosamines, polycyclic aromatic hydrocarbons, etc.) (Miller and Miller, 1977; Ashby, 1985). Indeed the 'structural alerts' for genotoxicity devised by Ashby (19851, can be used to recognize such electrophilic moieties (Ashby and Tennant, 19881. However, due to features unique to procaryotes (permeability, metabolism, physiology, etc.), the overlap between 'structural alerts' and mutagenieity is not perfect (i.e. there are 'cryptic' mutagens (Rosenkranz and Klopman, 1990e; Ashby, 1989)). Thus Michael-type acids may form electrophiles and indeed are thought to be 'genotoxic' carcinogens (Ashby et al., 1989) and yet they arc not mutagenie for Salmonella. On the other hand, there are Salmonella mutagens for which hcretoforc neither a structural nor a mechanistic basis has been found (e.g. diethylene glycol monoethylether). The present method permits an analysis of the relationship between mutagenicity in Salmonella and the presence of structural alerts using a simulated population consisting of molecules more representative of the 'chemical universe' than the existing data bases which may be largely congeneric or which were selected for specific protocols, i.e. NTP carcinogenicity assay, or testing in short-term tests. Our analysis reveals (Table 1, Number 1 and Fig. 1) that 12.5% of the molecules possess structural alerts yet are not predicted to be mutagens; as expected the molecules among this group include acrylates, methacrylates, as well as halogenated arylamines. On the other hand, 34.4% of the molecules which are mutagenic do not possess recognizable structural alerts (e.g. halogenated alkanes). Yet our analysis indicates that
80
....
.......
J
i
....
i
70 60 5(3
12-
4O 30
ii
10a
0:
0
.
.[. ,,
,2
10
20
_
1 .... ,'t
30 40 50 60 70 Mutagenicity in Salmonella (%)
80
90
1O0
Fig. 1. The effect of the prevalence of mutagens on the expected response of chemicals to other measures for genestoxicity. II, 'structural alerts' lbr gcnot~xicity: +. sistcr-chromatid exchanges; ~'. chromosomal aberrations: LJ, the formation of genotoxic diazonium ions following nitrosation.
this is the greatest overlap (53.1%, i.c. slope × 100) among the toxicological cndpoints that were compared in this study (Table 11.
Mutagenicity in Salmonella and carcinogenicity in rodents Based upon the NTP rodent carcinogenicity data base, we find that in a population of chemicals devoid of mutagens (i.e. prevalence = 0%), 22.5% of the molecules are predicted to be carcinogens (Table 1, Number 2), which may provide an approximation of the prevalence of 'nongenotoxic' carcinogens. On the other hand, the linear relationship between prevalence of expected mutagens and rodent carcinogens (Fig. 2), confirms the accepted relationship between mutagenicity and carcinogenicity, although the overlap is only 32%. However, for a population of pure mutagens, i.e. 1(X1% prevalence, only 55~7~ of the chemicals are predicted to be rodent carcinogens, i.e. 45% of the mutagens are not carcinogens, i.e. the rate of 'false-positive' predictions of carcinogenicity, based upon the results of the Salmonella assay, is 45%, This, in fact, is consistent with the analysis based upon the mutagenicity of a population of only carcinogens (above and Tablc 1, Number 10, Column 3 and [Zig. 3). A similar analysis of the relationship between prevalence of mutagens and the predicted pro-
29 10o
8o 70 60 n
....a r - ~ f
40 30
i ~
~
_....~,,~ ~
201
10-~ 0
,....~,~ e - - ~ ' ~ "-"me'~ ~
r lie "--'''-~
~
~ 10
"-'+"20
30 40 50 60 70 80 Mutagenicityin Salmonella(%)
90
100
Fig. 2. The effect of the prevalence of mutagens on the expected response of chemicals in rodent bioassays. II, carcinogenicity in rodents (rat and mouse, combined); *, carcinogenicity in rats; + , carcinogenicity in mice; [], 'non-genotoxic' rodent carcinogens.
portion of 'non-genotoxic' carcinogens (Table 1, Number 5) shows no increase, i.e. the two phenomena appear to be unrelated (slope = -0.014, Figure 2). Obviously, this is to be expected for mechanistic reasons, but it is not a tautology, as the CASE analyses were performed independently of one another using different data bases. This lack of relationship between mutagenicity and 'non-genotoxic' carcinogenicity in rodents also serves to establish the validity of the analytic methodology used herein. 100 90 80 70
401 30" 20~
0
0
Tb ~
~
4b 5b e,b io
Cardnogenlcltyin Rodent(%)
8b
9b Too
Fig. 3. The effect of the prevalence of carcinogens on the expected response of chemicals in genotoxicity assays. *, mutagenicity in Salmonella; II, induction of chromosomal aberrations; +, induction of sister-chromatid exchanges.
As expected from the results of the analyses of the NTP rodent carcinogenicity, the predicted proportion of rat- and mouse-specific carcinogens also increases as the prevalence of mutagens increases (Table 1, Numbers 3 and 4 and Fig. 2), i.e. a proportion of chemical-caused cancers in these species is due to a mutagenic ('genotoxic') mechanism. However, in these individual species the proportion of non-carcinogenic mutagens ('false-positives') is very high, i.e. - 7 0 % and 73% for rat and mouse, respectively. Actually, this conforms to previous analyses which indicated that chemicals carcinogenic for one species only are more likely to be non-mutagens (Ashby and Tennant, 1988; Ashby et al., 1989; Gold et al., 1989). It is puzzling, however, that the mouse-specific and rat-specific dose-response curves are additive; together they correspond to the rodent carcinogenicity curve (Fig. 2 and Table 1, Numbers 3 plus 4 = Number 2). This would suggest that the ability to cause cancers in the two species results from two species-specific independent processes and that it does not reflect a unique mechanism for multiple-species carcinogenicity. Obviously, this is an unexpected conclusion which merits further investigation.
Mutagenieity in Salmonella and other biological endpoints m rodents By and large, mutagenicity in Salmonella may reflect actual or potential electrophilic moieties in molecules (and see above). Such reactivities may endow molecules with a number of potentially toxic attributes which may not necessarily reflect actual mechanistic similarities yet could lead to fortuitous associations. In order to test this possibility, we undertook an analysis of the possible relationship between prevalence of mutagens and ability to induce sensory irritation in mice. The latter toxic end-point is known to be influenced by the presence of reactive moieties (Nielsen et al., 1990; Roberts, 1985). The analysis (Table 1, Number 11) shows that mutagenicity in Salmonella is unrelated to sensory irritation (as evidenced by a slope of 0.009; Fig. 4). In order to evaluate this possibility further and in view of the postulated existence of putative 'cryptic' mutagens, we also examined the relationship between the potential for sensory irritation and 'structural
30
tO0 _
90
t
I
..
i
i
v
;
I
80 70
...........
60 50
#.
40
t
30~
....
20
0: 0
10
20
.
.
.
.
.
.
30 40 50 60 70 Mutagenicily in Salmonella (%)
'i
!i .
80
90
~
basis of the pathogenesis of some renal tumors in male rats which occur by ,t "non-genotoxic" mechanism (Swcnbcrg et al., 1989). Indeed. inducers of this nephropathy appear to be cxclusixcly nonmutagens (2,2,4-trimcthylpcntanc, ¢~-limoncnc, etc.) (Goldworthy et al., 1988: Lchman-McKccman et al., 1989). As expected from these mechanistic considerations, there was no increase in the proportion of chemicals predicted as capable of inducing this effect with increase in the prevalence of mutagcns (Fig. 4: Table I, Number 12: slope = - 0.003).
100
Fig. 4. The effect of the p r e v a l e n c e of m u t a g c n s o n t h e e x p e c t e d r e s p o n s e of c h e m i c a l s in w h o l e a n i m a l assays. II, i n d u c t i o n of n e p h r o t o x i c i t y in male rats due to b i n d i n g to ~ 2 / z - g l o b u l i n . + , m a x i m u m t o l e r a t e d dose ( M T I ) ) in mice; * M T D in rats: LJ. induction of sensory irritation in mice. For the p r e d i c t i o n of the M T D , a positive p r e d i c t i o n was arbitrarily set at ~ l(]~l m g / k g / d a y .
alerts' for genotoxicity (i.e. electrophilicity). Again the analysis indicated no significant rclationship between sensory irritation and 'structural alcrts" for genotoxicity (i.e. electrophilicity) (Table 1, II1, Numbcr 11 and Fig. 5). One of the data bases available to us was of particular interest with rcspect to the prcscnt analyses, i.e. the ability to induce hyaline droplets (nephrotoxicity) in male rats duc to binding to a2#-globulin. This has been proposed as thc
loo I 90
80 70
g~
Mutagenicio" m Salmonella attd the maximum tolerated dose A numbcr of relationships have been postulated between the ability to induce cancers in rodents and the maximum tolerated dose (MTD) (Crouch ct al., 1987; Bernstcin c t a l . , 1985a, b: Hocl ct al., 1988; Zcise et al., 1986). in ordcr to explore this possibility, wc acquired the NTP M T D data (scc also Brown and Ashby, 19901 and, using an arbitrary cut-off of 100 m g / k g / d a y (i.e. < 100 m g / k g / d a y is activc; > 1(10 mg/kg/day, inactive), wc performed separate CASE analyscs of the structural basis of M T D in mice and in rats. Although the results of these analyses are as yet only preliminary, we performed a sufficicnt number of analyses to determine the effect of prevalence of mutagens on the proportion of chemicals with M T D values Icss than 100 m g / k g / d a y . Thc rcsults (Fig. 4; Table 1, Numbers 6 and 7) suggest only a minimal relationship between mutagcnicity in Salmonella and potency in the M T D protocol. The possiblc significance of this relationship is as yet tmknown and must await the results of detailed analyses including the use of cut-off values lower than the arbitrary cut-off of 100 m g / k g / d a y .
,o
30 20 10:~ ~
~
. . . . . .
_
~ _
J
m -
-
-
/
0
0
10
20
30
40 50 60 70 Structural Alerts (%)
80
90
100
Fig. 5. l,ack of r e l a t i o n s h i p b e t w e e n the p r e v a l e n c e of chemicals with "structural alerts" for genotoxicity and the induction of sensory irritation in mice.
Mutagenicity in Salmonella and other genotoxic assays A relationship between carcinogenicity in rodents and the induction of both chromosomal (Ivt) aberrations and sistcr-chromatid exchanges (SEE) was flmnd. It is to bc noted, however, that a significant number of non-carcinogens (20.1 and
31 36.8%, respectively) are expected to respond positively and between 52% and 35% are expected to be non-cytogenotoxic carcinogens (Fig. 3 and Table 1, II, Numbers 8 and 9). In other words, a relationship exists but it is far from an exact one; indeed the slopes for both curves are only 0.28. Additionally, it can be deduced that these assays are not independent in view of the fact that the expected dose-response curves of combinations of tests (multiplicative of individual probabilities) are less than the curves representing the actual results of the frequency of chemicals positive in two or three assays (Fig. 6 and Table 1, Numbers 14-16). In the present study we show that indeed both Cvt and SeE are related to the induction of mutations in Salmonella although 23% and 35% of Cvt and SCE positive chemicals are predicted to be non-mutagens and 52% and 30% of mutagens are predicted to respond negatively in Cvt and SCE tests, respectively (Fig. 1, Table 1, I, Numbers 8 and 9). Thus the overlap is only 26% and 35%, respectively. However, even though there is little overlap between the structural determinants of Cvt and SCE (Rosenkranz et al., 1990a,b), those few common determinants must represent principal biophore species, as on the basis of the present analysis there is significant
100 9O 8O
70 1E
6O
0
10
20
30 40 50 60 70 Carctnog~icity in Rodents (%)
80
90
100
Fig. 6. The effect of the prevalence of carcinogens on the expected positive response of chemicals in combinations of short-term tests, m, mutagenicity and chromosomal aberrations; +, mutagenicity and SeE; *, SeE and chromosomal aberrations; D, mutagenicity, SeE and chromosomal aberrations.
relationship between SCE and Cvt (Table 1, IV, Number 9; overlap = 42.4%). An analysis of the relationship between mutagenicity in Salmonella and predicted ability of forming DNA-reactive diazonium ions following nitrosation (Rosenkranz et al., 1990b), shows no commonality (Fig. 1 and Table 1, Number 13; slope = -0.006). This is not unexpected given the fact that the ability to form such reactive ions is associated with substituted phenols (Rosenkranz et al., 1990b), a species that is not inherently mutagenic in Salmonella. Conclusions
The present analyses have provided a measure not only of overlap between bioassays, but also of the extent of non-association, e.g. while the overlap between carcinogenicity in rodents and response in the Salmonella mutagenicity assay is 32%, 22% of rodent carcinogens are predicted to be mutagens and 45% of mutagens are projected to be non-carcinogens. Such analyses may provide a framework for developing a strategy for deploying single and combinations of tests, interpreting results of such tests and generating mechanistic hypotheses. Additionally the method may have other applications; thus, for example, we might interpret the finding that in Japan approximately 12% of chemicals tested for mutagenicity in Salmonella are found to be positive (Matsushima, 1987), as suggesting a prevalence of 26.4% [22.5 + (12 × 0.324)] carcinogens (i.e. 22.5% non-mutagenic carcinogens and 3.9% mutagenic carcinogens) among the population of chemicals tested. If we accept the premise that 'mutagenic' carcinogens present a greater risk to humans (Williams, 1987, 1990; Wilson, 1989; Netherlands, 1980), then the 4% prevalence suggests a situation where alternative strategies may be acceptable (Lave et al., 1988). Obviously, the type of studies described herein are preliminary in nature; they do suggest, however, that overall the currently used short-term tests are not highly predictive individually and that they reflect a spectrum of partially overlapping mechanisms, presumably involving mutagenicity/electrophilicity. However, because there
32
arc significant non-overlapping domains, the possibility to identify batteries of complementary tests for subclasses of chemicals exists. Such a hypothesis can be tested by using the method described herein on specific chemical classes or groups of chemicals sharing CASE-generated biophores. Additionally, the present approach can be used to test mechanistic hypotheses, such as the relationships between MTD and genotoxicity or between nephrotoxicity and carcinogenicity. One advantage of the present method is that it does not deal with restricted (and therefore biased) subsets of data such as alkylating agents or nitrosamines, but with populations of molecules representing a spectrum of origins and uses. Acknowledgements This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and the U.S. Environmental Protection Agency (R8154881. References Alarie, Y, (19731 Sensory irritation by airborne chemicals, CRC Crit. Rcv. Toxicol.. 299-363. Alarie, Y.. and J.E. Luo, 119861 Sensory irritation by airborne chemicals: A basis to establish acceptable levels of exposure, in: C.S. Barrow lEd.J, Toxicology of the Nasal Passages, }lemispherc Publishing Corporation. Washington. pp. 91- IIXI. Ashby, J. (1985) Fundamental structural alerts to potential careinogenicity or non-earcinogenicity, Environ. Mutagen., 7, 919-921. Ashby, J. 119891 Are non-mutagenic carcinogens merely crypto-mutagens'? Mutation Res., 216, 267. Ashby, J.. and R.W. Tennant (19881 Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S, N C I / N T P , Mutation Rcs., 204, 17-115. Ashby, J., and R.W. Tennant 119911 Definitive relationships among chemical structure carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP, Mutation) Rcs., 257, 229-3116. Ashby, J., R.W. Tennant. E. Zeiger and S. Stasiewicz (19891 Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the II.S. National Toxicology Program, Mutation Res., 223, 73-1113. Bernstein, L., L.S. Gold, B.N. Ames, M.C. Pike and D.G. l'loel (1985a) Some taut,alogous aspects of the comparis(m
of carcinogenic I~)tency m rats and mice. I-und. A p p l Toxicol., 5, 79 N6. Bernstein, I,., L.S. Gold, B.N. Ames. M.('. Pike and I).(L Hoel (1985h1 Toxicity and carcinogenic potency, Risk Anal., 5, 263-264. Brown. I,.P.. and J. Ashby ( 19901 Correlations between hioassay dose-level, mutagenicity to Salmonella, chemical structure and sites of carcinogenesis among 226 chemical', evaluated for carcinogenicity by the tr.S. NTI', Mutation Res., 244.67 76. Crouch. I'L, R. Wilson and L. Zcisc (19871 Tautology or not tautology'? J. Toxicol. Environ. Hllh., 21), I -li). Galloway, S.M.. A.D. Bloom, M. Resnick, B.ll. Margolin. F. Nakamura, P, Archer and IZ. Zeiger (19851 i)evelopmcrtt of a standard protocol for m vitro cytogcnetic testing with ('hincsc hamster ovary cells: ('omparison of results for 22 cornp()unds ill two laboratories, Environ. Mutagen., 7. 1 51. Galloway, S.M.. M.J. Armstrong, C. Reuben. S. ('olman, B. Brown. C. Cantata. A.D. Bloom, F. Nakamura. M. Ahmcd. S. Duk, J. Rimpo, B.II. Margolin, M.A. Resnick, B. Anderson and E. Zcigcr (19871 Chromosome abcrrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Fmviron. Mol. Mutagcn., 10 (Suppl. 10). I 175. Gold. I..S.. L. Bernstcin, R. Magav, and T.II. Shmc 119891 Intcrspccics extrapolation in carcinogenesis: F'rcdiction between rats and mice, Environ. llcalth Perspcct.. ~1, 211-219. Goldsworthy. T.L., O. Lyght, V.[,. Burnctt artd J.A. Popp 119881 Potential role of ~,-2~-globulin, protein droplet accurnulation, and cell replication in the renal carcino. gcnicity of rat~, exposed to trichloroethylcnc, perchlorocthylcne and pentachlorocthanc. Toxicol. Appl. Pharmacol., 96. 367 37cL Gulati, I)K., K. Witt, 13, Anderson, E. Zciger and M.I). Shelby (19891 Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells m vitr¢~. Ill. Results with 27 chemicals. Environm. Mtfl. Mutagcn.. 13, 133 193. Itoel, I).G., J K . Ilascman, M.D. Hogan, J. Iluff and t.Lf-. Me('onnell (19881 The impact of toxicity on carcinogenicity studies: Implications tor risk assessment, f'arcinogcnicity. 9, 21145-2052. Klopman, (L, and tt.S. Roscnkranz ( 19911 Structure--activity, relati(ms: Maximizing the usefulness of mutagenicity and carcinogcnicity data bases. Environ. Health Perspect.. m press. Klopman, (L, M . R Frierson and I t.S. Rosenkranz (19901 The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: "]'he Genc-Tox Data Base, Mutation Res., 228, 1-51). l,ehman-McKeeman, I,.D.. P.A. Rodriguez, R. Takigiku. D. ('audill and M.I,. Fcy (19801 d-lJmonenc-induccd ratspecific ncphrotoxieity: Evaluation of the association between d-limonene and ~,2/J,-globulin. Toxicol. Appl. Pharmacol., 9¢,L 250-259. l,(wcday, K.S., M.tt. Lugo, M.A. Resnick, B.I-. Anderson and E. Zcigcr (1989) Chromosome aberration and sister chromatid exchange tests in Chine~,c hamstcs ovary cells in
33 vitro, II. Results with 20 chemicals, Environ. Mol. Mutagen., 13, 60-94. Matsushima, T. (1987) Chemical Safety in Japan, pp. 175-178. Miller, J.A., and E.C. Miller (1977) Ultimate chemical carcinogens as reactive mutagenic electrophiles, in: H.H. Hiart, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. 605-627. Netherlands (1980) Health Council of The Netherlands, Report of the Evaluation of the Carcinogenicity of Chemical Substances, Government Printing Office, The Hague. Pet-Edwards, J., Y.Y. Haimes, V. Chankong, H.S. Rosenkranz and F.K. Ennever (1989) Risk Assessment and Decision Making Using Test Results: The Carcinogenicity Prediction and Battery Selection Approach, Plenum, New York, 221 pp. Roberts, D.W. (1986) QSAR for upper-respiratory tract irritation, Chem.-Biol. Interact., 57, 325-345. Rosenkranz, H.S., and F.K. Ennever (1988) Quantifying genotoxicity and non-genotoxicity, Mutation Res., 205, 59-67. Rosenkranz, H.S., and G. Klopman (1989) Structural basis of the mutagenicity of phenylazoaniline dyes, Mutation Res., 221, 217-239. Rosenkranz, H.S., and G. Klopman (1990a) Structural basis of carcinogenicity in rodents of genotoxicants and nongenotoxicants, Mutation Res., 228, 105-124. Rosenkranz, H.S., and G. Klopman (1990b) The structural basis of the mutagenicity of chemicals in Salmonella typhimuriurn: The National Toxicology Program data base, Mutation Res., 228, 51-80. Rosenkranz, H.S., and G. Klopman (1990c) Natural pesticides present in edible plants are predicted to be carcinogenic, Carcinogenesis, 11,349-353. Rosenkranz, H.S., and G. Klopman (1990d) Prediction of the carcinogenicity in rodents of chemicals currently being tested by the U.S. National Toxicology Program: Structure-activity correlations, Mutagenesis, 5, 425-432. Rosenkranz, H.S., and G. Klopman (1990e) 'Cryptic' mutagens and carcinogenicity, Mutagenesis, 5, 199-202. Rosenkranz, H.S., F.K. Ennever, M. Dimayuga and G. Klopman (1990a) Significant differences in the structural basis
of the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells, Environ. Mol. Mutagen., 16, 149-177. Rosenkranz, H.S., G. Klopman, H. Ohshima and H. Bartsch (1990b) Structural basis of the genotoxicity of nitrosatable phenols and derivatives present in smoked food products, Mutation Res., 230, 9-27. Swenberg, J.A., B. Short, S. Borghoff, J. Strasser and M. Charbonneau (1989) The comparative pathobiology of a2p.-globulin nephropathy, Toxicol. Appl. Pharmacol., 97, 35-46. Tennant, R.W., B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. tlaseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson and R. Minor (1987) Prediction of chemical carcinogenicity in rodents from in vitro genotoxicity assays, Science, 236, 933-941. Tennant, R.W., and J. Ashby (1991) Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program, Mutation Res., 257, 209-227. Williams, G.M. (1987) Definition of a human cancer hazard, in: B.E. Butterworth an,d T.J. Slaga (Eds.), Nongenotoxic Mechanisms in Carcinogenesis, Banbury Report 25 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 367-38{). Williams, G.M. (1990) Screening procedures for evaluating the potential carcinogenicity of food-packaging chemicals, Reg. Toxicol. Pharmacol., 12, 30-40. Wilson, J.D. (1989) Assessment of low-exposure risk from carcinogenesis: Implications of the Knudson-Moolgavkar Two-Critical Mutation Theory, in: C.C. Travis (Ed.), Biologically-Based Methods for Cancer Risk Assessment Plenum, New York, pp. 275-287. Zeiger, E. (1987) Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity, Cancer Res.. 47, 1287-1296. Zeise, L., E.A.C. Crouch and R. Wilson (1986) A possible relationship between toxicity and carcinogenicity, J. Am. Coll. Toxicol., 5, 137-151.
25
MUT 02530
Implications of newly recognized relationships between mutagenicity, genotoxicity and carcinogenicity of molecules Herbert S. Rosenkranz a, Ying Ping Zhang and Gilles Klopman b a
Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 and b Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106 (U.S.A.) (Accepted 16 April 1991)
Keywords: Relationships, between molecules; CASE structure-activity relational method
Summary The CASE structure-activity relational method was used to predict the mutagenicity, cytogenotoxicity, carcinogenicity, sensory irritation, male rat-specific a2/z-nephrotoxicity and maximum tolerated dose of a population of molecules (N >_ 1300). These chemicals were then sorted out by their predicted responses to specific tests and sub-populations of molecules with different prevalence with respect to described endpoints were constructed, i.e. 0-100% prevalences of mutagens, rodent carcinogens and SCE inducers. The predicted properties of these populations were analyzed and the overlap among tests was determined. The method also permits the determination of the dependence among assays and the level of false-positive and false-negative predictions.
The recent realization that a number of shortterm tests used to predict potential carcinogenicity may not be highly predictive has raised some questions regarding the selections of combinations of tests (batteries) for predicting carcinogenicity, the mechanisms of carcinogenicity itself and the significance of a finding of mutagenicity and/or genotoxicity in the absence of knowledge regarding carcinogenicity. Recently we have developed a method for addressing these problems. We have taken advantage of the ability of the CASE structure-activity
Correspondence: Prof. Herbert S. Rosenkranz, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261 (U.S.A.).
relational system (Klopman et al., 1990; Rosenkranz and Klopman, 1989) to predict the carcinogenic, mutagenic and genotoxic properties of molecules to simulate the properties of populations of molecules containing different proportions (prevalences) of chemicals with specific properties. Moreover, by comparing experimental with simulated results we have established the validity of the procedure. In a preliminary analysis we showed (Klopman and Rosenkranz, 1991) that, in simulated populations of molecules in which the prevalence of carcinogens ranged from 0% to 100%, mutagenicity ranged from 14% to 44% suggesting (a) that mutagenicity in Salmonella when used as a surrogate for carcinogenicity, may yield 14% false-positive predictions and (b) that 44% of carcinogens may be 'nongenotoxic' (see also Table 1, Number 10). How-
26 ever, that analysis did not address the usual situa-
primarily the relationships between the projected
t i o n , i.e. t h e s i g n i f i c a n c e o f t h e r e s u l t s o f s h o r t -
r e s u l t s o f a g r o u p o f in v i t r o a n d c a n c e r b i o a s s a y s ,
t e r m t e s t s in t h e a b s e n c e o f r o d e n t c a n c e r b i o a s -
on the one hand, and the results of the Salmonella
say d a t a .
m u t a g e n i c i t y assay, p r o b a b l y t h e m o s t w i d e l y u s e d
In t h e
present
analysis we
consider
TABLE 1 SUMMARY OF THE OVERLAP OF POSITIVE RESPONSES b No.
1 2 3 4 5
6
7 8 9
10 11 12 13
14 15 16 17 18
Biological system
Extent of overlaps I With Salmonella
II With carcinogenicity in rodents
(1%
10(1
0%
100 %
Slope
Structural alerts Carcinogenicity: rodents Carcinogenicity: mice
12.5 22.5
65.6 54.9
0.531 0.324
9.2
26.6
0.174
Carcinogenicity: rats Non-genotoxic Rodent Carcinogens MTD: Mice
15.4
30.6
0.152
9.2
7.8
- 11.1114
6.4
9.5
(I.(131
MTD: Rats Sister-chromatid exchanges (SCE) Chromosomal aberrations (Cvt)
6.8 35.0
11.0 69.9
0.043 0.349
36.8
64.9
0.281
22.8
48.3
11.255
2(1. I
47.7
0.276
12.6
39.9
0.273
7.2
8.1
11.11tl9
3.5
3.2
- 0.003
1.2
11.6
- 0.006
Salmonella (Sty) Sensory irritation a2gNephrotoxicity Formation of genotoxic diazonium ion Sty and Cvt Sty and SCE SCE and Cvt SCE, Cvt and Sty Sty, SCE and Cvt, All Negative
Slope
III With structural alerts
IV With SCE
11%
(1%
100
Slope
9.9
52.3
0.424
7.7
5.5 8.1
23.5 33.8
0.181 ;' 11.257~'
15.1 4.1
40.5 21.9
0.254 " 0.178
7.9
29.8
11.219
I(XI
9.0
Slope
I).1113
Abbreviations: Sty, Salmonella; SCE: sister-chromatid exchanges; (5,'t, chromosomal aberrations. The slopes expected if the tests were independent is 11.08 for each pair of tests. The significance of the differences between the actual and expected results is p < 0.(XI01. , Only prevalences of 0 and 100% are shown for illustrative purposes. Complete distributions are shown in Figs. 1-6.
27 short-term test, on the other. Such analyses may lead to a better understanding of the significance of these assays with respect to carcinogenic mechanisms as well as provide knowledge of the relationships of surrogates to one another (e.g. dependence, partial dependence, independence) which is necessary for the rational selection of predictive batteries of short-term tests (Rosenkranz and Ennever, 1988; Pet-Edwards et al., 1989).
Methodology The CASE method has been described on a number of occasions (Klopman et al., 1990; Rosenkranz and Klopman, 1989, 1990a,b). CASE selects its own descriptors from a learning set composed of active as well as inactive molecules. These descriptors are readily recognized as continuous structural fragments embedded in the molecule. The descriptors consist of either activating (biophore) or deactivating (biophobe) fragments. Each biophobe and biophore is characterized by its distribution among active and inactive molecules and the associated p value. Once biophores and biophobes have been identified, unknown molecules may be analyzed. Upon submission of such a molecule, the CASE program will generate all possible fragments ranging from 2 to 10 'heavy' atoms accompanied by their hydrogens and these will be compared to the previously identified biophores and biophobes. On the basis of the presence a n d / o r absence of these descriptors, CASE predicts activity or lack thereof.
Ddta bases A number of the data bases used by CASE as 'learning sets' were generated under the aegis of the U.S. National Toxicology Program: Mutagenicity in Salmonella typhimurium, induction of sister-chromatid exchanges and chromosomal aberrations in cultured CHO cells carcinogenicity in rodents (Ashby et al., 1989; Ashby and Tennant, 1988, 1991; Galloway et al., 1985, 1987; Gulati et al., 1989; Loveday et al., 1989; Tennant and Ashby, 1991; Tennant et al., 1987; Zeiger, 1987). Data on the maximum tolerated doses of
the chemicals used in the NTP cancer bioassays were kindly provided by Dr. R.W. Tennant, National Institute of Environmental Health Sciences. A data base related to binding to a2/~ in male rats was made available through the generosity of Dr. Lois D. Lehman-McKeeman, The Procter and Gamble Company. For the generation of a data base on non-genotoxic carcinogens, the results of the NTP cancer bioassays and Salmonella tests were used: A chemical was considered positive if it was carcinogenic in either the rat or the mouse or both and not mutagenic in Salmonella. Non-carcinogens or carcinogens which were also Salmonella mutagens were designated as negative. A data base of sensory irritants (Alarie, 1973; Alarie and Luo, 1986) was kindly assembled by Dr. Michelle Schaper of this Department. Some of the biophores and biophobes associated with individual endpoints have been described previously: Carcinogenicity in rodents (combined rats and mice), separately rats and mice (Rosenkranz and Klopman, 1990a,c,d), mutagenicity in Salmonella (Rosenkranz and Klopman, 1990b), induction of chromosomal aberrations and sister chromatid exchanges in cultured cells (Rosenkranz et al., 1990a), genotoxic diazonium ions following nitrosation of phenols (Rosenkranz et al., 1990b).
Simulation studies In order to construct a population of molecules which is representative of the universe of chemicals, chemicals were selected randomly from the Merck Index (10th edn.), the Handbook of Biochemistry (CRC) and various other publications and data bases. Our final population was composed of approximately 19% therapeutic agents, 29% natural products, 6% physiological chemicals, 9, 4, 17% respectively nitro-, amino- and halogen-containing substances. The potential toxicological properties of these molecules were determined using the above described data bases and the CASE program. Populations of varying prevalences with respect to specific properties were constructed by selecting active and inactive molecules at random.
28 100 ¸
Results and discussion
i
i---t-
Mutagenicity in Salmonella and structural alerts for genotoxicity The basis of the mutagenicity of chemicals is derived from their ability to interact with a n d / o r modify cellular DNA. These interactions include intercalation between DNA base pairs, (e.g. proflavin, ethidium bromide) and oxidative damage (e.g. peroxides). However, by far the largest group of chemicals derive their DNA-modifying ability from their actual or potential electrophilicity (e.g. alkylating agents, nitrosamines, polycyclic aromatic hydrocarbons, etc.) (Miller and Miller, 1977; Ashby, 1985). Indeed the 'structural alerts' for genotoxicity devised by Ashby (19851, can be used to recognize such electrophilic moieties (Ashby and Tennant, 19881. However, due to features unique to procaryotes (permeability, metabolism, physiology, etc.), the overlap between 'structural alerts' and mutagenieity is not perfect (i.e. there are 'cryptic' mutagens (Rosenkranz and Klopman, 1990e; Ashby, 1989)). Thus Michael-type acids may form electrophiles and indeed are thought to be 'genotoxic' carcinogens (Ashby et al., 1989) and yet they arc not mutagenie for Salmonella. On the other hand, there are Salmonella mutagens for which hcretoforc neither a structural nor a mechanistic basis has been found (e.g. diethylene glycol monoethylether). The present method permits an analysis of the relationship between mutagenicity in Salmonella and the presence of structural alerts using a simulated population consisting of molecules more representative of the 'chemical universe' than the existing data bases which may be largely congeneric or which were selected for specific protocols, i.e. NTP carcinogenicity assay, or testing in short-term tests. Our analysis reveals (Table 1, Number 1 and Fig. 1) that 12.5% of the molecules possess structural alerts yet are not predicted to be mutagens; as expected the molecules among this group include acrylates, methacrylates, as well as halogenated arylamines. On the other hand, 34.4% of the molecules which are mutagenic do not possess recognizable structural alerts (e.g. halogenated alkanes). Yet our analysis indicates that
80
....
.......
J
i
....
i
70 60 5(3
12-
4O 30
ii
10a
0:
0
.
.[. ,,
,2
10
20
_
1 .... ,'t
30 40 50 60 70 Mutagenicity in Salmonella (%)
80
90
1O0
Fig. 1. The effect of the prevalence of mutagens on the expected response of chemicals to other measures for genestoxicity. II, 'structural alerts' lbr gcnot~xicity: +. sistcr-chromatid exchanges; ~'. chromosomal aberrations: LJ, the formation of genotoxic diazonium ions following nitrosation.
this is the greatest overlap (53.1%, i.c. slope × 100) among the toxicological cndpoints that were compared in this study (Table 11.
Mutagenicity in Salmonella and carcinogenicity in rodents Based upon the NTP rodent carcinogenicity data base, we find that in a population of chemicals devoid of mutagens (i.e. prevalence = 0%), 22.5% of the molecules are predicted to be carcinogens (Table 1, Number 2), which may provide an approximation of the prevalence of 'nongenotoxic' carcinogens. On the other hand, the linear relationship between prevalence of expected mutagens and rodent carcinogens (Fig. 2), confirms the accepted relationship between mutagenicity and carcinogenicity, although the overlap is only 32%. However, for a population of pure mutagens, i.e. 1(X1% prevalence, only 55~7~ of the chemicals are predicted to be rodent carcinogens, i.e. 45% of the mutagens are not carcinogens, i.e. the rate of 'false-positive' predictions of carcinogenicity, based upon the results of the Salmonella assay, is 45%, This, in fact, is consistent with the analysis based upon the mutagenicity of a population of only carcinogens (above and Tablc 1, Number 10, Column 3 and [Zig. 3). A similar analysis of the relationship between prevalence of mutagens and the predicted pro-
29 10o
8o 70 60 n
....a r - ~ f
40 30
i ~
~
_....~,,~ ~
201
10-~ 0
,....~,~ e - - ~ ' ~ "-"me'~ ~
r lie "--'''-~
~
~ 10
"-'+"20
30 40 50 60 70 80 Mutagenicityin Salmonella(%)
90
100
Fig. 2. The effect of the prevalence of mutagens on the expected response of chemicals in rodent bioassays. II, carcinogenicity in rodents (rat and mouse, combined); *, carcinogenicity in rats; + , carcinogenicity in mice; [], 'non-genotoxic' rodent carcinogens.
portion of 'non-genotoxic' carcinogens (Table 1, Number 5) shows no increase, i.e. the two phenomena appear to be unrelated (slope = -0.014, Figure 2). Obviously, this is to be expected for mechanistic reasons, but it is not a tautology, as the CASE analyses were performed independently of one another using different data bases. This lack of relationship between mutagenicity and 'non-genotoxic' carcinogenicity in rodents also serves to establish the validity of the analytic methodology used herein. 100 90 80 70
401 30" 20~
0
0
Tb ~
~
4b 5b e,b io
Cardnogenlcltyin Rodent(%)
8b
9b Too
Fig. 3. The effect of the prevalence of carcinogens on the expected response of chemicals in genotoxicity assays. *, mutagenicity in Salmonella; II, induction of chromosomal aberrations; +, induction of sister-chromatid exchanges.
As expected from the results of the analyses of the NTP rodent carcinogenicity, the predicted proportion of rat- and mouse-specific carcinogens also increases as the prevalence of mutagens increases (Table 1, Numbers 3 and 4 and Fig. 2), i.e. a proportion of chemical-caused cancers in these species is due to a mutagenic ('genotoxic') mechanism. However, in these individual species the proportion of non-carcinogenic mutagens ('false-positives') is very high, i.e. - 7 0 % and 73% for rat and mouse, respectively. Actually, this conforms to previous analyses which indicated that chemicals carcinogenic for one species only are more likely to be non-mutagens (Ashby and Tennant, 1988; Ashby et al., 1989; Gold et al., 1989). It is puzzling, however, that the mouse-specific and rat-specific dose-response curves are additive; together they correspond to the rodent carcinogenicity curve (Fig. 2 and Table 1, Numbers 3 plus 4 = Number 2). This would suggest that the ability to cause cancers in the two species results from two species-specific independent processes and that it does not reflect a unique mechanism for multiple-species carcinogenicity. Obviously, this is an unexpected conclusion which merits further investigation.
Mutagenieity in Salmonella and other biological endpoints m rodents By and large, mutagenicity in Salmonella may reflect actual or potential electrophilic moieties in molecules (and see above). Such reactivities may endow molecules with a number of potentially toxic attributes which may not necessarily reflect actual mechanistic similarities yet could lead to fortuitous associations. In order to test this possibility, we undertook an analysis of the possible relationship between prevalence of mutagens and ability to induce sensory irritation in mice. The latter toxic end-point is known to be influenced by the presence of reactive moieties (Nielsen et al., 1990; Roberts, 1985). The analysis (Table 1, Number 11) shows that mutagenicity in Salmonella is unrelated to sensory irritation (as evidenced by a slope of 0.009; Fig. 4). In order to evaluate this possibility further and in view of the postulated existence of putative 'cryptic' mutagens, we also examined the relationship between the potential for sensory irritation and 'structural
30
tO0 _
90
t
I
..
i
i
v
;
I
80 70
...........
60 50
#.
40
t
30~
....
20
0: 0
10
20
.
.
.
.
.
.
30 40 50 60 70 Mutagenicily in Salmonella (%)
'i
!i .
80
90
~
basis of the pathogenesis of some renal tumors in male rats which occur by ,t "non-genotoxic" mechanism (Swcnbcrg et al., 1989). Indeed. inducers of this nephropathy appear to be cxclusixcly nonmutagens (2,2,4-trimcthylpcntanc, ¢~-limoncnc, etc.) (Goldworthy et al., 1988: Lchman-McKccman et al., 1989). As expected from these mechanistic considerations, there was no increase in the proportion of chemicals predicted as capable of inducing this effect with increase in the prevalence of mutagcns (Fig. 4: Table I, Number 12: slope = - 0.003).
100
Fig. 4. The effect of the p r e v a l e n c e of m u t a g c n s o n t h e e x p e c t e d r e s p o n s e of c h e m i c a l s in w h o l e a n i m a l assays. II, i n d u c t i o n of n e p h r o t o x i c i t y in male rats due to b i n d i n g to ~ 2 / z - g l o b u l i n . + , m a x i m u m t o l e r a t e d dose ( M T I ) ) in mice; * M T D in rats: LJ. induction of sensory irritation in mice. For the p r e d i c t i o n of the M T D , a positive p r e d i c t i o n was arbitrarily set at ~ l(]~l m g / k g / d a y .
alerts' for genotoxicity (i.e. electrophilicity). Again the analysis indicated no significant rclationship between sensory irritation and 'structural alcrts" for genotoxicity (i.e. electrophilicity) (Table 1, II1, Numbcr 11 and Fig. 5). One of the data bases available to us was of particular interest with rcspect to the prcscnt analyses, i.e. the ability to induce hyaline droplets (nephrotoxicity) in male rats duc to binding to a2#-globulin. This has been proposed as thc
loo I 90
80 70
g~
Mutagenicio" m Salmonella attd the maximum tolerated dose A numbcr of relationships have been postulated between the ability to induce cancers in rodents and the maximum tolerated dose (MTD) (Crouch ct al., 1987; Bernstcin c t a l . , 1985a, b: Hocl ct al., 1988; Zcise et al., 1986). in ordcr to explore this possibility, wc acquired the NTP M T D data (scc also Brown and Ashby, 19901 and, using an arbitrary cut-off of 100 m g / k g / d a y (i.e. < 100 m g / k g / d a y is activc; > 1(10 mg/kg/day, inactive), wc performed separate CASE analyscs of the structural basis of M T D in mice and in rats. Although the results of these analyses are as yet only preliminary, we performed a sufficicnt number of analyses to determine the effect of prevalence of mutagens on the proportion of chemicals with M T D values Icss than 100 m g / k g / d a y . Thc rcsults (Fig. 4; Table 1, Numbers 6 and 7) suggest only a minimal relationship between mutagcnicity in Salmonella and potency in the M T D protocol. The possiblc significance of this relationship is as yet tmknown and must await the results of detailed analyses including the use of cut-off values lower than the arbitrary cut-off of 100 m g / k g / d a y .
,o
30 20 10:~ ~
~
. . . . . .
_
~ _
J
m -
-
-
/
0
0
10
20
30
40 50 60 70 Structural Alerts (%)
80
90
100
Fig. 5. l,ack of r e l a t i o n s h i p b e t w e e n the p r e v a l e n c e of chemicals with "structural alerts" for genotoxicity and the induction of sensory irritation in mice.
Mutagenicity in Salmonella and other genotoxic assays A relationship between carcinogenicity in rodents and the induction of both chromosomal (Ivt) aberrations and sistcr-chromatid exchanges (SEE) was flmnd. It is to bc noted, however, that a significant number of non-carcinogens (20.1 and
31 36.8%, respectively) are expected to respond positively and between 52% and 35% are expected to be non-cytogenotoxic carcinogens (Fig. 3 and Table 1, II, Numbers 8 and 9). In other words, a relationship exists but it is far from an exact one; indeed the slopes for both curves are only 0.28. Additionally, it can be deduced that these assays are not independent in view of the fact that the expected dose-response curves of combinations of tests (multiplicative of individual probabilities) are less than the curves representing the actual results of the frequency of chemicals positive in two or three assays (Fig. 6 and Table 1, Numbers 14-16). In the present study we show that indeed both Cvt and SeE are related to the induction of mutations in Salmonella although 23% and 35% of Cvt and SCE positive chemicals are predicted to be non-mutagens and 52% and 30% of mutagens are predicted to respond negatively in Cvt and SCE tests, respectively (Fig. 1, Table 1, I, Numbers 8 and 9). Thus the overlap is only 26% and 35%, respectively. However, even though there is little overlap between the structural determinants of Cvt and SCE (Rosenkranz et al., 1990a,b), those few common determinants must represent principal biophore species, as on the basis of the present analysis there is significant
100 9O 8O
70 1E
6O
0
10
20
30 40 50 60 70 Carctnog~icity in Rodents (%)
80
90
100
Fig. 6. The effect of the prevalence of carcinogens on the expected positive response of chemicals in combinations of short-term tests, m, mutagenicity and chromosomal aberrations; +, mutagenicity and SeE; *, SeE and chromosomal aberrations; D, mutagenicity, SeE and chromosomal aberrations.
relationship between SCE and Cvt (Table 1, IV, Number 9; overlap = 42.4%). An analysis of the relationship between mutagenicity in Salmonella and predicted ability of forming DNA-reactive diazonium ions following nitrosation (Rosenkranz et al., 1990b), shows no commonality (Fig. 1 and Table 1, Number 13; slope = -0.006). This is not unexpected given the fact that the ability to form such reactive ions is associated with substituted phenols (Rosenkranz et al., 1990b), a species that is not inherently mutagenic in Salmonella. Conclusions
The present analyses have provided a measure not only of overlap between bioassays, but also of the extent of non-association, e.g. while the overlap between carcinogenicity in rodents and response in the Salmonella mutagenicity assay is 32%, 22% of rodent carcinogens are predicted to be mutagens and 45% of mutagens are projected to be non-carcinogens. Such analyses may provide a framework for developing a strategy for deploying single and combinations of tests, interpreting results of such tests and generating mechanistic hypotheses. Additionally the method may have other applications; thus, for example, we might interpret the finding that in Japan approximately 12% of chemicals tested for mutagenicity in Salmonella are found to be positive (Matsushima, 1987), as suggesting a prevalence of 26.4% [22.5 + (12 × 0.324)] carcinogens (i.e. 22.5% non-mutagenic carcinogens and 3.9% mutagenic carcinogens) among the population of chemicals tested. If we accept the premise that 'mutagenic' carcinogens present a greater risk to humans (Williams, 1987, 1990; Wilson, 1989; Netherlands, 1980), then the 4% prevalence suggests a situation where alternative strategies may be acceptable (Lave et al., 1988). Obviously, the type of studies described herein are preliminary in nature; they do suggest, however, that overall the currently used short-term tests are not highly predictive individually and that they reflect a spectrum of partially overlapping mechanisms, presumably involving mutagenicity/electrophilicity. However, because there
32
arc significant non-overlapping domains, the possibility to identify batteries of complementary tests for subclasses of chemicals exists. Such a hypothesis can be tested by using the method described herein on specific chemical classes or groups of chemicals sharing CASE-generated biophores. Additionally, the present approach can be used to test mechanistic hypotheses, such as the relationships between MTD and genotoxicity or between nephrotoxicity and carcinogenicity. One advantage of the present method is that it does not deal with restricted (and therefore biased) subsets of data such as alkylating agents or nitrosamines, but with populations of molecules representing a spectrum of origins and uses. Acknowledgements This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and the U.S. Environmental Protection Agency (R8154881. References Alarie, Y, (19731 Sensory irritation by airborne chemicals, CRC Crit. Rcv. Toxicol.. 299-363. Alarie, Y.. and J.E. Luo, 119861 Sensory irritation by airborne chemicals: A basis to establish acceptable levels of exposure, in: C.S. Barrow lEd.J, Toxicology of the Nasal Passages, }lemispherc Publishing Corporation. Washington. pp. 91- IIXI. Ashby, J. (1985) Fundamental structural alerts to potential careinogenicity or non-earcinogenicity, Environ. Mutagen., 7, 919-921. Ashby, J. 119891 Are non-mutagenic carcinogens merely crypto-mutagens'? Mutation Res., 216, 267. Ashby, J.. and R.W. Tennant (19881 Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S, N C I / N T P , Mutation Rcs., 204, 17-115. Ashby, J., and R.W. Tennant 119911 Definitive relationships among chemical structure carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP, Mutation) Rcs., 257, 229-3116. Ashby, J., R.W. Tennant. E. Zeiger and S. Stasiewicz (19891 Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the II.S. National Toxicology Program, Mutation Res., 223, 73-1113. Bernstein, L., L.S. Gold, B.N. Ames, M.C. Pike and D.G. l'loel (1985a) Some taut,alogous aspects of the comparis(m
of carcinogenic I~)tency m rats and mice. I-und. A p p l Toxicol., 5, 79 N6. Bernstein, I,., L.S. Gold, B.N. Ames. M.('. Pike and I).(L Hoel (1985h1 Toxicity and carcinogenic potency, Risk Anal., 5, 263-264. Brown. I,.P.. and J. Ashby ( 19901 Correlations between hioassay dose-level, mutagenicity to Salmonella, chemical structure and sites of carcinogenesis among 226 chemical', evaluated for carcinogenicity by the tr.S. NTI', Mutation Res., 244.67 76. Crouch. I'L, R. Wilson and L. Zcisc (19871 Tautology or not tautology'? J. Toxicol. Environ. Hllh., 21), I -li). Galloway, S.M.. A.D. Bloom, M. Resnick, B.ll. Margolin. F. Nakamura, P, Archer and IZ. Zeiger (19851 i)evelopmcrtt of a standard protocol for m vitro cytogcnetic testing with ('hincsc hamster ovary cells: ('omparison of results for 22 cornp()unds ill two laboratories, Environ. Mutagen., 7. 1 51. Galloway, S.M.. M.J. Armstrong, C. Reuben. S. ('olman, B. Brown. C. Cantata. A.D. Bloom, F. Nakamura. M. Ahmcd. S. Duk, J. Rimpo, B.II. Margolin, M.A. Resnick, B. Anderson and E. Zcigcr (19871 Chromosome abcrrations and sister chromatid exchanges in Chinese hamster ovary cells: Evaluation of 108 chemicals. Fmviron. Mol. Mutagcn., 10 (Suppl. 10). I 175. Gold. I..S.. L. Bernstcin, R. Magav, and T.II. Shmc 119891 Intcrspccics extrapolation in carcinogenesis: F'rcdiction between rats and mice, Environ. llcalth Perspcct.. ~1, 211-219. Goldsworthy. T.L., O. Lyght, V.[,. Burnctt artd J.A. Popp 119881 Potential role of ~,-2~-globulin, protein droplet accurnulation, and cell replication in the renal carcino. gcnicity of rat~, exposed to trichloroethylcnc, perchlorocthylcne and pentachlorocthanc. Toxicol. Appl. Pharmacol., 96. 367 37cL Gulati, I)K., K. Witt, 13, Anderson, E. Zciger and M.I). Shelby (19891 Chromosome aberration and sister chromatid exchange tests in Chinese hamster ovary cells m vitr¢~. Ill. Results with 27 chemicals. Environm. Mtfl. Mutagcn.. 13, 133 193. Itoel, I).G., J K . Ilascman, M.D. Hogan, J. Iluff and t.Lf-. Me('onnell (19881 The impact of toxicity on carcinogenicity studies: Implications tor risk assessment, f'arcinogcnicity. 9, 21145-2052. Klopman, (L, and tt.S. Roscnkranz ( 19911 Structure--activity, relati(ms: Maximizing the usefulness of mutagenicity and carcinogcnicity data bases. Environ. Health Perspect.. m press. Klopman, (L, M . R Frierson and I t.S. Rosenkranz (19901 The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: "]'he Genc-Tox Data Base, Mutation Res., 228, 1-51). l,ehman-McKeeman, I,.D.. P.A. Rodriguez, R. Takigiku. D. ('audill and M.I,. Fcy (19801 d-lJmonenc-induccd ratspecific ncphrotoxieity: Evaluation of the association between d-limonene and ~,2/J,-globulin. Toxicol. Appl. Pharmacol., 9¢,L 250-259. l,(wcday, K.S., M.tt. Lugo, M.A. Resnick, B.I-. Anderson and E. Zcigcr (1989) Chromosome aberration and sister chromatid exchange tests in Chine~,c hamstcs ovary cells in
33 vitro, II. Results with 20 chemicals, Environ. Mol. Mutagen., 13, 60-94. Matsushima, T. (1987) Chemical Safety in Japan, pp. 175-178. Miller, J.A., and E.C. Miller (1977) Ultimate chemical carcinogens as reactive mutagenic electrophiles, in: H.H. Hiart, J.D. Watson and J.A. Winsten (Eds.), Origins of Human Cancer, Cold Spring Harbor Laboratory, Cold Spring Harbor, pp. 605-627. Netherlands (1980) Health Council of The Netherlands, Report of the Evaluation of the Carcinogenicity of Chemical Substances, Government Printing Office, The Hague. Pet-Edwards, J., Y.Y. Haimes, V. Chankong, H.S. Rosenkranz and F.K. Ennever (1989) Risk Assessment and Decision Making Using Test Results: The Carcinogenicity Prediction and Battery Selection Approach, Plenum, New York, 221 pp. Roberts, D.W. (1986) QSAR for upper-respiratory tract irritation, Chem.-Biol. Interact., 57, 325-345. Rosenkranz, H.S., and F.K. Ennever (1988) Quantifying genotoxicity and non-genotoxicity, Mutation Res., 205, 59-67. Rosenkranz, H.S., and G. Klopman (1989) Structural basis of the mutagenicity of phenylazoaniline dyes, Mutation Res., 221, 217-239. Rosenkranz, H.S., and G. Klopman (1990a) Structural basis of carcinogenicity in rodents of genotoxicants and nongenotoxicants, Mutation Res., 228, 105-124. Rosenkranz, H.S., and G. Klopman (1990b) The structural basis of the mutagenicity of chemicals in Salmonella typhimuriurn: The National Toxicology Program data base, Mutation Res., 228, 51-80. Rosenkranz, H.S., and G. Klopman (1990c) Natural pesticides present in edible plants are predicted to be carcinogenic, Carcinogenesis, 11,349-353. Rosenkranz, H.S., and G. Klopman (1990d) Prediction of the carcinogenicity in rodents of chemicals currently being tested by the U.S. National Toxicology Program: Structure-activity correlations, Mutagenesis, 5, 425-432. Rosenkranz, H.S., and G. Klopman (1990e) 'Cryptic' mutagens and carcinogenicity, Mutagenesis, 5, 199-202. Rosenkranz, H.S., F.K. Ennever, M. Dimayuga and G. Klopman (1990a) Significant differences in the structural basis
of the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells, Environ. Mol. Mutagen., 16, 149-177. Rosenkranz, H.S., G. Klopman, H. Ohshima and H. Bartsch (1990b) Structural basis of the genotoxicity of nitrosatable phenols and derivatives present in smoked food products, Mutation Res., 230, 9-27. Swenberg, J.A., B. Short, S. Borghoff, J. Strasser and M. Charbonneau (1989) The comparative pathobiology of a2p.-globulin nephropathy, Toxicol. Appl. Pharmacol., 97, 35-46. Tennant, R.W., B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. tlaseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson and R. Minor (1987) Prediction of chemical carcinogenicity in rodents from in vitro genotoxicity assays, Science, 236, 933-941. Tennant, R.W., and J. Ashby (1991) Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals tested for carcinogenicity by the U.S. National Toxicology Program, Mutation Res., 257, 209-227. Williams, G.M. (1987) Definition of a human cancer hazard, in: B.E. Butterworth an,d T.J. Slaga (Eds.), Nongenotoxic Mechanisms in Carcinogenesis, Banbury Report 25 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 367-38{). Williams, G.M. (1990) Screening procedures for evaluating the potential carcinogenicity of food-packaging chemicals, Reg. Toxicol. Pharmacol., 12, 30-40. Wilson, J.D. (1989) Assessment of low-exposure risk from carcinogenesis: Implications of the Knudson-Moolgavkar Two-Critical Mutation Theory, in: C.C. Travis (Ed.), Biologically-Based Methods for Cancer Risk Assessment Plenum, New York, pp. 275-287. Zeiger, E. (1987) Carcinogenicity of mutagens: Predictive capability of the Salmonella mutagenesis assay for rodent carcinogenicity, Cancer Res.. 47, 1287-1296. Zeise, L., E.A.C. Crouch and R. Wilson (1986) A possible relationship between toxicity and carcinogenicity, J. Am. Coll. Toxicol., 5, 137-151.
