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The relationship between carcinogenicity and mutagenicity of some polynuclear hydrocarbons
311
Mutation Research, 51 (1978) 311--318 © Elsevier/North-Holland Biomedical Press
THE RELATIONSHIP BETWEEN CARCINOGENICITY AND MUTAGENICITY OF SOME POLYNUCLEAR HYDROCARBONS
A.W. ANDREWS, LENITA H. THIBAULT and W. LIJINSKY
Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.) (Received 6 December 1977) (Revision received 9 March 1978) (Accepted 17 March 1978)
Summary 25 polynuclear hydrocarbons were tested for mutagenicity using the Ames Assay and the results were compared with existing carcinogenicity data. The assessment of the predictive value of this particular short-term test showed a 58% positive and a 41% negative correlation.
Introduction The search for abbreviated tests of the carcinogenic potential of chemicals has led to several which appear promising as partial replacements for the traditional chronic bioassay in rodents. Of these tests, the most commonly applied and the one with the broadest acceptance, has been the Salmonella/microsome Mutagenicity Test developed by Ames et al. [2]. The overall correlation between the results of this test and carcinogenicity has usually been qualitatively quite good. Our facility for the large-scale screening of compounds for mutagenicity using the Ames assay allowed us to further evaluate this test as a predictor of Carcinogenic activity. Mutagenicity tests of several of the compounds have already been conducted [13]. We tested large representative samples from two groups of compounds, polynuclear hydrocarbons and nitrosamines (see accompanying paper), each of which include a large number of carcinogens. In this paper, we describe results with a variety of polynuclear hydrocarbons and some of their partially-hydrogenated derivatives. R e s e a r c h s p o n s o r e d b y the N a t i o n a l C a n c e r I n s t i t u t e u n d e r C o n t r a c t L i t t o n Bionetics, Inc.
No. NO1-CO-75380 with
312 Materials and Methods
Chemicals The c o m p o u n d s tested were selected on the basis of their chemical structure and because they encompass most carcinogenic types within the group. Some are partially hydrogenated derivatives. All have been tested for carcinogenic activity by painting on mouse skin or by subcutaneous injection. The parent hydrocarbons were: dibenz(aj)anthracene, dibenz(ah)anthracene, dibenz(ac)anthracene, 7,12-dimethylbenz(a)anthracene, anthanthrene, 3methylcholanthrene, benzo(ghi)perylene, 9,10-dimethylanthracene, benzo(a)pyrene and benzo(e)pyrene. The hydrogenated derivatives were: dibenz(a])anthracene : 5,6-dihydro- and 1,2,3,4,8,9-hexahydro-; dibenz(ah)anthracene: 1,2,7,8-tetrahydro-, 5,6-dihydro-, 7,14-dihydro-and 1,2,3,4, 12,13-hexahydro-; dibenz(ac)anthracene: 9,14-dihydro- and 10,11,12,13-tetrahydro-; 7,12-dimethylbenz(a)anthracene: 5,6-dihydro- and 8,9,10,11-tetrahydro-; anthanthrene: 4,5-dihydro- and 1,2,3,7,8,9-hexahydro-; 3-methylcholanthrene: 11,12-dihydro- and 6,7,8,9,10,12b-hexahydro-; benzo(ghi)perylene : 5,6,7,8,9,10-hexahydro-. The preparation, physical properties, and determination of the structures of these c o m p o u n d s have been described [5,6]. All were at least 99% pure. The testing of the c o m p o u n d s for carcinogenic activity and the results thereof have been reported [4,8--10,12]. Mutagenesis assay The plate test assays were performed essentially as described by Ames et al. [3]. Dimethylsulfoxide was used as the solvent for all the test compounds. Variations were: 0.2 ml of the tester strain instead of 0.1 ml, 20 ml of the VBE medium instead of 30 ml, the Aroclor 1254-stimulated microsome $9 mix was used at a level of 75 pl of $9 fraction/test. Test c o m p o u n d s were initially screened using at least two of the Salmonella tester strains. Selection of the strain used was based in part on the literature. Where little information was available, five strains (TA 1535, 1537, 1538, 98 and 100) were used. All c o m p o u n d s were tested with and w i t h o u t added $9. When phenobarbital-induced enzymes were used, the final concentration of $9 fraction was 150 pl/test. All c o m p o u n d s were tested with both types of $9 preparations. If all plate assays were negative, the liquid test was used by incubation of the tester strains and test c o m p o u n d , with and without added $9 mix, at 37°C in a Dri Block heater for 30 min. Top agar was then added and the combination poured on to VBE plates. All testing was carried o u t under yellow fluorescent lighting (Sylvania F15T8-G0). Statistical analysis o f data Dose--response curves were constructed using the plate incorporation method, tester strain and $9 mix which gave maximum mutagenesis. For each c o m p o u n d , the data were first analyzed to determine whether or not a dose--response relationship existed. The data were graphed on semi-log paper, plotting the dose (X) b y the number of revertants (Y). The dosages, at 1--1000 pg/plate range, were 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0, 250.0,
313 500.0 and 1000.0 pg. For virtually all of the c o m p o u n d s listed as mutagenic, a dose--response relationship was seen. The majority of these were statistically significant (i.e., there was a significant linear regression), and for those that were n o t [e.g., 5,6
Several of our test c o m p o u n d s displayed unique characteristics. Anthanthrene was mutagenic with strain TA 1535 only with Aroclor-stimulated microsomes. Benzo(ghi)perylene exhibited the same dependence with strain TA 100, suggesting a need for the c y t o c h r o m e P-448 system [1]. 6,7,8,9,10,12b-Hexahydromethylcholanthrene was a direct acting mutagen. 8,9,10,11-Tetrahydrodimethylbenz(a)anthracene, in contrast with the other hydrocarbons examined, was c y t o t o x i c at all levels tested unless the $9 mix was added. We have called 5,6-dihydrodimethylbenz(a)anthracene nonmutagenic based on our statistical procedures. It should be noted that during preliminary screening this c o m p o u n d , at a corresponding dose, gave revertant numbers that fell above the
314 Table 1. The Comparison of the Chemical Structure, Carcinogenicity and Mutagenicity of Polynuclear Hydrocarbons. 12 Test Chemical dibenz(aj)anthracene 5,6.dihydrodibenz(aj)ant hracene 1,2,3,4,8,9-hexahydrodibenz(aj)anthracene
Abbreviation
Car.
DB(aj)A DB(aj)A.H 2 DB(aj)A.H 6
+ +
Mut. + + +*
2
11
13
10 1
~
1
3 4
8
7
6 2
DB(ah)A DB(ah)A-H 4 5,6-DB(ah)A.H 2 7,14*DB(ah)A-H2 DB(ah)A-H 6
dibenz(ah)anthracene 1,2,7,8-tetrahydrodlbenz(ah)anthracene 5,6.dihyd r odibenz(ah)an th recene 7,14.dihydrodlbenz(eh)anthracene 1,2,3,4,12,13-hexahydrodibenz(ah)ant hracene
+
+ + + + +
+ + +
13
14
11
4
7
6
10 9
DB(ac)A DB(ac)A-H 2 DB(ac)A.H 4
dibenz(ec)anthracene 9,14-dihydrodibenz(ec)an th racene 10,11,12,13.let rahydrodibenz(ac)ant hracene
+ ?
+ + +
13
14
t0
9
12 11
CH 3 7,12-dimet hylbenz(e)ant hrecene 5,8.d| hydrodlmet hylbenzOl)anthracene 8,9,10,11 .t et rehydtodlmethy|benz(a)ant hracene
DMBA DMBA.H 2 DMBA-H 4
+
+
+
-,,
+
-
1
3
10 ••
4
9
5 CH 3 11
anthanthrene 4,5-dthydroant hant hren e 1,2,3,7,8,g.hexahydroant hanthrene
AA AA.H 2 AA-H 6
+
+ + +
12
1
9
3
8 7
6
5 1
3.methylcholanthrene 11,12.dihydromethylcholanthrene 6,7,8,9,10,12b.hexahydromethylcholanthrene
3MC 3MC-H 2 3MC-H 6
+ + +
+ + +
11
1
~
C
10
2 H
6
3
5
9 8 9 7 benzo(ghi)perylene 5,6,7,8,9,10-hexahyd robenzo(ghi)peryle ne
B(ghi)P B(ghi)P-H 6
+ +
64
~
8
10
1
0
12
3
9,10-dlmet hylanthracene
DMA
+
2
8
9
1
5
10
4
+ 8
3
10 benzo(a)pyrene benzo(e)pyrene
11
5
B(a)P
+
B(e)P
+ +
8'
i 6
"Only in liquid test • "Not mutagenic in plate or liquid test
1
5
315 calculated 95% tolerance limit and that it may therefore be an equivocal mutagen. Table 1 compares the carcinogenicity, mutagenicity, and the chemical structures of the parent compounds and their derivatives. Discussion Only one of the 25 hydrocarbons was mutagenic when tested without the $9 mix. The remainder were mutagenic in at least one strain of Salmonella when activated by a crude microsomal preparation from rat liver, even though polynuclear hydrocarbons do not induce liver tumors in adult rodents. Many of these compounds are skin carcinogens in mice. In the experiments reported here, all of the parent hydrocarbons were mutagenic, even though two of them, benzo(e}pyrene and benzo(ghi)perylene have not shown carcinogenic activity. Anthanthrene was recently shown to be a carcinogen on mouse skin [4], although previous tests had indicated that it was inactive [8]. Two partiallyhydrogenated derivatives of dimethylbenz(a)anthracene (5,6-dihydro- and 8,9, 10,11-tetrahydro-) were nonmutagenic, even though carcinogenic. 1,2,3,4,8,9Hexahydrodibenz(a])anthracene, 1,2,7,8-tetrahydrodibenz(ah)anthracene, 10, ll,12,13-tetrahydrodibenz(ac)anthracene, 4,5-dihydroanthanthrene and 1,2,3, 7,8,9-hexahydroanthanthrene were mutagenic although they were not carcinogenic. Both benzo(a)pyrene and the isomeric benzo(e)pyrene were mutagenic; the former is a potent skin and lung carcinogen in mice, while the latter is not carcinogenic. 9,14
TA100 + Aroclor S 9 Control Mean Value Based on 68 Tests 3000 - DB(ac)A
• 3MC-H 6
2700 2400 •DMBA = 2100 'E 0
1800 1500 --
AA-H 6
s OMA
•AA OB(a)P
•AA_H2
"6 1200
Z
) DB(ac)A-H2 •3MC ;'DB(aj)A )DB(aj)A-H2 • DB(ah)A 6OO --
900
• B(ghi)P
• 7,14DB(ah)A-H2 B(e)P•
3MC-H,)O eDB(ac)A'H4 300 -" 6 DB(ah)A-H 4 _ • B(ghi)P-H 6 95% 101
I 25
I 50
I 102
I 250
I 103
.g F i g . 1. T h e c o m p a r a t i v e m u t a g e n i c i t y
o f p o l y n u c l e a r h y d r o c a r b o n s using t e s t e r strain T A 1 0 0 w i t h an A r o c l o r 1 2 5 4 - s t i m u l a t e d m i c r o s o m a l p r e p a r a t i o n s h o w i n g t h e c o n t r o l m e a n value (X---) and the u p p e r 9 5 % t o l e r a n c e limit.
316 TA1537 + Aroclor S9 Control Mean Value Based on 39 Tests
60 5,6-DB(ah)A-H 2 •
'~
50
c
40 DB(ah)A- H6 •
~" 95%
DMBA-H2e
"6
~
2o
E D z
10i
101
I
I
I
50
102
500
~,g Fig. 2. T h e c o m p a r a t i v e m u t a g e n i c i t y o f p o l y n u c l e a r h y d r o c a r b o n s using tester strain T A 1 5 3 7 w i t h a n A r o c l o r 1 2 5 4 - s t i m u l a t e d m i c r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n v a l u e ( ~ ) and the u p p e r 95% tolerance limit.
Overall, of 24 hydrocarbons tested for carcinogenicity there was a correlation between mutagenicity in this system and carcinogenicity for 14 compounds while there was no correlation for 10 c o m p o u n d s (8 werefalse positives, 2 were false negatives). The failure to detect carcinogens as mutagens is troublesome, since it suggests the possibility of a delay in further testing of a c o m p o u n d which is carcinogenic. The 8 false positives are also disturbing. One suggested use of the Salmonella system is the screening of mixtures containing polynuclear compounds, such as products of coal liquefaction or petroleum processing. In this TA1538 + Phenobarbital S9 Control Mean Value Based on 54 Tests • DB(aj)A-H 6
._~
120 100
8o a:
60
"3 ~95%
z
2~
ODMBA-H 4
I 25
i 50
• 100
~g Fig. 3. T h e c o m p a r a t i v e m u t a g e n i c i t y Of p o l y n u c l e a r h y d r o c a r b o n s using tester stxain T A 1 5 3 8 w i t h a p h e n o b a r b i t a l - s t i m u l a t e d m i e r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n value ( ~ ) and the u p p e r 95% tolerance limit.
317 way the products which are not mutagenic could be used with more assurance of safety from carcinogenic effects than those which showed mutagenicity. The finding that many non-carcinogenic polynuclear hydrocarbons, for example benzo(e)pyrene, benzo(ghi)perylene, 10,11,12,13-tetrahydrodibenz(ac)anthracene and 1,2 ,7 ,8-tetrahydrodibenz(ah )anthracene, are mutagenic does not support such an assumption. Benzo(ghi)perylene is almost as mutagenic as benzo(a)pyrene and is even more widespread in petroleum and coal tar products than the latter. A finding of mutagenicity in such a mixture would give a wrong indication of carcinogenicity. In particular, benzo(ghi)perylene is frequently found in petroleum products, while benzo(a)pyrene is usually absent or at a much lower level. This makes it probable that incorrect assumptions would be made about the possible carcinogenicity of such materials as petrolatums and petroleum asphalt [7,11,14] based on this type of mutagenesis test. It must be noted that very few of the compounds examined have been tested other than by skin painting on mice. However, there have been very few observations of carcinogenicity in other organs by polynuclear hydrocarbons, including those cited in the early evaluation of the Salmonella test by Ames, in which a correlation between mutagenicity and carcinogenicity was noted [13]. In the case of the hydrocarbons in our study all of the carcinogenicity tests were conducted under very similar conditions so that their carcinogenicity could be compared. This does not preclude acceptance of the basic precept of the test as a good one, and the possibility exists that with further use, modification and improvement, the effectiveness in predicting carcinogenicity might increase. Acknowledgments The authors wish to thank Carl Valentine, Corinthia Brown, Jan Reichard and James Fornwald for their expert technical assistance, and Charles W. Riggs for his valuable advice with the statistical analysis. References 1 Alvares, A.P., D.R. Bickers a n d A. ~ a p p a s , P o l y c h l o r i n a t e d b i p h e n y l s : A new type of inducer of cytochrome P-448 in the liver, Proc. Natl. A c a d . Sci. (U.S.A.), 70 ( 1 9 7 3 ) 1 3 2 1 - - 1 3 2 5 . 2 Ames, B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F.D. Lee, C a r c i n o g e n s are m u t a g e n s : A simple test syst e m c o m b i n i n g liver h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , Proc. Natl. Acad. Sci. (U.S.A.), 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 3 Ames, B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for detecting carcinogens and mutagens w i t h the S a l m o n e l l a / m a m m a l i a n m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 4 Cavalieri, E., P. M a l l a n d e r a n d A. Pelfrene, C a r c i n o g e n i c activity o f a n t h a n t h r e n e o n m o u s e skin, Z. Krebsforsch. 89 ( 1 9 7 7 ) 1 1 3 - - 1 1 8 . 5 Lijinsky, W., The c a t a l y t i c h y d r o g e n a t i o n o f d i b e n z ( a h ) a n t h r a c e n e , J. Org. Chem., 26 ( 1 9 6 1 ) 3 2 3 0 - 3237. 6 Lijinsky, W., G. Advani, L. Keefer, H.Y. R a m a h i a n d L. Staeh, C a t a l y t i c h y d r o g e n a t i o n of p o l y n u c l e a r h y d r o c a r b o n s , J. C h e m . Eng. Data, 17 ( 1 9 7 2 ) 1 0 0 - - 1 0 4 . 7 Lijinsky, W., I. D o m s k y , G. Mason, H.Y. R a m a h i a n d T. Safari, The c h r o m a t o g r a p h i c d e t e r m i n a t i o n of t r a c e a m o u n t s of polynuclear h y d r o c a r b o n s in p e t r o l a t u m , m i n e r a l oil a n d coal tar, Anal. Chem., 35 (1963) 952--956. 8 L i j i n s k y , W., a n d H. Garcia, Skin carcinogenesis o f hydrogenated derivatives of a n t h a n t h r e n e a n d other polynuclear hydrocarbons in Swiss mice, Z. Krebsforsch., 77 ( 1 9 7 2 ) 2 2 6 - - 2 3 0 . 9 L i j i n s k y , W., H. O a r c l a a n d U. S a f f i o t t i , Structure---activity relationships among some polynuclear h y d r o c a r b o n s and their hydrogenated derivatives, J. Natl. C a n c e r Inst., 4 4 ( 1 9 7 0 ) 6 4 1 - - 6 4 9 .
318
10 Lijinsky, W,, H. Garcia, Bo Terracini a n d U. S a f f i o t t i , T u m o r i g e n i c activity o f h y d r o g e n a t e d derivatives of d i b e n z ( a h ) a n t h r a c e n e , J. NatL C a n c e r Inst., 3 4 ( 1 9 6 5 ) 1--6. 11 Lijinsky, W., a n d C.R. R a h a , P o l y c y c l i c a r o m a t i c h y d r o c a r b o n s in c o m m e r c i a l solvents, Toxieol. AppL Pharmacol., 3 (1961) 469--473. 1 2 Lijinsky, W., a n d U. S a f f i o t t i , R e l a t i o n s h i p s b e t w e e n s t r u c t u r e a n d skin t u m o r i g e n i c activity a m o n g h y d r o g e n a t e d derivatives o f several p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s , Ann. It. Derm. Clin. Sper. 19 (1965) 34--44. 13 McCann, J., E. Choi, E. Y a m a s a k i a n d B.N. Ames, 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 the Salm o n e l l a / m i c r o s o m e test: Assay of 3 0 0 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 14 WMlcave, L., H. Garcia, R. F e l d m a n , W. Lijinsky a n d P. S h u b i k , Skin t u m o r i g e n e s i s in mice b y p e t r o l e u m a s p h a l t s a n d coal-tar p i t c h e s of k n o w n p o l y n u c l e a r a r o m a t i c h y d r o c a r b o n c o n t e n t , Toxicol. Appl. P h a r m a c o L , 18 ( 1 9 7 1 ) 4 1 - - 5 2 .
Mutation Research, 51 (1978) 311--318 © Elsevier/North-Holland Biomedical Press
THE RELATIONSHIP BETWEEN CARCINOGENICITY AND MUTAGENICITY OF SOME POLYNUCLEAR HYDROCARBONS
A.W. ANDREWS, LENITA H. THIBAULT and W. LIJINSKY
Chemical Carcinogenesis Program, Frederick Cancer Research Center, Frederick, MD 21701 (U.S.A.) (Received 6 December 1977) (Revision received 9 March 1978) (Accepted 17 March 1978)
Summary 25 polynuclear hydrocarbons were tested for mutagenicity using the Ames Assay and the results were compared with existing carcinogenicity data. The assessment of the predictive value of this particular short-term test showed a 58% positive and a 41% negative correlation.
Introduction The search for abbreviated tests of the carcinogenic potential of chemicals has led to several which appear promising as partial replacements for the traditional chronic bioassay in rodents. Of these tests, the most commonly applied and the one with the broadest acceptance, has been the Salmonella/microsome Mutagenicity Test developed by Ames et al. [2]. The overall correlation between the results of this test and carcinogenicity has usually been qualitatively quite good. Our facility for the large-scale screening of compounds for mutagenicity using the Ames assay allowed us to further evaluate this test as a predictor of Carcinogenic activity. Mutagenicity tests of several of the compounds have already been conducted [13]. We tested large representative samples from two groups of compounds, polynuclear hydrocarbons and nitrosamines (see accompanying paper), each of which include a large number of carcinogens. In this paper, we describe results with a variety of polynuclear hydrocarbons and some of their partially-hydrogenated derivatives. R e s e a r c h s p o n s o r e d b y the N a t i o n a l C a n c e r I n s t i t u t e u n d e r C o n t r a c t L i t t o n Bionetics, Inc.
No. NO1-CO-75380 with
312 Materials and Methods
Chemicals The c o m p o u n d s tested were selected on the basis of their chemical structure and because they encompass most carcinogenic types within the group. Some are partially hydrogenated derivatives. All have been tested for carcinogenic activity by painting on mouse skin or by subcutaneous injection. The parent hydrocarbons were: dibenz(aj)anthracene, dibenz(ah)anthracene, dibenz(ac)anthracene, 7,12-dimethylbenz(a)anthracene, anthanthrene, 3methylcholanthrene, benzo(ghi)perylene, 9,10-dimethylanthracene, benzo(a)pyrene and benzo(e)pyrene. The hydrogenated derivatives were: dibenz(a])anthracene : 5,6-dihydro- and 1,2,3,4,8,9-hexahydro-; dibenz(ah)anthracene: 1,2,7,8-tetrahydro-, 5,6-dihydro-, 7,14-dihydro-and 1,2,3,4, 12,13-hexahydro-; dibenz(ac)anthracene: 9,14-dihydro- and 10,11,12,13-tetrahydro-; 7,12-dimethylbenz(a)anthracene: 5,6-dihydro- and 8,9,10,11-tetrahydro-; anthanthrene: 4,5-dihydro- and 1,2,3,7,8,9-hexahydro-; 3-methylcholanthrene: 11,12-dihydro- and 6,7,8,9,10,12b-hexahydro-; benzo(ghi)perylene : 5,6,7,8,9,10-hexahydro-. The preparation, physical properties, and determination of the structures of these c o m p o u n d s have been described [5,6]. All were at least 99% pure. The testing of the c o m p o u n d s for carcinogenic activity and the results thereof have been reported [4,8--10,12]. Mutagenesis assay The plate test assays were performed essentially as described by Ames et al. [3]. Dimethylsulfoxide was used as the solvent for all the test compounds. Variations were: 0.2 ml of the tester strain instead of 0.1 ml, 20 ml of the VBE medium instead of 30 ml, the Aroclor 1254-stimulated microsome $9 mix was used at a level of 75 pl of $9 fraction/test. Test c o m p o u n d s were initially screened using at least two of the Salmonella tester strains. Selection of the strain used was based in part on the literature. Where little information was available, five strains (TA 1535, 1537, 1538, 98 and 100) were used. All c o m p o u n d s were tested with and w i t h o u t added $9. When phenobarbital-induced enzymes were used, the final concentration of $9 fraction was 150 pl/test. All c o m p o u n d s were tested with both types of $9 preparations. If all plate assays were negative, the liquid test was used by incubation of the tester strains and test c o m p o u n d , with and without added $9 mix, at 37°C in a Dri Block heater for 30 min. Top agar was then added and the combination poured on to VBE plates. All testing was carried o u t under yellow fluorescent lighting (Sylvania F15T8-G0). Statistical analysis o f data Dose--response curves were constructed using the plate incorporation method, tester strain and $9 mix which gave maximum mutagenesis. For each c o m p o u n d , the data were first analyzed to determine whether or not a dose--response relationship existed. The data were graphed on semi-log paper, plotting the dose (X) b y the number of revertants (Y). The dosages, at 1--1000 pg/plate range, were 1.0, 2.5, 5.0, 10.0, 25.0, 50.0, 100.0, 250.0,
313 500.0 and 1000.0 pg. For virtually all of the c o m p o u n d s listed as mutagenic, a dose--response relationship was seen. The majority of these were statistically significant (i.e., there was a significant linear regression), and for those that were n o t [e.g., 5,6
Several of our test c o m p o u n d s displayed unique characteristics. Anthanthrene was mutagenic with strain TA 1535 only with Aroclor-stimulated microsomes. Benzo(ghi)perylene exhibited the same dependence with strain TA 100, suggesting a need for the c y t o c h r o m e P-448 system [1]. 6,7,8,9,10,12b-Hexahydromethylcholanthrene was a direct acting mutagen. 8,9,10,11-Tetrahydrodimethylbenz(a)anthracene, in contrast with the other hydrocarbons examined, was c y t o t o x i c at all levels tested unless the $9 mix was added. We have called 5,6-dihydrodimethylbenz(a)anthracene nonmutagenic based on our statistical procedures. It should be noted that during preliminary screening this c o m p o u n d , at a corresponding dose, gave revertant numbers that fell above the
314 Table 1. The Comparison of the Chemical Structure, Carcinogenicity and Mutagenicity of Polynuclear Hydrocarbons. 12 Test Chemical dibenz(aj)anthracene 5,6.dihydrodibenz(aj)ant hracene 1,2,3,4,8,9-hexahydrodibenz(aj)anthracene
Abbreviation
Car.
DB(aj)A DB(aj)A.H 2 DB(aj)A.H 6
+ +
Mut. + + +*
2
11
13
10 1
~
1
3 4
8
7
6 2
DB(ah)A DB(ah)A-H 4 5,6-DB(ah)A.H 2 7,14*DB(ah)A-H2 DB(ah)A-H 6
dibenz(ah)anthracene 1,2,7,8-tetrahydrodlbenz(ah)anthracene 5,6.dihyd r odibenz(ah)an th recene 7,14.dihydrodlbenz(eh)anthracene 1,2,3,4,12,13-hexahydrodibenz(ah)ant hracene
+
+ + + + +
+ + +
13
14
11
4
7
6
10 9
DB(ac)A DB(ac)A-H 2 DB(ac)A.H 4
dibenz(ec)anthracene 9,14-dihydrodibenz(ec)an th racene 10,11,12,13.let rahydrodibenz(ac)ant hracene
+ ?
+ + +
13
14
t0
9
12 11
CH 3 7,12-dimet hylbenz(e)ant hrecene 5,8.d| hydrodlmet hylbenzOl)anthracene 8,9,10,11 .t et rehydtodlmethy|benz(a)ant hracene
DMBA DMBA.H 2 DMBA-H 4
+
+
+
-,,
+
-
1
3
10 ••
4
9
5 CH 3 11
anthanthrene 4,5-dthydroant hant hren e 1,2,3,7,8,g.hexahydroant hanthrene
AA AA.H 2 AA-H 6
+
+ + +
12
1
9
3
8 7
6
5 1
3.methylcholanthrene 11,12.dihydromethylcholanthrene 6,7,8,9,10,12b.hexahydromethylcholanthrene
3MC 3MC-H 2 3MC-H 6
+ + +
+ + +
11
1
~
C
10
2 H
6
3
5
9 8 9 7 benzo(ghi)perylene 5,6,7,8,9,10-hexahyd robenzo(ghi)peryle ne
B(ghi)P B(ghi)P-H 6
+ +
64
~
8
10
1
0
12
3
9,10-dlmet hylanthracene
DMA
+
2
8
9
1
5
10
4
+ 8
3
10 benzo(a)pyrene benzo(e)pyrene
11
5
B(a)P
+
B(e)P
+ +
8'
i 6
"Only in liquid test • "Not mutagenic in plate or liquid test
1
5
315 calculated 95% tolerance limit and that it may therefore be an equivocal mutagen. Table 1 compares the carcinogenicity, mutagenicity, and the chemical structures of the parent compounds and their derivatives. Discussion Only one of the 25 hydrocarbons was mutagenic when tested without the $9 mix. The remainder were mutagenic in at least one strain of Salmonella when activated by a crude microsomal preparation from rat liver, even though polynuclear hydrocarbons do not induce liver tumors in adult rodents. Many of these compounds are skin carcinogens in mice. In the experiments reported here, all of the parent hydrocarbons were mutagenic, even though two of them, benzo(e}pyrene and benzo(ghi)perylene have not shown carcinogenic activity. Anthanthrene was recently shown to be a carcinogen on mouse skin [4], although previous tests had indicated that it was inactive [8]. Two partiallyhydrogenated derivatives of dimethylbenz(a)anthracene (5,6-dihydro- and 8,9, 10,11-tetrahydro-) were nonmutagenic, even though carcinogenic. 1,2,3,4,8,9Hexahydrodibenz(a])anthracene, 1,2,7,8-tetrahydrodibenz(ah)anthracene, 10, ll,12,13-tetrahydrodibenz(ac)anthracene, 4,5-dihydroanthanthrene and 1,2,3, 7,8,9-hexahydroanthanthrene were mutagenic although they were not carcinogenic. Both benzo(a)pyrene and the isomeric benzo(e)pyrene were mutagenic; the former is a potent skin and lung carcinogen in mice, while the latter is not carcinogenic. 9,14
TA100 + Aroclor S 9 Control Mean Value Based on 68 Tests 3000 - DB(ac)A
• 3MC-H 6
2700 2400 •DMBA = 2100 'E 0
1800 1500 --
AA-H 6
s OMA
•AA OB(a)P
•AA_H2
"6 1200
Z
) DB(ac)A-H2 •3MC ;'DB(aj)A )DB(aj)A-H2 • DB(ah)A 6OO --
900
• B(ghi)P
• 7,14DB(ah)A-H2 B(e)P•
3MC-H,)O eDB(ac)A'H4 300 -" 6 DB(ah)A-H 4 _ • B(ghi)P-H 6 95% 101
I 25
I 50
I 102
I 250
I 103
.g F i g . 1. T h e c o m p a r a t i v e m u t a g e n i c i t y
o f p o l y n u c l e a r h y d r o c a r b o n s using t e s t e r strain T A 1 0 0 w i t h an A r o c l o r 1 2 5 4 - s t i m u l a t e d m i c r o s o m a l p r e p a r a t i o n s h o w i n g t h e c o n t r o l m e a n value (X---) and the u p p e r 9 5 % t o l e r a n c e limit.
316 TA1537 + Aroclor S9 Control Mean Value Based on 39 Tests
60 5,6-DB(ah)A-H 2 •
'~
50
c
40 DB(ah)A- H6 •
~" 95%
DMBA-H2e
"6
~
2o
E D z
10i
101
I
I
I
50
102
500
~,g Fig. 2. T h e c o m p a r a t i v e m u t a g e n i c i t y o f p o l y n u c l e a r h y d r o c a r b o n s using tester strain T A 1 5 3 7 w i t h a n A r o c l o r 1 2 5 4 - s t i m u l a t e d m i c r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n v a l u e ( ~ ) and the u p p e r 95% tolerance limit.
Overall, of 24 hydrocarbons tested for carcinogenicity there was a correlation between mutagenicity in this system and carcinogenicity for 14 compounds while there was no correlation for 10 c o m p o u n d s (8 werefalse positives, 2 were false negatives). The failure to detect carcinogens as mutagens is troublesome, since it suggests the possibility of a delay in further testing of a c o m p o u n d which is carcinogenic. The 8 false positives are also disturbing. One suggested use of the Salmonella system is the screening of mixtures containing polynuclear compounds, such as products of coal liquefaction or petroleum processing. In this TA1538 + Phenobarbital S9 Control Mean Value Based on 54 Tests • DB(aj)A-H 6
._~
120 100
8o a:
60
"3 ~95%
z
2~
ODMBA-H 4
I 25
i 50
• 100
~g Fig. 3. T h e c o m p a r a t i v e m u t a g e n i c i t y Of p o l y n u c l e a r h y d r o c a r b o n s using tester stxain T A 1 5 3 8 w i t h a p h e n o b a r b i t a l - s t i m u l a t e d m i e r o s o m a l p r e p a r a t i o n s h o w i n g the c o n t r o l m e a n value ( ~ ) and the u p p e r 95% tolerance limit.
317 way the products which are not mutagenic could be used with more assurance of safety from carcinogenic effects than those which showed mutagenicity. The finding that many non-carcinogenic polynuclear hydrocarbons, for example benzo(e)pyrene, benzo(ghi)perylene, 10,11,12,13-tetrahydrodibenz(ac)anthracene and 1,2 ,7 ,8-tetrahydrodibenz(ah )anthracene, are mutagenic does not support such an assumption. Benzo(ghi)perylene is almost as mutagenic as benzo(a)pyrene and is even more widespread in petroleum and coal tar products than the latter. A finding of mutagenicity in such a mixture would give a wrong indication of carcinogenicity. In particular, benzo(ghi)perylene is frequently found in petroleum products, while benzo(a)pyrene is usually absent or at a much lower level. This makes it probable that incorrect assumptions would be made about the possible carcinogenicity of such materials as petrolatums and petroleum asphalt [7,11,14] based on this type of mutagenesis test. It must be noted that very few of the compounds examined have been tested other than by skin painting on mice. However, there have been very few observations of carcinogenicity in other organs by polynuclear hydrocarbons, including those cited in the early evaluation of the Salmonella test by Ames, in which a correlation between mutagenicity and carcinogenicity was noted [13]. In the case of the hydrocarbons in our study all of the carcinogenicity tests were conducted under very similar conditions so that their carcinogenicity could be compared. This does not preclude acceptance of the basic precept of the test as a good one, and the possibility exists that with further use, modification and improvement, the effectiveness in predicting carcinogenicity might increase. Acknowledgments The authors wish to thank Carl Valentine, Corinthia Brown, Jan Reichard and James Fornwald for their expert technical assistance, and Charles W. Riggs for his valuable advice with the statistical analysis. References 1 Alvares, A.P., D.R. Bickers a n d A. ~ a p p a s , P o l y c h l o r i n a t e d b i p h e n y l s : A new type of inducer of cytochrome P-448 in the liver, Proc. Natl. A c a d . Sci. (U.S.A.), 70 ( 1 9 7 3 ) 1 3 2 1 - - 1 3 2 5 . 2 Ames, B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F.D. Lee, C a r c i n o g e n s are m u t a g e n s : A simple test syst e m c o m b i n i n g liver h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , Proc. Natl. Acad. Sci. (U.S.A.), 70 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 3 Ames, B.N., J. M c C a n n a n d E. Y a m a s a k i , M e t h o d s for detecting carcinogens and mutagens w i t h the S a l m o n e l l a / m a m m a l i a n m i c r o s o m e m u t a g e n i c i t y test, M u t a t i o n Res., 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 4 Cavalieri, E., P. M a l l a n d e r a n d A. Pelfrene, C a r c i n o g e n i c activity o f a n t h a n t h r e n e o n m o u s e skin, Z. Krebsforsch. 89 ( 1 9 7 7 ) 1 1 3 - - 1 1 8 . 5 Lijinsky, W., The c a t a l y t i c h y d r o g e n a t i o n o f d i b e n z ( a h ) a n t h r a c e n e , J. Org. Chem., 26 ( 1 9 6 1 ) 3 2 3 0 - 3237. 6 Lijinsky, W., G. Advani, L. Keefer, H.Y. R a m a h i a n d L. Staeh, C a t a l y t i c h y d r o g e n a t i o n of p o l y n u c l e a r h y d r o c a r b o n s , J. C h e m . Eng. Data, 17 ( 1 9 7 2 ) 1 0 0 - - 1 0 4 . 7 Lijinsky, W., I. D o m s k y , G. Mason, H.Y. R a m a h i a n d T. Safari, The c h r o m a t o g r a p h i c d e t e r m i n a t i o n of t r a c e a m o u n t s of polynuclear h y d r o c a r b o n s in p e t r o l a t u m , m i n e r a l oil a n d coal tar, Anal. Chem., 35 (1963) 952--956. 8 L i j i n s k y , W., a n d H. Garcia, Skin carcinogenesis o f hydrogenated derivatives of a n t h a n t h r e n e a n d other polynuclear hydrocarbons in Swiss mice, Z. Krebsforsch., 77 ( 1 9 7 2 ) 2 2 6 - - 2 3 0 . 9 L i j i n s k y , W., H. O a r c l a a n d U. S a f f i o t t i , Structure---activity relationships among some polynuclear h y d r o c a r b o n s and their hydrogenated derivatives, J. Natl. C a n c e r Inst., 4 4 ( 1 9 7 0 ) 6 4 1 - - 6 4 9 .
318
10 Lijinsky, W,, H. Garcia, Bo Terracini a n d U. S a f f i o t t i , T u m o r i g e n i c activity o f h y d r o g e n a t e d derivatives of d i b e n z ( a h ) a n t h r a c e n e , J. NatL C a n c e r Inst., 3 4 ( 1 9 6 5 ) 1--6. 11 Lijinsky, W., a n d C.R. R a h a , P o l y c y c l i c a r o m a t i c h y d r o c a r b o n s in c o m m e r c i a l solvents, Toxieol. AppL Pharmacol., 3 (1961) 469--473. 1 2 Lijinsky, W., a n d U. S a f f i o t t i , R e l a t i o n s h i p s b e t w e e n s t r u c t u r e a n d skin t u m o r i g e n i c activity a m o n g h y d r o g e n a t e d derivatives o f several p o l y c y c l i c a r o m a t i c h y d r o c a r b o n s , Ann. It. Derm. Clin. Sper. 19 (1965) 34--44. 13 McCann, J., E. Choi, E. Y a m a s a k i a n d B.N. Ames, 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 the Salm o n e l l a / m i c r o s o m e test: Assay of 3 0 0 chemicals, Proc. Natl. Acad. Sci. (U.S.A.), 72 ( 1 9 7 5 ) 5 1 3 5 - 5139. 14 WMlcave, L., H. Garcia, R. F e l d m a n , W. Lijinsky a n d P. S h u b i k , Skin t u m o r i g e n e s i s in mice b y p e t r o l e u m a s p h a l t s a n d coal-tar p i t c h e s of k n o w n p o l y n u c l e a r a r o m a t i c h y d r o c a r b o n c o n t e n t , Toxicol. Appl. P h a r m a c o L , 18 ( 1 9 7 1 ) 4 1 - - 5 2 .