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NTP
Mutation Research, 291 (1993) 61-77
61
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
MUTENV 08858
Validation of a novel molecular orbital approach (COMPACT) for the prospective safety evaluation of chemicals, by comparison with rodent carcinogenicity and Salmonella mutagenicity data evaluated by the U.S. NCI/NTP David F.V. Lewis, C. Ioannides and D.V. Parke Molecular Toxicology Group, School of Biological Sciences, Unit,ersityof Surrey, Guildford, Surrey, GU2 5XH, UK (Received 7 July 1992) (Revision received 14 October 1992) (Accepted 15 October 1992)
Keywords: Molecular structure; Cytochromes P450; Computer graphics; Ames test; Safety evaluation; Rodent carcinogenicity
Summary The molecular dimensions and electronic structures of 100 chemicals of structural diversity have been determined from molecular orbital calculations and molecular mechanics. From these parameters of molecular structure, those chemicals that are likely substrates of cytochromes P4501 and P4502E have been identified by the computer-optimized molecular parametric analysis of chemical toxicity (COMPACT) programme, and their potential toxicity, mutagenicity and carcinogenicity evaluated. The degree of correlation between COMPACT prediction of toxicity and rodent two species life-span carcinogenicity data is estimated to be 92%, and between COMPACT and Salmonella mutagenicity (Ames test) data is 64%. Anomalous rodent carcinogens are rationalized on the basis of biochemical mechanisms of metabolism, genotoxicity and carcinogenicity. Correlation of the Ames test data with rodent carcinogenicity data was 64%, but correlation of COMPACT plus Ames data versus rodent carcinogenicity data provided the highest correlation of 94%.
The need for the safety evaluation of chemicals, especially novel entities to be used as medicines or pesticides, became apparent nearly half a century ago with the human tragedies following the use of the drugs chloramphenicol, diethylstilboestrol, thalidomide, etc. (Parke et al.,
Correspondence: Dr. D.V. Parke, Molecular Toxicology Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH, UK.
1990). Current safety evaluation procedures, based largely on the use of rodents and other experimental animals as surrogates for man, are mostly empirical and when ultimately validated, as in the prescription o~f new medicines for humans, have frequently b~en found to be inadequate (Eason et al., 1990) as in the case of perhexiline (Amoah et al., 1986), benoxaprofen (Griest et al., 1982; Aronoff et al., 1982) and tienilic acid (Neuberger and Williams, 1989). The validity of the rodent life-span bioassay for poten-
62
tial carcinogenicity, used for the prediction of toxicity/carcinogenicity of chemicals in humans, has recently been questioned, and has been considered by experts to be 'unscientific' and 'essentially ritualistic' (Ames and Gold, 1990; Ennever et al., 1987; Gori, 1991a and b). From knowledge of mechanisms of toxicity/carcinogenicity it is no longer considered valid to assume that an animal-based observation will always be relevant to humans, for some carcinogens appear to be species-specific because of differences in metabolism, receptor response, genetic regulation, etc. (Clayson and Clegg, 1991). Three major factors appear to determine chemical toxicity/carcinogenicity, namely, (1) the molecular structure and properties of the chemical, upon which its biological behaviour depends, (2) the genetic complement of the exposed biological species, and (3) the environment of the exposed species, especially diet and exposure to
other chemicals. Of these, the first, namely, molecular structure is the only relevant determinant in the safety evaluation of chemicals. Moreover, because of variations in species/genetics and environment/diet, current safety evaluations in experimental animals as human surrogates frequently lead to spurious decision making. The direct determination of potential toxicity/carcinogenicity of a chemical from its molecular structure requires detailed knowledge of (i) the molecular conformation and electronic structure of the chemical, and (ii) its metabolism and interactions with numerous biological receptors, and the toxicological consequences of these phenomena. Recent advances in computer technology have now made feasible the determination of molecular structure from energy calculations by molecular mechanics and electronic structure by molecular orbital (MO) methods for relatively large molecules, and the present extensive libraries of
TABLE 1 METABOLIC ACTIVATION OF CHEMICAL CARCINOGENS BY RAT HEPATIC CYTOCHROME P450 PROTEINS
Carcinogen
Cytochrome P450 protein
A a C (2-amino-9H-pyrido[2,3-b]indole) 2-Acetylaminofluorene Aflatoxin B1 2-Aminoanthracene
1AI, 1A2 1AI, IA2,2B1 1A2,2BI, 2Cll, 2C12 1AI, 1A2
o-Aminoazotoluene 4-Aminobiphenyl 2-Aminofluorene Benzo[a]pyrene
1A2 1A2 1AI, IA2 1A1,2BI
4,4'-(bis)Methylene chloroaniline (MOCA) 1,2,3,4-Dibenzanthracene 7,12-Dimethylbenz[a]anthracene N, N-Dimethylnitrosamine
1A2,2B1 1AI, IA2,2B1 1AI 2El
GIu-P- 1 (2-amino-6-methylpyrido[ 1,2-a : 3',2'-d] imidazole) GIu-P-2 (2-amidopyrido[1,2-a : 3',2'-d] imidazole) IQ (2-amino-3-methylimidazo [4,5-f]-quinoline) MeA o~C (2-amino-3-methyl-9H-pyrido[2,3-b] indole)
1A2 1AI, IA2 1AI, 1A2 1AI, IA2
MeIQ (2-amino-3,4-dimethylimidazo[4,5-f ] quinoline) MeIQx (2-amino-3,8-dimethylimidazo[4,5-f ] quinoxaline) 3-Methyleholanthrene N-Methyl-4-aminoazobenzene
1AI, IA2 1AI, IA2 1A1 1AI,1A2
2-Naphthylamine Trp-P-1 (3-amino-l,4-dimethyl-5H-pyrido[4,3-b] indole) Trp-P-2 (3-amino-l-methyl-5H-pyrido 14,3-b] indole)
1A2 1AI, 1A2 1AI, 1A2
Data from Guengerich (1988).
63
knowledge on xenobiotic metabolism, mechanisms of toxicity, and the molecular biology of enzymes, receptors and DNA, have now made possible the prediction of potential toxicity/carcinogenicity of known chemical structures from fundamental scientific principles. Chemicals manifesting overt toxicity, and direct-acting mutagens/carcinogens, e.g. alkylating agents, are readily identified from chemical structure, but those chemicals that require metabolic activation to electrophilic reactive intermediates to manifest carcinogenicity/toxicity are more difficult to recognise. Much of the metabolic activation of chemicals to toxic electrophiles that covalently interact with DNA, enzymes and proteins to cause mutations and malignancy, acute lethal injury and tissue necrosis, and neoantigen formation and immunotoxicity, respectively, is the result of metabolic oxygenation by the cytochrome P450 superfamily and other enzymes (Lewis et al., 1989b; Parke et al., 1988; Parke et al., 1991). Of the cytochromes P450, some families more than others are associated with metabolic activation and the formation of reactive intermediates. Cytochrome P4501 (CYP1) is such a family and leads to chemical toxicity (Ioannides and Parke, 1990) by (i) oxygenating planar molecules, that can intercalate with DNA (Ioannides and Parke,
1987) and thus function as genotoxic alkylating agents, and (ii) by its genomal regulation via the cytosolic Ah receptor which results also in the initiation of the protein kinase c cascade, DNA replication and cellular proliferation (Parke et al., 1988; Ioannides and Parke, 1990). MO determination of the electronic structures, and molecular mechanics determination of the molecular conformations, of substrates of CYP1, and of inducers of CYP1 that interact with the Ah receptor, show that these are very similar (Lewis et ai., 1986). Thus many chemicals that interact with CYP1 to produce genotoxic reactive intermediates will also interact with the Ah receptor resulting in the induction of CYP1 and associated enzymes, and the initiation of the protein kinase c cascade culminating in non-genotoxic, mitotic events (Ioannides and Parke, 1987; Ioannides and Parke, 1990). Lists of known chemical carcinogens reveal that more than 90% of the compounds are substrates of rat CYP1 (see Table 1) (Guengerich, 1988; Ioannides and Parke, 1987) although in man several carcinogens are activated by CYP3 (cytochrome P4503) enzymes (Guengerich, 1992). For this reason, a procedure known as COMPACT (Computer Optimised Molecular Parametric Analysis of Chemical Toxicity) was developed to recognise potential car-
TABLE 2 CORRELATION OF MOLECULAR ORBITAL CHRARACTERISTICS OF SOME CHEMICALS WITH CYTOCHROME P450 SPECIFICITY AND TOXICITY Chemical
Major interacting CYP
area/depth 2
AE
COMPACT ratio (area/(depth 2 × zaE))
Collision diameter
Toxicity
1. Dibenzanthracene 2. Benzo[a]pyrene 3. 3-Methylcholanthrene
1A1 1A1 1A1
14.4 12.0 7.9
10.8 9.3 7.8
1.33 1.29 1.01
7.8 7.6 10.1
C,M C,M C,M
4. 5. 6. 7.
2-Aminoanthracene Trp-P-1 4-Aminobiphenyl 2-Acetamidofluorene
1A2 1A2 1A2 1A2
9.2 8.5 8.3 5.0
10.0 12.8 12.9 12.8
0.92 0.66 0.64 0.39
7.0 7.2 6.8 7.3
C,M C,M C,M C,M
8. Benzene 9. Dimethylnitrosamine 10. Halothane
2El 2El 2El
5.4 2.9 2.0
17.9 14.5 10.8
0.30 0.20 0.19
5.4 5.1 5.6
L C,M,H H
11. Hexobarbital 12. Phenobarbital 13. DDT
2B 2B 2B
1.8 1.1 0.8
15.8 15.8 13.5
0.11 0.07 0.06
7.4 7.3 7.9
N N N
C, carcinogenic; M, mutagenic; L, leukaemogenic; H, hepatotoxic; N, no overt toxicity.
64 COMPACT
%
I0
%
D 7
t 12 Delta
r _ 17 E
Fig. 1. S e p a r a t i o n o f C Y P 1 s u b s t r a t e s in a p l o t o f a / d 2 against A E for some miscellaneous chemicals. CYP1 substrates are Nos. 1-7. The identities of the other compounds, a n d t h e s t r u c t u r a l p a r a m e t e r s a n d i n t e r a c t i n g C Y P s a r e given in T a b l e 2. S u b s t r a t e s o f C Y P I a r e a b o v e t h e line.
cinogens/toxic chemicals from their molecular structure by identification of the CYP family with which they are most likely to interact and undergo metabolic activation. Substrates of CYP1 are characterised by their high degree of planarity, which is expressed as high values of a r e a / d e p t h s (a/d2), and by their electronic structure, which facilitates oxygenation (Lewis et al., 1989b), and is expressed as low values of A E ( A E = energy of the lowest empty molecular orbital - energy of the highest occupied molecular orbital, or A E = E ( L U M O ) E ( H O M O ) ) . The ratio of a / ( d 2 × AE) is termed the C O M P A C T ratio, which is used as a criterion in the identification of CYP1 substrates. A plot of a / d 2 against AE provides a more precise means for the separation of CYP1 substrates from other chemicals (Fig. 1), and the structural characteristics of these substrates are shown in Table 2. Criteria for potential CYP1 substrates are as follows: a / d 2 = > 5.0, AE = < 14.0, and COMP A C T ratio = > 0.15, of which the C O M P A C T ratio ( a / d 2) is the most valuable. However, it is more precise to examine the position of the chemical on the graph of a / d 2 against AE Experience has shown that although the C O M P A C T procedure for CYP1 substrates identifies most potential carcinogens, mutagens and
toxic chemicals, such as polycyclic aromatic hydrocarbons, aromatic amines and heterocyclic amines, it does not identify all chemicals characterised as carcinogens by the rodent, two-species, lifespan assay, for example some short-chain dialkyi nitrosamines and haloalkanes. Consideration of these exceptions led to the conclusion that many are substrates/inducers of cytochrome P4502E1 (CYP2E1). Induction of this cytochrome results in the generation of reactive oxygen species, ROS, (Ingelman-Sundberg and Hagbj6rk, 1982) which can result in oxidative stress culminating in tissue necrosis, mutations and malignancy (Halliwell and Gutteridge, 1990; Sies, 1991) especially in rodents (Parke and Ioannides, 1990). It therefore became desirable to define the molecular structure characteristics of CYP2E substrates, the most characteristic of which is a low collision diameter. Criteria for CYP2E substrates are therefore as follows: a / d 2 = 2.0-6.0, AE = > 10.0; C O M P A C T ratio = < 0.35; collision diameter = < 6.5, of which the most critical is the collision diameter. A plot of collision diameter against C O M P A C T ratio provides good separation of CYP2E substrates from other chemicals (Fig. 2). The advantages of the C O M P A C T procedure are that (i) unlike the Ames test, it is not limited
15 []
.o
1.0
ev < 12. x o
0.5 [] []
0.0
i
6
r
r
7 B 9 10 11 Collision Diameter Fig, 2. Separation of CYP2E substrates in a plot of COMPACT ratio against collision diameter for some miscellaneous chemicals. CYP2E substrates are Nos. 8-10. The identities of the other compounds, and the structural parameters and interacting CYPs are given in Table 2. Chemicals with collision diameters of < 6.5 (Nos. 8-10) are CYP2E substrates.
65 TABLE 3 M O L E C U L A R PARAMETERS, AMES TEST A N D R O D E N T LIFE-SPAN C A R C I N O G E N I C I T Y ASSAY F O R 100 MISCELL A N E O U S CHEMICALS Key to Carcinogenicity data: R, rat; M, mouse; m, male; f, female; P, positive; N, negative; E, equivocal; IS, inadequate study; CE, clear evidence; NE, no evidence; SE, some evidence; EE, equivocal evidence; NT, not tested; * difficult to evaluate. In the COMPACT assay (CYP1), values of a r e a / d e p t h 2 x AE of 0.15 or greater are taken as ( + ) ; and values < 0.15 are taken as ( - ) . In the COMPACT assay (CYP2E) a collision diameter of 6.5 or less is taken as ( + ) . Code
Compound name
Area/
Collision
Salmonella
Rodent carcinogenicity
COMPACT
depth2xAE
diameter
assay
R(m)
R(f)
M(m)
M(f)
(CYP1)
(CYP2E)
0.13
6.5
-
P
N
N
P
-
+
12.3 0.84
+
P
P
P
+
4.4
12.6
0.35
+
P
P
P
P
+
6.2 4.3 4.8
12.0 14.7 17.9
0.52 0.30 0.27
+ + -
P P CE
P P CE
N P CE
P P CE
+ + +
2.2
14.9
0.14
+
P
E *
P
P
-
5.1
14.3
0.36
+
P
N
N
P
+
5.7
13.8
0.41
+
P
P
P
P
+
CI basic red 9 CI disperse yellow 3 cinnamyl anthranilate
3.8
8.6
0.44
+
CE
CE
CE
CE
+
8.4
10.5 0.80
+
P
N
N
P
+
7.3
14.1 0.52
-
P
N
P
P
+
p-cresidine cupferron 2,4-diaminoanisole
4.6 8.6
14.4 13.2
0.32 0.65
+ +
P P
P P
P P
P P
+ +
3.7
14.1 0.26
+
P
P
P
P
+
4.0
15.0
0.26
+
P
P
N
P
+
1.5
10.0
0.15
+
P
P
P
P
+
2.4
12.4
0.19
+
P
P
P
P
+
2.2 1.7
15.7 0.14 15.5 0.11
5.2 4.7
+ NT
P SE
P CE
P CE
P CE
-
+ +
1.3
14.7
0.09
5.4
+
CE
SE
IS
CE
-
+
5.3
+
P P EE
P P SE
P P CE
P P CE
+ +
+
No. 1. 2. 3.
4. 5. 6. 7. 8. 9.
10. 11. 12.
13. 14. 15.
16. 17. 18.
19. 20. 21.
Area/
AE
depth 2 allyl isovalerate 2-aminoanthraquinone 3-amino 2-ethylcarbazole 1-amino 2-methylanthraquinone o-anisidine benzene 3-chloromethyl pyridine 4-chloro-m-phenylene diamine 4-chloro-o-phenylene diamine
2,4-diaminotoluene 1,3-dibromo3-chloropropane 1,2-dibromoethane 1,2-dichloroethane dichloromethane 1,3-dichloropropene
2.3 10.3
17.6
5.9
22. 23. 24.
DEHP 1,4-dioxane HC blue 1
3.1 1.6 3.7
14.4 0.22 19.5 0.08 1t.6 0.32
25. 26.
hydrazobenzene 4,4'-methylenebis(N,N-dimethyl) aniline
9.9
14.2
0.70
+
P
P
N
P
+
2.9
14.4
0.20
-
P
P
E
P
+
+
66 T A B L E 3 (continued) Code
Compound name
No. 27.
28. 29. 30.
31. 32. 33.
34. 35. 36.
37.
Area/
~E
depth 2 4,4'-methylenedianiline 2-methyl-l-nitroanthraq uinone Michler's ketone 1,5-naphthalenediamine nithiazide nitrilotriacetic acid 5-nitroacenaphthene 5-nitroo-anisidine nitrofen p-nitrosophenylamine
Area/
Collision
Salmonella
R ode nt carcinogenicity
COMPACT
depth2xaE
diameter
assay
R(m)
R(f)
M(m)
M(f)
(CYP1)
7.2
+
P
P
P
P
-
2.0
14.7
0.14
6.3 4.8
12.2 13.0
0.52 0.37
+ +
P P
P P
NT P
NT P
+ +
7.8
12.0
0.65
+
N
P
P
P
+
4.0
12.5
0.32
+
N
P
P
E
+
1.1
15.5
0.07
-
P
P
N
N
-
5.0
13.5
0.37
+
P
P
N
P
+
5.0 2.2
12.8 13.2
0.38 0.17
+ +
P N
P N
E * P
P P
+ +
3.2
I1.4
0.28
+
P
N
P
N
+
3.1 11.0 4.0
13.7 10.7 13.6
0.23 1.03 0.30
+ + -
P P N
P P P
P N P
P P P
+ + +
+ +
SE P P
SE N P
CE P P
CE P P
+ +
6.6
38. 39.
4,4'-oxydianiline phenazopyridine phenesterin
40. 41. 42.
1,2-propene oxide 1.6 reserpine 3.4 sulfallate 2.5
19.3 12.8 12.4
0.08 0.26 0.20
43. 44.
TCDD tetrachlorovinphos 4,4'-thiodianiline
7.8
12.7
0.61
-
P
P
P
P
+
2.3
12.4
0.18
-
N
P *
P
P *
+
2.9
13.0
0.22
+
P
P
P
P
+
o-toluidine TCP 2,4,5-trimethylaniline
3.7 5.9
15.1 13.8
0.24 0.43
+ -
P P
P N
P P
P P
+ +
3.7
14.4
0.26
+
P
P
E *
P
+
1.3
15.6
0.08
+
P *
N
N
P
-
1.4
8.4
0.16
+
P
P
P
P
+
3.2
17.5
0.18
-
P
N
E *
N
+
aniline 5.4 azobenzene 9.7 bis (2-chloromethyl ethyl) ether 1.7
15.4 11.2
0.35 0.80
+
P P
P P
N N
N N
+ +
16.1
0.10
+
N
N
P
P
-
4.4 5.2
14.3 9.9
0.31 0.52
+
N P
N E *
P N
P N
+ +
3.7
11.1
0.33
+
P
P
N
N
+
45.
46. 47. 48.
49. 50.
51.
52. 53. 54.
55. 56. 57.
trimethyl phosphate tris (2,3-dibromopropyl) phosphate 11-amino-undecanoic acid
5-chloroo-toluidine C red 9 3,3 '-dimethoxybenzidine diisocyanate
4.7
5.9
6.5
(CYP2E)
+
+
67 TABLE 3 (continued) Code No.
Compound name
58.
dimethyl hydrogen phosphite 2,4-dinitrotoluene 1,2-butene oxide
Area/
dE
depth 2
Area/
Collision
Salmonella
Rodent carcinogenicity
COMPACT
depth2×AE
diameter
assay
R(m)
R(f)
M(m)
M(f)
(CYP1)
(CYP2E)
1.4
15.7
0.09
5.5
+
CE
EE
NE
NE
-
+
4.4 1.3
13.3 19.2
0.33 0.06
5.1
+ +
P CE
P EE
N NE
N NE
+ -
+
6.4
13.4
0.48
NT
N
N
P
P
+
4.2
13.1
0.32
+
N
N
P
P
+
2.5
13.0
0.19
-
P
N
N
N
+
65.
1,1,2-trichloroethane trifuralin
1.6 1.7
15.1 12.1
0.11 0.14
+
N N
N N
P N
P P
-
66.
zearalenone
4.0
13.9
0.29
-
N
N
P
P
+
67. 68. 69.
captan chlordane chlorobenzilate
1.7 1.2 2.4
12.9 13.0 14.3
0.13 0.09 0.17
+ -
N N E
N N E
P * P P
P * P P
+
70. 71.
chlorotalonil 4-chloroo-toluidine CI disperse blue 1
6.1
13.6
0.45
-
P
P
N
N
+
4.0
14.3
0.28
-
N
N
P
P
+
10.5
10.9
0.91
+
CE
CE
EE
NE
+
4.4
14.0
0.32
+
P
P
N
+
1.7 2.5
11.0 12.1
0.15 0.21
-
SE N
SE N
EE P
NE P
+ +
6.3
12.9
0.49
+
N
N
P
P
+
3.9
17.5
0.22
-
N
N
P
P
+
3.8
12.9
0.29
-
P
P
N
N
+
1.6 1.2
14.1 13.0
0.11 0.09
6.6 7.5
-
SE N
SE N
NE P
NE P
-
-
1.3
14.0
0.09
6.3
-
N
N
P
P
-
+
6.4
13.3
0.48
+
N
N
P
P
+
2.1
12.6
0.18
-
P
P
N
N
+
1.3
14.2
0.09
6.0
-
E *
N
P
P
-
+
1.1
15.5
0.07
5.6
+
P
P
N
N
-
+
1.5
14.5
0.01
5.5
-
E *
N
P
P
-
+
59. 60. 61. 62. 63.
64.
72.
73. 74. 75. 76.
77. 78.
79.
80. 81.
82. 83. 84.
85. 86.
estradiol mustard 5-nitroo-toluidine 4,4'-sulphonyldianiline
rn-cresidine decabromodiphenyloxide DDE 2,6-dichlorop-phenylene diamine di(2-ethylhexyl)adipate N,N-diethylthiourea dimethylmorpholinophosphoramidate heptachlor hexachloroethane 5-nitrobenzimidazole N-nitrosodiphenylalanine pentachloroethane pivalolactone 1,1,1,2-tetrachloroethane
5.5 7.4
7.4 7.6
+ -
-
68
TABLE 3 (continued) Code
Compound name
No. 87.
88. 89. 90.
91. 92. 93.
94. 95. 96.
Area/
zaE
depth 2 1,1,2,2-tetrachloroethane trichloroethene aldrin allylisothiocyanate 3-amino 4-ethoxyacetanilide 4-amino-2-nitrophenol 2-amino-5-nitrothiazole p-benzoquinone dioxime 2-aminobiphenyl butylbenzylphthalate
Area/
Salmonella
Rodent carcinogenicity
COMPACT
R(m) R(f) M(m) M(f) (CYP1) (CYP2E)
1.9
14.9
0.13
5.8
-
E *
N
P
P
-
+
3.5 1.1
14.2 12.7
0.24 0.08
7.7
NT
N E *
N E *
P P
P N
+ -
-
1.9
12.1
0.16
+
P
E
N
N
+
3.2
14.2
0.22
+
N
N
P
N
+
7.0
12.2
0.57
+
P
E
N
N
+
4.1
12.7
0.32
+
P
N
N
N
+
6.7 6.0
9.0 13.0
0.74 0.46
+ +
N N
P N
N E
N P
+ +
1.3
14.4
0.09
P *
N
N
-
0.51
97.
chloramben
6.8
13.5
98.
chlorodibromoethane CI vat yellow 4 dicofol
1.5 14.4 2.2
10.4 0.14 9.4 1.54 13.1 0.17
99. 100.
Collision
depthZxAE diameter assay
8.1
5.3
to identification of genotoxic carcinogens since it also identifies chemicals that activate the Ah receptor resulting in CYP1 induction and tissue proliferation, or stabilize CYP2E with increased generation of ROS, (ii) it also identifies chemicals that are metabolically activated to electrophiles which bind to enzymes and proteins and hence are cytotoxic (e.g. paracetamol) or immunotoxic (e.g. halothane), (iii) it is unique in that it requires no biological or chemical materials and hence can be applied even before the particular chemical has been synthesized. This makes the procedure truly predictive, so that safety evaluation may be applied at the design stage with the potential of integrating desired pharmacological activity with the absence of toxicity at the earliest stages of product development, thus facilitating major savings in time and capital investment. To validate this novel safety evaluation procedure of C O M P A C T , which is based on molecular structure rather than biological events, correlations of the C O M P A C T identification of CYP1
+
N
N
E
P
+
-
NE N N
NE N N
EE P P
SE N N
+ +
-
+
and CYP2E substrates as potential carcinogens with those identified as carcinogens in the rodent carcinogenicity assay, or as mutagens in the Salmonella mutagenicity (Ames) assay, was undertaken utilising the first 100, structurally diverse, organic chemicals of the U.S. N C I / N T P database (Ashby and Tennant, 1988). Methods
Determination of molecular and electronic structural parameters. All 100 chemicals listed in Table 3 were taken from the data of Ashby and Tennant (1988) and their molecular structures analysed for CYP1 and CYP2E specificity using the C O M P A C T procedure as described previously (Lewis et al., 1989a). Molecular structures were constructed from first principles using the C O S M I C modelling package (Vinter et al., 1987) and minimised to give optimised geometries by the conjugate gradient method. Molecular shape
69
parameters ( a r e a / d e p t h z) were calculated from overall molecular dimensions using° the following van dero Waals radii: ocarb°n = 1.6 A, hyodrogen = 1.2 A, oxygen = 1.4 A, nitrogen = 1.4 A, sulphur = 1.85 A, chlorine = 1.8 A, bromine = 1.95 A, phosphorus = 1.9 A and fluorine = 1.35 A. The energy minimised molecular structures were subjected to molecular orbital (MO) calculations by the C N D O / 2 procedure (Pople et al., 1965) via the COSMIC software framework. Compound 'length' was the longest molecular dimension, 'width' was the longest molecular dimension at right angles to the 'length', and 'depth' was the longest molecular dimension at right angles to the 'length' and 'width'; 'diameter' is the collision diameter which is equivalent to the diameter of a sphere traced out by the free rotation of the molecule about its geometric centre along three mutually orthogonal (x,y and z) axes. The collision diameter is related to the van der Waals volume and is proportional to the cross-sectional area perpendicular to the main molecular plane. The difference between frontier orbital energy levels was taken as the alE electronic structural p a r a m e t e r (where alE = E ( L U M O ) E(HOMO)). All calculations and measurements were performed on a Sigmex $6130 graphics terminal connected to a MicroVAX II computer running the COSMIC software package. o
o
o
Determination of CYP1 substrates. As previously described (Lewis et al., 1989a; Lewis et al., 1990), the values for the a r e a / d e p t h 2 for the molecular dimensions of the 100 chemicals were plotted against the electronic structural parameter, AE. The objective was to identify substrates of CYP1, which can be activated to reactive intermediates and function as mutagens, carcinogens, neoantigens and other cytotoxic agents. CYP1 substrates are characterised by high values of a r e a / d e p t h 2 (planarity) and by low values of A E (ease of oxygenation). CYP1 substrates may thus be identified by reading from the plot of a r e a / d e p t h 2 vs. alE (see Fig. 1). However, to avoid the subjective nature of graphic interpretation, values of area/(depth2 x alE) ( C O M P A C T ratio) were calculated and used to identify CYP1 substrates. From previous C O M P A C T studies of more than 200 miscellaneous chemicals and
chemical carcinogens it was considered that chemicals with C O M P A C T ratios of > 0.15 were potential CYP1 substrates, i.e. ( + ) ; those with values of < 0.15 are not likely substrates of CYP1, i.e. ( - ) . It is appreciated that this simplistic numerical approach will incur errors, as the boundary defining the characterisitic spatial and electronic parameters of CYP1 substrates is nonlinear. Hence, for compounds with very low values of alE, C O M P A C T calculations will have an increased number of false negatives, and for compounds with very high values of d E there will be an increased number of false positives. In these two categories, inspection of the graph may be necessary.
Determination of CYP2E substrates. Similarly, CYP2E substrates were identified primarily from measurement of the collision diameter. Known substrates of CYP2E were all found to have collision diameters of < 6.5, to be small molecules (low a / d 2 values), high values of AE (difficult to oxygenate), and therefore low values for the C O M P A C T ratio. Chemicals with collision diameters of < 6.5 were designated CYP2E substrates, i.e. ( + ) . Inspection of the graph (Fig. 2) shows that a plot of collision diameter against C O M P A C T ratio readily segregates CYP2E substrates from those of CYP1, CYP2B and other cytochromes P450. The precision of this segregation of CYP2E substrates from molecular structural parameters has been extended even further by the use of a 3-dimensional plot (diameter vs. a / d 2 vs. AE), which can readily be viewed on the computer screen. Validation procedures. The identification of CYP1 and CYP2E substrates as potential carcinogens was essentially unequivocal, as was the identification of Salmonella mutagenicity (with and without metabolic activation), but rodent carcinogenicity is dependent on species and sex, and was therefore dependent on the evaluation of the data. In the study of both sexes of rats and mice only 21/100 compounds were positive for carcinogenicity in both sexes of both species (4/4), and no compounds were negative in both sexes of both species. The problem was therefore how to define a positive result. Enquiries of those regula-
70 TABLE 4 COMPARISON OF COMPACT NEOUS CHEMICALS
ASSAY WITH AMES TEST AND RODENT
LIFE-SPAN ASSAY FOR
100 M I S C E L L A -
In the r o d e n t ( 1 / 4 ) assay, o n e positive result in e i t h e r or b o t h sexes of o n e o r b o t h species is t a k e n as ( + ) a n d no positive in e i t h e r species is t a k e n as ( - ) . I n t h e r o d e n t ( 3 / 4 ) assay, if 3 o f the 4 p a r a m e t e r s o f 2 sexes o f 2 species are positive the result is t a k e n as ( + ) , if less t h a n this the result is t a k e n as ( - ). In t h e C O M P A C T assay, 91 c o m p o u n d s w e r e ( + ) a n d 9 c o m p o u n d s w e r e ( - ) . In t h e A m e s test, 59 c o m p o u n d s w e r e ( + ) , 38 c o m p o u n d s w e r e ( - ) , a n d 3 c o m p o u n d s w e r e n o t studied. In the r o d e n t assay, 97 c o m p o u n d s w e r e ( + ) a n d 3 c o m p o u n d s w e r e not sufficiently t e s t e d w h e r e t h e c r i t e r i o n was 1 / 4 o f the two species two sexes b e i n g positive; a n d 38 c o m p o u n d s w e r e ( + ) a n d 48 c o m p o u n d s w e r e ( - )
a n d 14 w e r e not sufficiently t e s t e d
w h e r e the c r i t e r i o n was 3 / 4 o f the two species two sexes b e i n g positive. Compound No.
Ames test
Rat
Mouse
Rat and Mouse r o d e n t assay
COMPACT assay
A m e s plus rodent 1/4
1/4
3/4
(CYP1 & CYP2E)
assay
A m e s plus COMPACT
1.
-
-+ ++ ++
-
+
+ +
++? ++
+
2. 3.
+ +
+ +
+ +
_+ + +
_+ + +
4. 5.
+ +
++ ++
-+ ++
+ +
+ +
+ +
+ +
+ +
6.
-
++
++
+
+
+
_
_+
7.
+
+?
++
+
+
+
+
+
8.
+
-
+
+
+
+
-+ ++
+
9.
+++
+
+
+
+
+
10.
+
++
++
+
+
+
+
+
11. 12.
+ -
++-
-+ + +
+ +
+
+ +
+ _+
+ +
13. 14. 15.
+ + +
++ ++ ++
++ ++ ++
+ + +
+ + +
+ + +
+ + +
+ + +
16. 17. 18.
+ + +
++ ++ ++
-+ ++ ++
+ +
+ +
+ +
+ +
+ +
+
+
+
+
+
19. 20.
+ NT
++ ?+
++ + +
+ +
+ +
+ +
+ NT
+ NT
21. 22.
+ -
+? ++
?+ + +
+ +
? +
+ +
+ _-2-
+ _+
23. 24. 25.
+ +
+ + ?? + +
++ + + - +
+ + +
+ ? +
+ + +
_+ + +
_+ + +
26. 27. 28. 29. 30.
+ + +
+ + ++ + + ++ - +
?+ ++ NT NT + +
+ + + +
+ + ? +
+ + +
_+ + + +
_+ + + +
++
+
+
+
+
+
31. 32. 33. 34. 35.
+ + + +
- + + + ++ + +
+? -+ ?+ + +
+ + + + +
? + + -
+ + + +
+ + + + +
+ + + +
36. 37. 38. 39. 40.
+ + + +
+++ ++ -+ ??
+++ -+ ++ + +
+ + + + +
+ + + 9
+ + + + +
+ + + _+ +
+ + + +_ +
+
71
TABLE
4 (continued)
Compound
Ames
No.
test
Mouse
Rat
Rat
and
rodent
-
Mouse assay
1/4
3/4
COMPACT
Ames
assay
rodent
(CYP1
& CYP2E)
plus 1/4
Ames
assay
!k
41.
-
+
+ +
+
+
+
42.
+
++
++
+
+
+
43.
-
++
+
+
+
+
+
44.
-
-+
++
+
+
+
±
45.
+
+ +
+ +
+
+
+
+
+
46,
+
+ +
+
+
+
+
+
+
+
47,
-
+ -
+ +
+
+
+
48,
+
+
.9+
+
+
+
+
+
49,
+
+ -
-
+
-
+
4-
+
50,
+
++
++
+
+
+
+
-/-
+
+
+
+
51.
_
+_
9_
+
_
+
±
52.
-
+ +
-
-
+
-
+
±
+
53.
+
+
+
-
-
+
-
+
+
+
54.
+
-
-
+ +
+
-
+
+
÷
55.
-
-
-
+ +
+
-
+
__+_
56.
+
+?
-
-
+
-
+
+
+
57.
+
+ +
-
-
+
-
+
÷
+
58.
+
+ ?
-
-
+
-
+
+
-t-
59.
+
+ +
-
-
+
-
+
+
÷
60.
+
+ ?
-
-
+
-
+
+
+
61.
NT
-
-
+ +
+
-
+
NT
NT
62.
+
-
-
+ +
+
-
+
+
+
63.
-
+-
- -
+
-
+
64.
-
-
-
+
+
+
-
+
65.
+
-
-
-
+
+
-
-
÷ +
66.
-
-
-
+ +
+
-
+
÷
67.
+
-
-
+
+
+
-
-
+
68.
-
-
-
+
+
+
-
-
÷
69.
-
??
+ +
+
?
+
70.
-
+ +
-
+
-
+
÷
71.
-
- -
+ +
+
-
+
-1-
72.
+
+ +
?-
+
?
+
+
+
73.
+
+ +
?-
+
?
+
+
+
74.
-
??
? -
?
?
+
9
÷
75.
-
-
+
-
+
÷
q-
+
+
-
+
-
+
76.
+
-
-
+ +
+
-
+
77.
-
-
-
+
+
+
-
+
78.
-
+ +
-
-
+
-
+
79.
-
??
-
-
?
?
-
80.
-
-
-
+ +
+
-
-
÷
81.
-
-
-
+
+
+
-
+
+
-
-
+
+
+
-
+
83.
-
+ +
+
-
+
84.
-
?-
+
+
+
?
+
85.
+
+ +
-
-
+
-
+
+
+
+
-1-
86.
-
?-
+ +
+
-
+
!-
87.
-
?-
+
+
+
?
+
÷
88.
-
-
+ +
+
-
+
-1-
-
÷
?
82.
plus
COMPACT
+
72 T A B L E 4 (continued) Compound No.
Ames test
Rat
89. 90.
NT +
?? + ?
91. 92. 93. 94. 95.
+ + + + +
+ ? +- +
96. 97. 98. 99. 100.
+ -
?+
Mouse
Rat and Mouse rodent assay
COMPACT assay
Ames plus rodent 1 / 4
1/4
3/4
(CYPI & CYP2E)
assay
+ -
+ +
9 -
+
NT +
NT +
+-
?+
+ + + + +
-
+ + + + +
+ + + + +
+ + + + +
?+ ?+ ?? +-
+ ? +
9 -
+ + +
+
--
+
+ + ? _+ _+
+ + _+
+
-
Ames plus COMPACT
TABLE 5 C O R R E L A T I O N O F D A T A F R O M T H E C O M P A C T ASSAY, R O D E N T C A R C I N O G E N I C I T Y ASSAY A N D A MES TEST F O R 100 M I S C E L L A N E O U S C H E M I C A L S (1) In the mouse vs. rat correlation this was considered to be positive when both sexes of both species were either ( + + ) or ( - - ); there were 21 compounds positive in both sexes of both species out of the 70 compounds for which there is firm evidence for both sexes of both species, and no compounds that were negative in both species, which gives an overall correlation of 30%. If the mouse vs. rat correlation was considered positive when 3 or more segments (male rat, female rat, male mouse, female mouse) were in agreement, then 38 compounds were positive in 3 / 4 animals, and 7 compounds were negative in 3 / 4 animals, out of a total of 86 chemicals for which firm evidence is available, giving a correlation of 45/ 86, i.e. 52%. In the correlation of the C O M P A C T assay vs. the rodent assay or vs. the Ames test, a ( + ) was correlated with ( + ) , and ( - ) with ( - ). (2) In the correlation of the rodent assay ( 1 / 4 ) with the Ames test, 59 were positively consonant, 1 was negatively consonant, and 35 were in disagreement; for 3 compounds no Ames data were available, and for another 3 carcinogenicity data were equivocal. Consonance was therefore 60/94, or 64%. (3) In the correlation of C O M P A C T with Ames, 56 were positively consonant, 6 were negatively consonant, and 35 were in disagreement; for 3 compounds no Ames data were available. Consonance was therefore 62/97, i.e. 64%. (4) In the correlation of C O M P A C T with the rodent assay (1/4), 89 were positively consonant, none were negatively consonant, and 8 were in disagreement. Consonance was therefore 89 out of 97, i.e. 92%. (5) In the correlation of C O M P A C T vs. the Ames plus the rodent assay, of the 91 compounds that were ( + ) in the COMPACT, 56 were ( + ) in both Ames and the rodent assay (1/4), and of the 9 compounds that were ( - ) in C O M P A C T none was ( - ) in both Ames and the rodent assay and 6 were not tested in both Ames and the rodent assay. The consonance was 56 out of 94, i.e. 60%. (6) In the correlation of C O M P A C T plus Ames vs. the rodent assay (1/4), of the 97 compounds that were ( + ) in the rodent assay, 56 were ( + ) in both C O M P A C T and the Ames and 33 more were ( + ) in one or the other. Three were not tested in the Ames, in one of which no firm carcinogenicity data were available. The consonance was therefore 89 out of 95, i.e. 94%.
%
1. 2. 3. 4. 5. 6.
Rat vs. Mouse Rodent assay vs. Ames C O M P A C T vs. Ames C O M P A C T vs. Rodent assay C O M P A C T vs. Ames plus Rodent assay C O M P A C T plus Ames vs. Rodent assay
52 64 64 92 60 94
Consonance
% False positives given by C O M P A C T
% False negatives given by C O M P A C T
33 0
3 8
73
Figs. 1 and 2 (Lewis et al., 1986, 1987; Parke et al., 1988). The rodent carcinogenicity data are evaluated as ( + ) where 1 or more of the 4 animal studies (rat M & F; mouse M & F) was positive, and ( - ) only where none of the 4 animal studies was positive. The original Ames and rodent carcinogenicity data, and Ames plus C O M P A C T data have been combined by assigning ( + ) , ( - ) or ( + ) values by means of a notional 50 : 50 weighting between the two methods of toxicity evaluation. The total refined data set is shown in Table 4 for purposes of comparison and for inspection of the degree of correlation between each method. In the Ames test 59 chemicals were positive, 38 were negative, and 3 were not tested. In the rodent assay ( 1 / 4 ) 97 chemicals were positive and 3 were not sufficiently tested. In the C O M P A C T evaluation 91 chemicals were positive and 9 were negative. Correlations between the various combinations of Ames test, rodent carcinogenicity assay and C O M P A C T were arrived at as defined in Table 5. The rodent assay versus the Ames test gave 64% consonance, the C O M P A C T versus Ames
tors making such decisions revealed that the practice varied in different countries and different agencies, and was often dependent on a variety of other factors. It was therefore decided to follow the general practice of NCI and to apply the most stringent standard of evaluating a single positive result ( 1 / 4 or more) as an indication of positive carcinogenicity ( + ) , and assigning a negative ( - ) value only where there was no positive result in any of the 4 (rat M & F, mouse M & F) segments of the rodent assay. Results
COMPACT procedure. The full data set for 100 chemicals together with carcinogenicity data are presented in Table 3 for comparison between COMPACT, the rodent two-species life-span carcinogenicity assay and the Ames test for mutagenicity. The raw structural data were analysed and compared against those of known substrates of CYP1 and CYP2E (see Table 2). The regions of molecular parametric space defining CYP1 and CYP2E substrate specificity are presented in
TABLE 6 COMPACT DATA ON KNOWN HUMAN CARCINOGENS AND TOXIC CHEMICALS Compound
Area/depth 2
AE
COMPACT ratio
Collision diameter
Toxicity
/3-naphthylamine diethylstilboestrol dimethylaminoazobenzene
7.9 4.8 3.8
12.5 13.0 10.6
0.63 0.37 0.36
6.4 7.9 7.4
bladder carcinogen transplacental carcinogen hepatocarcinogen
paracetamol tienilic acid afla/oxin B 1
4.8 3.6 3.2
14.4 12.0 11.9
0.33 0.30 0.27
6.4 7.7 7.7
fatal hepatotoxin fatal hepatotoxin hepatocarcinogen
benoxaprofen halothane chloramphenicol
2.5 2.0 2.2
12.0 10.8 13.6
0.21 0.19 0.16
7.7 5.6 7.6
fatal hepatotoxin fatal hepatotoxin acute neonatal toxicity
thalidomide a ethanol 4-aminobiphenyl
2.0 1.8 8.3
13.6 22.4 12.9
0.15 0.08 0.64
7.4 4.5 6.8
teratogen hepatocarcinogen bladder carcinogen
4-nitrobiphenyl benzidine vinyl chloride
8.4 7.3 2.8,
12.7 12.5 16.3
0.66 0.58 0.17
7.0 7.0 4.6
bladder carcinogen bladder carcinogen hepatocarcinogen
COMPACT ratio and collision diameter values in heavy type indicates that these chemicals will be substrates of CYP1 and CYP2E1 respectively. a The teratogenic enantiomer.
74 also gave 64% agreement, C O M P A C T versus the rodent assay gave 92%, and the highest of all was 94% for C O M P A C T plus Ames versus the rodent assay.
Determination of the sensitivity of Ames test and COMPACT procedure. On the basis of the 100 chemicals evaluated in the present study, the index of "sensitivity" was calculated as follows: Sensitivity Number of carcinogens positive in test Total number of carcinogens The total number of carcinogens on the basis of the rodent bioassay ( 1 / 4 ) is 97, but of these 2 were not tested in the Ames assay so that the total number of carcinogens evaluated in this test is 95. 57 Sensitivity for Ames test = - - × 100 = 60% 95 Sensitivity for C O M P A C T procedure 89 -
97
x
100
=
92%
Validation of COMPACT against human data. Much of the unequivocal human data on the toxicity/carcinogenicity of chemicals comes from the exposure to new drugs. Table 6 shows a list of chemicals known to be toxic to humans, and many of these are seen to be substrates of CYP1 a n d / o r CYP2E. However, there are some such as chloramphenicol which is activated via nitro reduction, and not oxidation, and tienilic acid which appears also to be activated by C Y P 2 C l l in addition to CYP1. Discussion
From recent studies of mechanisms of carcinogenicity and chemical toxicity it is apparent that there are two major pathways to malignancy, namely (i) the alkylation of D N A by electrophilic
metabolites (genotoxic), and (ii) nuclear damage by reactive oxygen species generated by futile cycling of CYP2E1 and possibly other P450 enzymes, redox cycling of quinones, peroxisomal proliferation, or transoxygenation in eicosanoid biosynthesis. Genotoxic reactive electrophiles which function as genotoxins are primarily formed through metabolism of essentially planar molecules, catalysed by the CYP1A family (Ioannides and Parke, 1990). Moreover, what also prompted emphasis on this cytochrome P-450 family is the fact that this activity is highly inducible by chemicals to which man is exposed frequently through the diet, such as the indoles in cruciferous vegetables, polycyclic aromatic hydrocarbons formed during the charcoal broiling of food, and heterocyclic amines formed during the frying and grilling of meat and fish. Even drugs such as omeprazole can induce CYP1 activity in humans at therapeutic doses (Diaz et al., 1990). Indeed, human studies support the premise that high CYP1 levels predispose to chemically-induced cancers and expression of CYP1 proteins is currently being evaluated as an index of lung cancer risk in individuals exposed to pulmonary carcinogens, such as those present in tobacco smoke (Petruzzelli et al., 1988; Bartsch et al., 1991). Furthermore, CYP1 activity appears also to correlate with increased risk of breast cancer (Pyykko et al., 1989). In human lung specimens CYP1 was only present in smokers with none of this protein being detectable in non-smokers, and recent studies revealed that smokers are at an increasing risk of developing lung peripheral adenocarcinoma if they display induced pulmonary CYP1 levels (Antilla et al., 1991). Interestingly pulmonary activity was higher in the lungs of cancer patients who had smoked within 30 days before surgery than in cancer-free patients with similar smoking" habits (Bartsch et al., 1991). These findings indicate that expression of CYP1 activity in lung, and thus the ability to metabolically-activate tobacco-derived carcinogens, provide a feasible explanation as to why only a fraction of tobacco smokers develop lung cancer. Finally, in agreement with this concept, lung cancer cell lines exhibit CYP1 m R N A levels, normally absent from normal cell lines (McLemore et al., 1989).
75 The CYP2E family has also been associated with a limited role in the activation of chemical carcinogens, such as certain nitrosamines (Yang et al., 1985).
COMPACT for CYP1 and CYP2E. As there was poor concordance between the sexes, and between the two species, in the rodent assay, when three or more segments (male rat, female rat, male mouse, female mouse) were in agreement, the only rational way forward was to accept a positive result for malignancy in only one or more segment as an overall positive result in the rodent carcinogenicity assay. This resulted in 97/100 chemicals being positive, whereas the remaining three were not sufficiently tested. If the criterion was changed to 3 / 4 segments being in agreement, then only 38/100 chemicals were rated positive (Table 4). In the Ames Salmonella mutagenicity assay, 59/100 chemical were positive, 38/100 negative, and 3 were not tested. In the C O M P A C T procedure 91/100 chemicals were positive and 9 / 1 0 0 negative. From this, it would appear that the C O M P A C T procedure can identify carcinogens/toxic chemicals not positive in the Ames test. This is not surprising since the C O M P A C T procedure identifies the generation of reactive intermediates which may manifest other forms of toxicity, e.g. hepatic necrosis in the case of paracetamol, whereas the Ames test picks up only genotoxins. On the basis of the 100 chemicals evaluated in the present study, the sensitivity for the Ames test is 60% whereas for the C O M P A C T procedure it is much higher at 92%, demonstrating the high capability of the latter procedure to identify carcinogens. Among these chemicals, the rodent bioassay ( 1 / 4 ) did not reveal any unequivocal non-carcinogens, precluding the calculation of "specificity" and "predictive value" and thus the ability of these tests to identify "non-carcinogens" and to distinguish between carcinogens and noncarcinogens. Anomalous rodent carcinogens. Of the 8 chemicals not rated as positive in the C O M P A C T procedure but rated as positive in the rodent ( 1 / 4 ) assay, trifluralin (65), captan (67), chlordane (68), heptachlor (80) and aldrin (89) (Table
3) are all "mouse carcinogens", that is are tumorigenic in one or both sexes of mice but not in either sexes of rat; they are all highly halogenated and consequently resistant to metabolism, and because of this they can initiate ROS generation by the redox cycling of any of the cytochromes P450 to which they bind as substrates. Hence, this is analogous to the generation of ROS, oxidative stress, and malignant transformation seen with CYP2E1 substrates. The greater susceptibility of rodents, especially mice, to the tumorigenicity of these chemicals (Parke and Ioannides, 1990) has long been recognised by the Joint W H O / F A O Meeting on Pesticide Residues (J.M.P.R., 1978) which recommends the study of such chemicals in several other animal species. Trifluralin, which contains the CF 3 group, and a dipropylamino group hindered by two vicinal nitro groups, is a similar case, being a positive tumorigen only in the female mouse, and likely to be resistant to metabolism. Nitrilotriacetic acid (32) is tumourigenic in both sexes of rat, but has a C O M P A C T ratio of 0.07 and a collision diameter of 6.6; it readily forms a ligand complex with iron which is tumorigenic and the mechanism probably involves the catalytic generation of reactive oxygen species by the iron (Preece et al., 1989). Butyl benzyl phthalate (96) is tumorigenic only in the female rat, and is likely to be a substrate of CYP4 and a hepatic peroxisomal proliferator, generating ROS in the liver of rats via excess production of hydrogen peroxide. 4,4'-Methylene dianiline (27), is not recognised as positive in the COMPACT procedure, having a C O M P A C T ratio of 0.14 and a collision diameter of 7.2, whereas a positive value for CYP1 is taken when the ratio is > 0.15, or the collision diameter is < 6.5, for CYP2E1. Finally, dimethylmorpholino phosphoramidate is also not recognised as positive in the C O M P A C T procedure, having a C O M P A C T ratio of 0.11 and a collision diameter of 6.6, but is equivocal in the rodent assay.
Conclusions. Ashby and Tennant's (1988) survey of the rodent carcinogenicity, Salmonella mutagenicity, and chemical structure (potential electrophilic sites) of 222 chemicals tested in rodents in the U.S. N C I / N T P programme, revealed an overall correlation between these three parame-
76
ters. The Salmonella assay was considered to be a sensitive method of detecting genotoxicity, and its use reveals two groups of carcinogens, namely, genotoxic and putatively non-genotoxic chemicals. These two groups show differences in species and tissue specificity, and only 30% of those carcinogens specific for mouse liver were found to be genotoxic. The high correlation of COMPACT with the rodent assay, namely 92%, indicates that this toxicity evaluation procedure, based on chemical structure and knowledge of metabolic activation and detoxication, is highly suitable for screening chemicals of diverse structure. Furthermore, as only the chemical structure has to be known, COMPACT can be used at the earliest stages of product development, thus avoiding costly mistakes of developing new drugs, food additives, or pesticides, that finally turn out to be carcinogenic, or seriously toxic in other ways. From the COMPACT data of a number of drugs and industrial chemicals that were originally considered to be safe, but were later found to be toxic in man, all would have given rise to alerts in the COMPACT procedure (see Table 6). Data obtained by the COMPACT procedure are relevant to all animal species that express the CYP1 and CYP2E families, assuming that their substrate specificity is very similar to the rat orthologous proteins. The cost of conducting the COMPACT procedure is < 1% of a rodent assay and can be completed in days instead of years, so is a small investment to make before undertaking new product development. Furthermore, for those chemicals that are marketed in small amounts, such as food flavours, the COMPACT procedure is ideal for safety evaluation. COMPACT studies may subsequently be confirmed by CYP enzyme induction studies in rodents and other species, which take into account pharmacokinetic aspects that can add a quantitative element to the safety evaluation procedure (Ayrton et al., 1990). Thorough validation of COMPACT is essential to instil public confidence in this new procedure, and unfortunately extensive validation against toxicity/carcinogenicity in humans is limited to the availability of toxicity data from accidental poisoning. Thus one is restricted to validation against experimental animal data, essentially
rodent toxicity and carcinogenicity studies. Validation against a further 100 chemicals studied in the U.S. N C I / N T P evaluation of toxicity/carcinogenicity, has recently been concluded and will be published in due course.
Acknowledgements The authors are most grateful to Mr. A. MacAlastair-Brown and the Humane Research Trust for their continued support over the past five years in the development of the COMPACT procedures, and to the Ministry of Agriculture, Fisheries and Food for support in the application of COMPACT procedures to the screening of food chemicals and flavours for potential chemical toxicity. Our grateful thanks also to Dr. John Ashby who initially suggested this approach for the validation of COMPACT and has given continued advice and encouragement.
References Ames, B.N. and L.S. Gold (1990) Chemical carcinogens: Too many rodent carcinogens, Science, 249, 970-971. Amoah, A.G.B., B.J. Gould and D.V. Parke (1986) Further studies on the pharmacokinetics of perhexiline maleate in humans, Xenobiotica, 16, 63-68. Antilla, S., E. Hietanen, H. Vainio, A.-M. Camus, H.V. Gelboin, S.S. Park, L. Heikkila, A. Karjalainen and H. Bartsch (1991) Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients, International Journal of Cancer, 47, 681685. Aronoff, G.R., T. Ozawa, K.A. DeSante, J.F. Nash and A.S. Ridolfo (1982) Benoxaprofen kinetics in renal impairment, Clinical Pharmacology and Therapeutics, 32, 190-194. Ashby, J. and R.W. Tennant (1988) Chemical structure. salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NC1/NTP, Mutation Research, 204, 17-115. Ayrton, A.D., S. Neville and C. Ioannides (1992) Cytosolic activation of 2-aminoanthracene: Implications in its use as diagnostic mutagen in the Ames test, Mutation Research, 265, 1-8. Bartsch, H., S. Petruzzelli, S. De Flora, E. Hietanen, A.M. Camus, M. Castegnaro, O. Geneste, A. Camoirano, R. Saracci and C. Giutini (1991) Carcinogen metabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control lung study on lung cancer patients, Mutation Research, 250, 103-114. Clayson, D.B. and D.J. Clegg (1991) Classification of carcinogens: Polemics, pedantics or progress?, Regulatory Toxicology and Pharmacology, 14, 147-166.
77 Diaz, O., I. Fabre, M. Daujat, B. Saint Aubert, P. Bories, H. Michel and P. Maurel (1990) Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450, Gastroenterology, 99, 737-747. Eason, C.T., F.W. Bonner and D.V. Parke (1990) The importance of pharmacokinetic and receptor studies in drug safety evaluation, Regulatory Toxicology and Pharmacology, 11,288-307. Ennever, F.K., T.J. Noonan and H.S. Rosenkranz (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans, Mutagenesis, 2, 73-78. Gori, G.B. (1991a) Are animal tests relevant in cancer risk assessment? A persistent issue becomes uncomfortable, Regulatory Toxicology and Pharmacology, 13, 225-227. Gori, G.B. (1991b) Adjudicating cancer causation: Scientific, legal and political conflicts, Regulatory Toxicology and Pharmacology, 13, 309-325. Griest, F.C., I.I. Ozols, A.S. Ridolfo and J.C. Muus (1982) The phototoxic effects of benoxaprofen and their management and prevention, European Journal of Rheumatology and Inflammation, 5, 138-147. Guengerich, F.P. (1988) Roles of cytochromes P-450 enzymes in chemical carcinogenesis and cancer chemotherapy, Cancer Research, 48, 2946-2954. Guengerich, F.P. (1992) Human cytochrome P-450 enzymes, Life Sciences, 50, 1471-1478. Halliwell, B., and J.M.C. Gutteridge (1990) Role of free radicals and catalytic metal ions in human diseases: an overview, Methods in Enzymology, 186, 1-85. Ingelman-Sundberg, M. and A.L. Hagbj6rk (1982) On the significance of cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism, Xenobiotica, 12, 673-686. Ioannides, C. and D.V. Parke (1987) The cytochromes P448 a unique family of enzymes involved in chemical toxicity and carcinogenesis, Biochemical Pharmacology, 36, 4197-4207. Ioannides, C. and D.V. Parke (1990) The cytochrome P4501 gene family of microsomal haemoproteins and their r61e in the metabolic activation of chemicals, Drug Metabolism ReL,iews, 22, 1-86. J.M.P.R. (1978) Etiology and pathogenesis of liver tumours. Joint Meeting on Pesticide Residues, Pesticide residues in food, 1977, FAO Plant Production and Protection Paper 10, Rev., Rome: FAO, pp. 7 and 8. Lewis, D.F.V., C. Ioannides and D.V. Parke (1986) Molecular dimensions of the substrate binding site of cytochrome P-448, Biochemical Pharmacology, 35, 2179-2185. Lewis, D.F.V., C. Ioannides and D.V. Parke (1987) Structural requirements for substrates of cytochromes P-450 and P-448, Chemico-Biological Interactions, 64, 39-60. Lewis, D.F.V., C. Ioannides and D.V. Parke (1989a) Prediction of chemical carcinogenicity from molecular and electronic structure, Toxicology Letters, 45, 1-13. Lewis, D.F.V., C. Ioannides and D.V. Parke (1989b) Molecular orbital studies of oxygen activation and mechanisms of -
-
cytochromes P-450-mediated oxidative metabolism of xenobiotics, Chemico-Biological Interactions, 263-280. Lewis, D.F.V., C. Ioannides and D.V. Parke (1990) A prospective toxicity evaluation (COMPACT) on 40 chemicals currently being tested by the National Toxicology Program, Mutagenesis, 5,433-435. McLemore, T.L., S. Adelberg, M. Czerwinki, W.C. Hubbard, S.J. Yu, R. Storeng, T.G. Wood, R.N. Hines and M.R. Bord (1989) Altered regulation of the cytochrome P450IA gene. Novel inducer-independent gene expression in pulmonary carcinoma cell lines, J. Natl. Cancer Inst., 81, 1787-1794. Neuberger, J. and R. Williams (1989) Immune mechanisms in tienilic acid associated hepatotoxicity, Gut, 30, 515-519. Parke, D.V., C. Ioannides and D.F.V. Lewis (1988) Metabolic activation of carcinogens and toxic chemicals, Human Toxicology, 7, 397-404. Parke, D.V. and C. Ioannides (1990) Role of cytochromes P-450 in mouse liver tumour production, pp. 215-230, In:
Mouse Lit,er Carcinogenesis, Mechanisms and Species Comparisons, Eds: D.E. Stevenson, R.M. McClain, J.A. Popp, T.A. Slaga, J.M. Ward and H.C. Pitot, New York: Alan R. Liss. Parke, D.V., C. Ioannides and D.F.V. Lewis (1990) The safety evaluation of drugs and chemicals by the use of Computer Optimised Molecular Parametric Analysis of Chemical Toxicity (COMPACT), ATLA, 18, 91-102. Parke, D.V., C. Ioannides and D.F.V. Lewis (1991) The role of the cytochromes P-450 in the detoxication and activation of drugs and other chemicals, Canadian Journal of Physiology and Pharmacology, 69, 537-549. Petruzzel|i, S., A.M. Camus, L. Carrozzi, L. Chelarducci, M. Rindi, G. Menconi, C.A. Angeletti, M. Ahotupa, E. Hietanen, A. Aito, R. Saracci, H. Bartsch and C. Giuntini (1988) Long-lasting effects of tobacco smoking on pulmonary drug-metabolising enzymes: a case-control study on lung cancer patients, Cancer Res., 48, 4695-4700. Pople, J.A., D.P. Santry and G.A. Segal (1965) Approximate self-consistent MO theory. 1. Invariant procedures, Journal of Chemical Physics, 43, S129-135. Preece, N.E., D.E. Hall, J.A. Howarth, L.J. King and D.V. Parke (1989) Effects of acute and sub-chronic administration of iron nitrilotriacetate in the rat, Toxicology, 59, 37-58. Pyykko, K., R. Tuimala, L. Aalto and T. Perkio (1989) Aryl hydrocarbon hydroxylase activity in human breast cancer tissue, In: I. Schuster (Ed.), Cytochrome P450 Biochemistry and Biophysics, Taylor and Francis, London, pp. 528-531. Sies, H. (1991) Role of reactive oxygen species in biological processes, Klinische Wochenschrift, 69, 965-968. Vinter, J.G., A. Davis and M.R. Saunders (1987) Strategic approaches to drug design. I. An integrated software framework for molecular modelling, Journal of ComputerAided Molecular Design, 1, 31-51. Yang, C.S., Y.Y. Tu, D.R. Koop and M.J. Coon (1985) Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes, Cancer Research, 45, 1140-1145.
61
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1161/93/$06.00
MUTENV 08858
Validation of a novel molecular orbital approach (COMPACT) for the prospective safety evaluation of chemicals, by comparison with rodent carcinogenicity and Salmonella mutagenicity data evaluated by the U.S. NCI/NTP David F.V. Lewis, C. Ioannides and D.V. Parke Molecular Toxicology Group, School of Biological Sciences, Unit,ersityof Surrey, Guildford, Surrey, GU2 5XH, UK (Received 7 July 1992) (Revision received 14 October 1992) (Accepted 15 October 1992)
Keywords: Molecular structure; Cytochromes P450; Computer graphics; Ames test; Safety evaluation; Rodent carcinogenicity
Summary The molecular dimensions and electronic structures of 100 chemicals of structural diversity have been determined from molecular orbital calculations and molecular mechanics. From these parameters of molecular structure, those chemicals that are likely substrates of cytochromes P4501 and P4502E have been identified by the computer-optimized molecular parametric analysis of chemical toxicity (COMPACT) programme, and their potential toxicity, mutagenicity and carcinogenicity evaluated. The degree of correlation between COMPACT prediction of toxicity and rodent two species life-span carcinogenicity data is estimated to be 92%, and between COMPACT and Salmonella mutagenicity (Ames test) data is 64%. Anomalous rodent carcinogens are rationalized on the basis of biochemical mechanisms of metabolism, genotoxicity and carcinogenicity. Correlation of the Ames test data with rodent carcinogenicity data was 64%, but correlation of COMPACT plus Ames data versus rodent carcinogenicity data provided the highest correlation of 94%.
The need for the safety evaluation of chemicals, especially novel entities to be used as medicines or pesticides, became apparent nearly half a century ago with the human tragedies following the use of the drugs chloramphenicol, diethylstilboestrol, thalidomide, etc. (Parke et al.,
Correspondence: Dr. D.V. Parke, Molecular Toxicology Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH, UK.
1990). Current safety evaluation procedures, based largely on the use of rodents and other experimental animals as surrogates for man, are mostly empirical and when ultimately validated, as in the prescription o~f new medicines for humans, have frequently b~en found to be inadequate (Eason et al., 1990) as in the case of perhexiline (Amoah et al., 1986), benoxaprofen (Griest et al., 1982; Aronoff et al., 1982) and tienilic acid (Neuberger and Williams, 1989). The validity of the rodent life-span bioassay for poten-
62
tial carcinogenicity, used for the prediction of toxicity/carcinogenicity of chemicals in humans, has recently been questioned, and has been considered by experts to be 'unscientific' and 'essentially ritualistic' (Ames and Gold, 1990; Ennever et al., 1987; Gori, 1991a and b). From knowledge of mechanisms of toxicity/carcinogenicity it is no longer considered valid to assume that an animal-based observation will always be relevant to humans, for some carcinogens appear to be species-specific because of differences in metabolism, receptor response, genetic regulation, etc. (Clayson and Clegg, 1991). Three major factors appear to determine chemical toxicity/carcinogenicity, namely, (1) the molecular structure and properties of the chemical, upon which its biological behaviour depends, (2) the genetic complement of the exposed biological species, and (3) the environment of the exposed species, especially diet and exposure to
other chemicals. Of these, the first, namely, molecular structure is the only relevant determinant in the safety evaluation of chemicals. Moreover, because of variations in species/genetics and environment/diet, current safety evaluations in experimental animals as human surrogates frequently lead to spurious decision making. The direct determination of potential toxicity/carcinogenicity of a chemical from its molecular structure requires detailed knowledge of (i) the molecular conformation and electronic structure of the chemical, and (ii) its metabolism and interactions with numerous biological receptors, and the toxicological consequences of these phenomena. Recent advances in computer technology have now made feasible the determination of molecular structure from energy calculations by molecular mechanics and electronic structure by molecular orbital (MO) methods for relatively large molecules, and the present extensive libraries of
TABLE 1 METABOLIC ACTIVATION OF CHEMICAL CARCINOGENS BY RAT HEPATIC CYTOCHROME P450 PROTEINS
Carcinogen
Cytochrome P450 protein
A a C (2-amino-9H-pyrido[2,3-b]indole) 2-Acetylaminofluorene Aflatoxin B1 2-Aminoanthracene
1AI, 1A2 1AI, IA2,2B1 1A2,2BI, 2Cll, 2C12 1AI, 1A2
o-Aminoazotoluene 4-Aminobiphenyl 2-Aminofluorene Benzo[a]pyrene
1A2 1A2 1AI, IA2 1A1,2BI
4,4'-(bis)Methylene chloroaniline (MOCA) 1,2,3,4-Dibenzanthracene 7,12-Dimethylbenz[a]anthracene N, N-Dimethylnitrosamine
1A2,2B1 1AI, IA2,2B1 1AI 2El
GIu-P- 1 (2-amino-6-methylpyrido[ 1,2-a : 3',2'-d] imidazole) GIu-P-2 (2-amidopyrido[1,2-a : 3',2'-d] imidazole) IQ (2-amino-3-methylimidazo [4,5-f]-quinoline) MeA o~C (2-amino-3-methyl-9H-pyrido[2,3-b] indole)
1A2 1AI, IA2 1AI, 1A2 1AI, IA2
MeIQ (2-amino-3,4-dimethylimidazo[4,5-f ] quinoline) MeIQx (2-amino-3,8-dimethylimidazo[4,5-f ] quinoxaline) 3-Methyleholanthrene N-Methyl-4-aminoazobenzene
1AI, IA2 1AI, IA2 1A1 1AI,1A2
2-Naphthylamine Trp-P-1 (3-amino-l,4-dimethyl-5H-pyrido[4,3-b] indole) Trp-P-2 (3-amino-l-methyl-5H-pyrido 14,3-b] indole)
1A2 1AI, 1A2 1AI, 1A2
Data from Guengerich (1988).
63
knowledge on xenobiotic metabolism, mechanisms of toxicity, and the molecular biology of enzymes, receptors and DNA, have now made possible the prediction of potential toxicity/carcinogenicity of known chemical structures from fundamental scientific principles. Chemicals manifesting overt toxicity, and direct-acting mutagens/carcinogens, e.g. alkylating agents, are readily identified from chemical structure, but those chemicals that require metabolic activation to electrophilic reactive intermediates to manifest carcinogenicity/toxicity are more difficult to recognise. Much of the metabolic activation of chemicals to toxic electrophiles that covalently interact with DNA, enzymes and proteins to cause mutations and malignancy, acute lethal injury and tissue necrosis, and neoantigen formation and immunotoxicity, respectively, is the result of metabolic oxygenation by the cytochrome P450 superfamily and other enzymes (Lewis et al., 1989b; Parke et al., 1988; Parke et al., 1991). Of the cytochromes P450, some families more than others are associated with metabolic activation and the formation of reactive intermediates. Cytochrome P4501 (CYP1) is such a family and leads to chemical toxicity (Ioannides and Parke, 1990) by (i) oxygenating planar molecules, that can intercalate with DNA (Ioannides and Parke,
1987) and thus function as genotoxic alkylating agents, and (ii) by its genomal regulation via the cytosolic Ah receptor which results also in the initiation of the protein kinase c cascade, DNA replication and cellular proliferation (Parke et al., 1988; Ioannides and Parke, 1990). MO determination of the electronic structures, and molecular mechanics determination of the molecular conformations, of substrates of CYP1, and of inducers of CYP1 that interact with the Ah receptor, show that these are very similar (Lewis et ai., 1986). Thus many chemicals that interact with CYP1 to produce genotoxic reactive intermediates will also interact with the Ah receptor resulting in the induction of CYP1 and associated enzymes, and the initiation of the protein kinase c cascade culminating in non-genotoxic, mitotic events (Ioannides and Parke, 1987; Ioannides and Parke, 1990). Lists of known chemical carcinogens reveal that more than 90% of the compounds are substrates of rat CYP1 (see Table 1) (Guengerich, 1988; Ioannides and Parke, 1987) although in man several carcinogens are activated by CYP3 (cytochrome P4503) enzymes (Guengerich, 1992). For this reason, a procedure known as COMPACT (Computer Optimised Molecular Parametric Analysis of Chemical Toxicity) was developed to recognise potential car-
TABLE 2 CORRELATION OF MOLECULAR ORBITAL CHRARACTERISTICS OF SOME CHEMICALS WITH CYTOCHROME P450 SPECIFICITY AND TOXICITY Chemical
Major interacting CYP
area/depth 2
AE
COMPACT ratio (area/(depth 2 × zaE))
Collision diameter
Toxicity
1. Dibenzanthracene 2. Benzo[a]pyrene 3. 3-Methylcholanthrene
1A1 1A1 1A1
14.4 12.0 7.9
10.8 9.3 7.8
1.33 1.29 1.01
7.8 7.6 10.1
C,M C,M C,M
4. 5. 6. 7.
2-Aminoanthracene Trp-P-1 4-Aminobiphenyl 2-Acetamidofluorene
1A2 1A2 1A2 1A2
9.2 8.5 8.3 5.0
10.0 12.8 12.9 12.8
0.92 0.66 0.64 0.39
7.0 7.2 6.8 7.3
C,M C,M C,M C,M
8. Benzene 9. Dimethylnitrosamine 10. Halothane
2El 2El 2El
5.4 2.9 2.0
17.9 14.5 10.8
0.30 0.20 0.19
5.4 5.1 5.6
L C,M,H H
11. Hexobarbital 12. Phenobarbital 13. DDT
2B 2B 2B
1.8 1.1 0.8
15.8 15.8 13.5
0.11 0.07 0.06
7.4 7.3 7.9
N N N
C, carcinogenic; M, mutagenic; L, leukaemogenic; H, hepatotoxic; N, no overt toxicity.
64 COMPACT
%
I0
%
D 7
t 12 Delta
r _ 17 E
Fig. 1. S e p a r a t i o n o f C Y P 1 s u b s t r a t e s in a p l o t o f a / d 2 against A E for some miscellaneous chemicals. CYP1 substrates are Nos. 1-7. The identities of the other compounds, a n d t h e s t r u c t u r a l p a r a m e t e r s a n d i n t e r a c t i n g C Y P s a r e given in T a b l e 2. S u b s t r a t e s o f C Y P I a r e a b o v e t h e line.
cinogens/toxic chemicals from their molecular structure by identification of the CYP family with which they are most likely to interact and undergo metabolic activation. Substrates of CYP1 are characterised by their high degree of planarity, which is expressed as high values of a r e a / d e p t h s (a/d2), and by their electronic structure, which facilitates oxygenation (Lewis et al., 1989b), and is expressed as low values of A E ( A E = energy of the lowest empty molecular orbital - energy of the highest occupied molecular orbital, or A E = E ( L U M O ) E ( H O M O ) ) . The ratio of a / ( d 2 × AE) is termed the C O M P A C T ratio, which is used as a criterion in the identification of CYP1 substrates. A plot of a / d 2 against AE provides a more precise means for the separation of CYP1 substrates from other chemicals (Fig. 1), and the structural characteristics of these substrates are shown in Table 2. Criteria for potential CYP1 substrates are as follows: a / d 2 = > 5.0, AE = < 14.0, and COMP A C T ratio = > 0.15, of which the C O M P A C T ratio ( a / d 2) is the most valuable. However, it is more precise to examine the position of the chemical on the graph of a / d 2 against AE Experience has shown that although the C O M P A C T procedure for CYP1 substrates identifies most potential carcinogens, mutagens and
toxic chemicals, such as polycyclic aromatic hydrocarbons, aromatic amines and heterocyclic amines, it does not identify all chemicals characterised as carcinogens by the rodent, two-species, lifespan assay, for example some short-chain dialkyi nitrosamines and haloalkanes. Consideration of these exceptions led to the conclusion that many are substrates/inducers of cytochrome P4502E1 (CYP2E1). Induction of this cytochrome results in the generation of reactive oxygen species, ROS, (Ingelman-Sundberg and Hagbj6rk, 1982) which can result in oxidative stress culminating in tissue necrosis, mutations and malignancy (Halliwell and Gutteridge, 1990; Sies, 1991) especially in rodents (Parke and Ioannides, 1990). It therefore became desirable to define the molecular structure characteristics of CYP2E substrates, the most characteristic of which is a low collision diameter. Criteria for CYP2E substrates are therefore as follows: a / d 2 = 2.0-6.0, AE = > 10.0; C O M P A C T ratio = < 0.35; collision diameter = < 6.5, of which the most critical is the collision diameter. A plot of collision diameter against C O M P A C T ratio provides good separation of CYP2E substrates from other chemicals (Fig. 2). The advantages of the C O M P A C T procedure are that (i) unlike the Ames test, it is not limited
15 []
.o
1.0
ev < 12. x o
0.5 [] []
0.0
i
6
r
r
7 B 9 10 11 Collision Diameter Fig, 2. Separation of CYP2E substrates in a plot of COMPACT ratio against collision diameter for some miscellaneous chemicals. CYP2E substrates are Nos. 8-10. The identities of the other compounds, and the structural parameters and interacting CYPs are given in Table 2. Chemicals with collision diameters of < 6.5 (Nos. 8-10) are CYP2E substrates.
65 TABLE 3 M O L E C U L A R PARAMETERS, AMES TEST A N D R O D E N T LIFE-SPAN C A R C I N O G E N I C I T Y ASSAY F O R 100 MISCELL A N E O U S CHEMICALS Key to Carcinogenicity data: R, rat; M, mouse; m, male; f, female; P, positive; N, negative; E, equivocal; IS, inadequate study; CE, clear evidence; NE, no evidence; SE, some evidence; EE, equivocal evidence; NT, not tested; * difficult to evaluate. In the COMPACT assay (CYP1), values of a r e a / d e p t h 2 x AE of 0.15 or greater are taken as ( + ) ; and values < 0.15 are taken as ( - ) . In the COMPACT assay (CYP2E) a collision diameter of 6.5 or less is taken as ( + ) . Code
Compound name
Area/
Collision
Salmonella
Rodent carcinogenicity
COMPACT
depth2xAE
diameter
assay
R(m)
R(f)
M(m)
M(f)
(CYP1)
(CYP2E)
0.13
6.5
-
P
N
N
P
-
+
12.3 0.84
+
P
P
P
+
4.4
12.6
0.35
+
P
P
P
P
+
6.2 4.3 4.8
12.0 14.7 17.9
0.52 0.30 0.27
+ + -
P P CE
P P CE
N P CE
P P CE
+ + +
2.2
14.9
0.14
+
P
E *
P
P
-
5.1
14.3
0.36
+
P
N
N
P
+
5.7
13.8
0.41
+
P
P
P
P
+
CI basic red 9 CI disperse yellow 3 cinnamyl anthranilate
3.8
8.6
0.44
+
CE
CE
CE
CE
+
8.4
10.5 0.80
+
P
N
N
P
+
7.3
14.1 0.52
-
P
N
P
P
+
p-cresidine cupferron 2,4-diaminoanisole
4.6 8.6
14.4 13.2
0.32 0.65
+ +
P P
P P
P P
P P
+ +
3.7
14.1 0.26
+
P
P
P
P
+
4.0
15.0
0.26
+
P
P
N
P
+
1.5
10.0
0.15
+
P
P
P
P
+
2.4
12.4
0.19
+
P
P
P
P
+
2.2 1.7
15.7 0.14 15.5 0.11
5.2 4.7
+ NT
P SE
P CE
P CE
P CE
-
+ +
1.3
14.7
0.09
5.4
+
CE
SE
IS
CE
-
+
5.3
+
P P EE
P P SE
P P CE
P P CE
+ +
+
No. 1. 2. 3.
4. 5. 6. 7. 8. 9.
10. 11. 12.
13. 14. 15.
16. 17. 18.
19. 20. 21.
Area/
AE
depth 2 allyl isovalerate 2-aminoanthraquinone 3-amino 2-ethylcarbazole 1-amino 2-methylanthraquinone o-anisidine benzene 3-chloromethyl pyridine 4-chloro-m-phenylene diamine 4-chloro-o-phenylene diamine
2,4-diaminotoluene 1,3-dibromo3-chloropropane 1,2-dibromoethane 1,2-dichloroethane dichloromethane 1,3-dichloropropene
2.3 10.3
17.6
5.9
22. 23. 24.
DEHP 1,4-dioxane HC blue 1
3.1 1.6 3.7
14.4 0.22 19.5 0.08 1t.6 0.32
25. 26.
hydrazobenzene 4,4'-methylenebis(N,N-dimethyl) aniline
9.9
14.2
0.70
+
P
P
N
P
+
2.9
14.4
0.20
-
P
P
E
P
+
+
66 T A B L E 3 (continued) Code
Compound name
No. 27.
28. 29. 30.
31. 32. 33.
34. 35. 36.
37.
Area/
~E
depth 2 4,4'-methylenedianiline 2-methyl-l-nitroanthraq uinone Michler's ketone 1,5-naphthalenediamine nithiazide nitrilotriacetic acid 5-nitroacenaphthene 5-nitroo-anisidine nitrofen p-nitrosophenylamine
Area/
Collision
Salmonella
R ode nt carcinogenicity
COMPACT
depth2xaE
diameter
assay
R(m)
R(f)
M(m)
M(f)
(CYP1)
7.2
+
P
P
P
P
-
2.0
14.7
0.14
6.3 4.8
12.2 13.0
0.52 0.37
+ +
P P
P P
NT P
NT P
+ +
7.8
12.0
0.65
+
N
P
P
P
+
4.0
12.5
0.32
+
N
P
P
E
+
1.1
15.5
0.07
-
P
P
N
N
-
5.0
13.5
0.37
+
P
P
N
P
+
5.0 2.2
12.8 13.2
0.38 0.17
+ +
P N
P N
E * P
P P
+ +
3.2
I1.4
0.28
+
P
N
P
N
+
3.1 11.0 4.0
13.7 10.7 13.6
0.23 1.03 0.30
+ + -
P P N
P P P
P N P
P P P
+ + +
+ +
SE P P
SE N P
CE P P
CE P P
+ +
6.6
38. 39.
4,4'-oxydianiline phenazopyridine phenesterin
40. 41. 42.
1,2-propene oxide 1.6 reserpine 3.4 sulfallate 2.5
19.3 12.8 12.4
0.08 0.26 0.20
43. 44.
TCDD tetrachlorovinphos 4,4'-thiodianiline
7.8
12.7
0.61
-
P
P
P
P
+
2.3
12.4
0.18
-
N
P *
P
P *
+
2.9
13.0
0.22
+
P
P
P
P
+
o-toluidine TCP 2,4,5-trimethylaniline
3.7 5.9
15.1 13.8
0.24 0.43
+ -
P P
P N
P P
P P
+ +
3.7
14.4
0.26
+
P
P
E *
P
+
1.3
15.6
0.08
+
P *
N
N
P
-
1.4
8.4
0.16
+
P
P
P
P
+
3.2
17.5
0.18
-
P
N
E *
N
+
aniline 5.4 azobenzene 9.7 bis (2-chloromethyl ethyl) ether 1.7
15.4 11.2
0.35 0.80
+
P P
P P
N N
N N
+ +
16.1
0.10
+
N
N
P
P
-
4.4 5.2
14.3 9.9
0.31 0.52
+
N P
N E *
P N
P N
+ +
3.7
11.1
0.33
+
P
P
N
N
+
45.
46. 47. 48.
49. 50.
51.
52. 53. 54.
55. 56. 57.
trimethyl phosphate tris (2,3-dibromopropyl) phosphate 11-amino-undecanoic acid
5-chloroo-toluidine C red 9 3,3 '-dimethoxybenzidine diisocyanate
4.7
5.9
6.5
(CYP2E)
+
+
67 TABLE 3 (continued) Code No.
Compound name
58.
dimethyl hydrogen phosphite 2,4-dinitrotoluene 1,2-butene oxide
Area/
dE
depth 2
Area/
Collision
Salmonella
Rodent carcinogenicity
COMPACT
depth2×AE
diameter
assay
R(m)
R(f)
M(m)
M(f)
(CYP1)
(CYP2E)
1.4
15.7
0.09
5.5
+
CE
EE
NE
NE
-
+
4.4 1.3
13.3 19.2
0.33 0.06
5.1
+ +
P CE
P EE
N NE
N NE
+ -
+
6.4
13.4
0.48
NT
N
N
P
P
+
4.2
13.1
0.32
+
N
N
P
P
+
2.5
13.0
0.19
-
P
N
N
N
+
65.
1,1,2-trichloroethane trifuralin
1.6 1.7
15.1 12.1
0.11 0.14
+
N N
N N
P N
P P
-
66.
zearalenone
4.0
13.9
0.29
-
N
N
P
P
+
67. 68. 69.
captan chlordane chlorobenzilate
1.7 1.2 2.4
12.9 13.0 14.3
0.13 0.09 0.17
+ -
N N E
N N E
P * P P
P * P P
+
70. 71.
chlorotalonil 4-chloroo-toluidine CI disperse blue 1
6.1
13.6
0.45
-
P
P
N
N
+
4.0
14.3
0.28
-
N
N
P
P
+
10.5
10.9
0.91
+
CE
CE
EE
NE
+
4.4
14.0
0.32
+
P
P
N
+
1.7 2.5
11.0 12.1
0.15 0.21
-
SE N
SE N
EE P
NE P
+ +
6.3
12.9
0.49
+
N
N
P
P
+
3.9
17.5
0.22
-
N
N
P
P
+
3.8
12.9
0.29
-
P
P
N
N
+
1.6 1.2
14.1 13.0
0.11 0.09
6.6 7.5
-
SE N
SE N
NE P
NE P
-
-
1.3
14.0
0.09
6.3
-
N
N
P
P
-
+
6.4
13.3
0.48
+
N
N
P
P
+
2.1
12.6
0.18
-
P
P
N
N
+
1.3
14.2
0.09
6.0
-
E *
N
P
P
-
+
1.1
15.5
0.07
5.6
+
P
P
N
N
-
+
1.5
14.5
0.01
5.5
-
E *
N
P
P
-
+
59. 60. 61. 62. 63.
64.
72.
73. 74. 75. 76.
77. 78.
79.
80. 81.
82. 83. 84.
85. 86.
estradiol mustard 5-nitroo-toluidine 4,4'-sulphonyldianiline
rn-cresidine decabromodiphenyloxide DDE 2,6-dichlorop-phenylene diamine di(2-ethylhexyl)adipate N,N-diethylthiourea dimethylmorpholinophosphoramidate heptachlor hexachloroethane 5-nitrobenzimidazole N-nitrosodiphenylalanine pentachloroethane pivalolactone 1,1,1,2-tetrachloroethane
5.5 7.4
7.4 7.6
+ -
-
68
TABLE 3 (continued) Code
Compound name
No. 87.
88. 89. 90.
91. 92. 93.
94. 95. 96.
Area/
zaE
depth 2 1,1,2,2-tetrachloroethane trichloroethene aldrin allylisothiocyanate 3-amino 4-ethoxyacetanilide 4-amino-2-nitrophenol 2-amino-5-nitrothiazole p-benzoquinone dioxime 2-aminobiphenyl butylbenzylphthalate
Area/
Salmonella
Rodent carcinogenicity
COMPACT
R(m) R(f) M(m) M(f) (CYP1) (CYP2E)
1.9
14.9
0.13
5.8
-
E *
N
P
P
-
+
3.5 1.1
14.2 12.7
0.24 0.08
7.7
NT
N E *
N E *
P P
P N
+ -
-
1.9
12.1
0.16
+
P
E
N
N
+
3.2
14.2
0.22
+
N
N
P
N
+
7.0
12.2
0.57
+
P
E
N
N
+
4.1
12.7
0.32
+
P
N
N
N
+
6.7 6.0
9.0 13.0
0.74 0.46
+ +
N N
P N
N E
N P
+ +
1.3
14.4
0.09
P *
N
N
-
0.51
97.
chloramben
6.8
13.5
98.
chlorodibromoethane CI vat yellow 4 dicofol
1.5 14.4 2.2
10.4 0.14 9.4 1.54 13.1 0.17
99. 100.
Collision
depthZxAE diameter assay
8.1
5.3
to identification of genotoxic carcinogens since it also identifies chemicals that activate the Ah receptor resulting in CYP1 induction and tissue proliferation, or stabilize CYP2E with increased generation of ROS, (ii) it also identifies chemicals that are metabolically activated to electrophiles which bind to enzymes and proteins and hence are cytotoxic (e.g. paracetamol) or immunotoxic (e.g. halothane), (iii) it is unique in that it requires no biological or chemical materials and hence can be applied even before the particular chemical has been synthesized. This makes the procedure truly predictive, so that safety evaluation may be applied at the design stage with the potential of integrating desired pharmacological activity with the absence of toxicity at the earliest stages of product development, thus facilitating major savings in time and capital investment. To validate this novel safety evaluation procedure of C O M P A C T , which is based on molecular structure rather than biological events, correlations of the C O M P A C T identification of CYP1
+
N
N
E
P
+
-
NE N N
NE N N
EE P P
SE N N
+ +
-
+
and CYP2E substrates as potential carcinogens with those identified as carcinogens in the rodent carcinogenicity assay, or as mutagens in the Salmonella mutagenicity (Ames) assay, was undertaken utilising the first 100, structurally diverse, organic chemicals of the U.S. N C I / N T P database (Ashby and Tennant, 1988). Methods
Determination of molecular and electronic structural parameters. All 100 chemicals listed in Table 3 were taken from the data of Ashby and Tennant (1988) and their molecular structures analysed for CYP1 and CYP2E specificity using the C O M P A C T procedure as described previously (Lewis et al., 1989a). Molecular structures were constructed from first principles using the C O S M I C modelling package (Vinter et al., 1987) and minimised to give optimised geometries by the conjugate gradient method. Molecular shape
69
parameters ( a r e a / d e p t h z) were calculated from overall molecular dimensions using° the following van dero Waals radii: ocarb°n = 1.6 A, hyodrogen = 1.2 A, oxygen = 1.4 A, nitrogen = 1.4 A, sulphur = 1.85 A, chlorine = 1.8 A, bromine = 1.95 A, phosphorus = 1.9 A and fluorine = 1.35 A. The energy minimised molecular structures were subjected to molecular orbital (MO) calculations by the C N D O / 2 procedure (Pople et al., 1965) via the COSMIC software framework. Compound 'length' was the longest molecular dimension, 'width' was the longest molecular dimension at right angles to the 'length', and 'depth' was the longest molecular dimension at right angles to the 'length' and 'width'; 'diameter' is the collision diameter which is equivalent to the diameter of a sphere traced out by the free rotation of the molecule about its geometric centre along three mutually orthogonal (x,y and z) axes. The collision diameter is related to the van der Waals volume and is proportional to the cross-sectional area perpendicular to the main molecular plane. The difference between frontier orbital energy levels was taken as the alE electronic structural p a r a m e t e r (where alE = E ( L U M O ) E(HOMO)). All calculations and measurements were performed on a Sigmex $6130 graphics terminal connected to a MicroVAX II computer running the COSMIC software package. o
o
o
Determination of CYP1 substrates. As previously described (Lewis et al., 1989a; Lewis et al., 1990), the values for the a r e a / d e p t h 2 for the molecular dimensions of the 100 chemicals were plotted against the electronic structural parameter, AE. The objective was to identify substrates of CYP1, which can be activated to reactive intermediates and function as mutagens, carcinogens, neoantigens and other cytotoxic agents. CYP1 substrates are characterised by high values of a r e a / d e p t h 2 (planarity) and by low values of A E (ease of oxygenation). CYP1 substrates may thus be identified by reading from the plot of a r e a / d e p t h 2 vs. alE (see Fig. 1). However, to avoid the subjective nature of graphic interpretation, values of area/(depth2 x alE) ( C O M P A C T ratio) were calculated and used to identify CYP1 substrates. From previous C O M P A C T studies of more than 200 miscellaneous chemicals and
chemical carcinogens it was considered that chemicals with C O M P A C T ratios of > 0.15 were potential CYP1 substrates, i.e. ( + ) ; those with values of < 0.15 are not likely substrates of CYP1, i.e. ( - ) . It is appreciated that this simplistic numerical approach will incur errors, as the boundary defining the characterisitic spatial and electronic parameters of CYP1 substrates is nonlinear. Hence, for compounds with very low values of alE, C O M P A C T calculations will have an increased number of false negatives, and for compounds with very high values of d E there will be an increased number of false positives. In these two categories, inspection of the graph may be necessary.
Determination of CYP2E substrates. Similarly, CYP2E substrates were identified primarily from measurement of the collision diameter. Known substrates of CYP2E were all found to have collision diameters of < 6.5, to be small molecules (low a / d 2 values), high values of AE (difficult to oxygenate), and therefore low values for the C O M P A C T ratio. Chemicals with collision diameters of < 6.5 were designated CYP2E substrates, i.e. ( + ) . Inspection of the graph (Fig. 2) shows that a plot of collision diameter against C O M P A C T ratio readily segregates CYP2E substrates from those of CYP1, CYP2B and other cytochromes P450. The precision of this segregation of CYP2E substrates from molecular structural parameters has been extended even further by the use of a 3-dimensional plot (diameter vs. a / d 2 vs. AE), which can readily be viewed on the computer screen. Validation procedures. The identification of CYP1 and CYP2E substrates as potential carcinogens was essentially unequivocal, as was the identification of Salmonella mutagenicity (with and without metabolic activation), but rodent carcinogenicity is dependent on species and sex, and was therefore dependent on the evaluation of the data. In the study of both sexes of rats and mice only 21/100 compounds were positive for carcinogenicity in both sexes of both species (4/4), and no compounds were negative in both sexes of both species. The problem was therefore how to define a positive result. Enquiries of those regula-
70 TABLE 4 COMPARISON OF COMPACT NEOUS CHEMICALS
ASSAY WITH AMES TEST AND RODENT
LIFE-SPAN ASSAY FOR
100 M I S C E L L A -
In the r o d e n t ( 1 / 4 ) assay, o n e positive result in e i t h e r or b o t h sexes of o n e o r b o t h species is t a k e n as ( + ) a n d no positive in e i t h e r species is t a k e n as ( - ) . I n t h e r o d e n t ( 3 / 4 ) assay, if 3 o f the 4 p a r a m e t e r s o f 2 sexes o f 2 species are positive the result is t a k e n as ( + ) , if less t h a n this the result is t a k e n as ( - ). In t h e C O M P A C T assay, 91 c o m p o u n d s w e r e ( + ) a n d 9 c o m p o u n d s w e r e ( - ) . In t h e A m e s test, 59 c o m p o u n d s w e r e ( + ) , 38 c o m p o u n d s w e r e ( - ) , a n d 3 c o m p o u n d s w e r e n o t studied. In the r o d e n t assay, 97 c o m p o u n d s w e r e ( + ) a n d 3 c o m p o u n d s w e r e not sufficiently t e s t e d w h e r e t h e c r i t e r i o n was 1 / 4 o f the two species two sexes b e i n g positive; a n d 38 c o m p o u n d s w e r e ( + ) a n d 48 c o m p o u n d s w e r e ( - )
a n d 14 w e r e not sufficiently t e s t e d
w h e r e the c r i t e r i o n was 3 / 4 o f the two species two sexes b e i n g positive. Compound No.
Ames test
Rat
Mouse
Rat and Mouse r o d e n t assay
COMPACT assay
A m e s plus rodent 1/4
1/4
3/4
(CYP1 & CYP2E)
assay
A m e s plus COMPACT
1.
-
-+ ++ ++
-
+
+ +
++? ++
+
2. 3.
+ +
+ +
+ +
_+ + +
_+ + +
4. 5.
+ +
++ ++
-+ ++
+ +
+ +
+ +
+ +
+ +
6.
-
++
++
+
+
+
_
_+
7.
+
+?
++
+
+
+
+
+
8.
+
-
+
+
+
+
-+ ++
+
9.
+++
+
+
+
+
+
10.
+
++
++
+
+
+
+
+
11. 12.
+ -
++-
-+ + +
+ +
+
+ +
+ _+
+ +
13. 14. 15.
+ + +
++ ++ ++
++ ++ ++
+ + +
+ + +
+ + +
+ + +
+ + +
16. 17. 18.
+ + +
++ ++ ++
-+ ++ ++
+ +
+ +
+ +
+ +
+ +
+
+
+
+
+
19. 20.
+ NT
++ ?+
++ + +
+ +
+ +
+ +
+ NT
+ NT
21. 22.
+ -
+? ++
?+ + +
+ +
? +
+ +
+ _-2-
+ _+
23. 24. 25.
+ +
+ + ?? + +
++ + + - +
+ + +
+ ? +
+ + +
_+ + +
_+ + +
26. 27. 28. 29. 30.
+ + +
+ + ++ + + ++ - +
?+ ++ NT NT + +
+ + + +
+ + ? +
+ + +
_+ + + +
_+ + + +
++
+
+
+
+
+
31. 32. 33. 34. 35.
+ + + +
- + + + ++ + +
+? -+ ?+ + +
+ + + + +
? + + -
+ + + +
+ + + + +
+ + + +
36. 37. 38. 39. 40.
+ + + +
+++ ++ -+ ??
+++ -+ ++ + +
+ + + + +
+ + + 9
+ + + + +
+ + + _+ +
+ + + +_ +
+
71
TABLE
4 (continued)
Compound
Ames
No.
test
Mouse
Rat
Rat
and
rodent
-
Mouse assay
1/4
3/4
COMPACT
Ames
assay
rodent
(CYP1
& CYP2E)
plus 1/4
Ames
assay
!k
41.
-
+
+ +
+
+
+
42.
+
++
++
+
+
+
43.
-
++
+
+
+
+
+
44.
-
-+
++
+
+
+
±
45.
+
+ +
+ +
+
+
+
+
+
46,
+
+ +
+
+
+
+
+
+
+
47,
-
+ -
+ +
+
+
+
48,
+
+
.9+
+
+
+
+
+
49,
+
+ -
-
+
-
+
4-
+
50,
+
++
++
+
+
+
+
-/-
+
+
+
+
51.
_
+_
9_
+
_
+
±
52.
-
+ +
-
-
+
-
+
±
+
53.
+
+
+
-
-
+
-
+
+
+
54.
+
-
-
+ +
+
-
+
+
÷
55.
-
-
-
+ +
+
-
+
__+_
56.
+
+?
-
-
+
-
+
+
+
57.
+
+ +
-
-
+
-
+
÷
+
58.
+
+ ?
-
-
+
-
+
+
-t-
59.
+
+ +
-
-
+
-
+
+
÷
60.
+
+ ?
-
-
+
-
+
+
+
61.
NT
-
-
+ +
+
-
+
NT
NT
62.
+
-
-
+ +
+
-
+
+
+
63.
-
+-
- -
+
-
+
64.
-
-
-
+
+
+
-
+
65.
+
-
-
-
+
+
-
-
÷ +
66.
-
-
-
+ +
+
-
+
÷
67.
+
-
-
+
+
+
-
-
+
68.
-
-
-
+
+
+
-
-
÷
69.
-
??
+ +
+
?
+
70.
-
+ +
-
+
-
+
÷
71.
-
- -
+ +
+
-
+
-1-
72.
+
+ +
?-
+
?
+
+
+
73.
+
+ +
?-
+
?
+
+
+
74.
-
??
? -
?
?
+
9
÷
75.
-
-
+
-
+
÷
q-
+
+
-
+
-
+
76.
+
-
-
+ +
+
-
+
77.
-
-
-
+
+
+
-
+
78.
-
+ +
-
-
+
-
+
79.
-
??
-
-
?
?
-
80.
-
-
-
+ +
+
-
-
÷
81.
-
-
-
+
+
+
-
+
+
-
-
+
+
+
-
+
83.
-
+ +
+
-
+
84.
-
?-
+
+
+
?
+
85.
+
+ +
-
-
+
-
+
+
+
+
-1-
86.
-
?-
+ +
+
-
+
!-
87.
-
?-
+
+
+
?
+
÷
88.
-
-
+ +
+
-
+
-1-
-
÷
?
82.
plus
COMPACT
+
72 T A B L E 4 (continued) Compound No.
Ames test
Rat
89. 90.
NT +
?? + ?
91. 92. 93. 94. 95.
+ + + + +
+ ? +- +
96. 97. 98. 99. 100.
+ -
?+
Mouse
Rat and Mouse rodent assay
COMPACT assay
Ames plus rodent 1 / 4
1/4
3/4
(CYPI & CYP2E)
assay
+ -
+ +
9 -
+
NT +
NT +
+-
?+
+ + + + +
-
+ + + + +
+ + + + +
+ + + + +
?+ ?+ ?? +-
+ ? +
9 -
+ + +
+
--
+
+ + ? _+ _+
+ + _+
+
-
Ames plus COMPACT
TABLE 5 C O R R E L A T I O N O F D A T A F R O M T H E C O M P A C T ASSAY, R O D E N T C A R C I N O G E N I C I T Y ASSAY A N D A MES TEST F O R 100 M I S C E L L A N E O U S C H E M I C A L S (1) In the mouse vs. rat correlation this was considered to be positive when both sexes of both species were either ( + + ) or ( - - ); there were 21 compounds positive in both sexes of both species out of the 70 compounds for which there is firm evidence for both sexes of both species, and no compounds that were negative in both species, which gives an overall correlation of 30%. If the mouse vs. rat correlation was considered positive when 3 or more segments (male rat, female rat, male mouse, female mouse) were in agreement, then 38 compounds were positive in 3 / 4 animals, and 7 compounds were negative in 3 / 4 animals, out of a total of 86 chemicals for which firm evidence is available, giving a correlation of 45/ 86, i.e. 52%. In the correlation of the C O M P A C T assay vs. the rodent assay or vs. the Ames test, a ( + ) was correlated with ( + ) , and ( - ) with ( - ). (2) In the correlation of the rodent assay ( 1 / 4 ) with the Ames test, 59 were positively consonant, 1 was negatively consonant, and 35 were in disagreement; for 3 compounds no Ames data were available, and for another 3 carcinogenicity data were equivocal. Consonance was therefore 60/94, or 64%. (3) In the correlation of C O M P A C T with Ames, 56 were positively consonant, 6 were negatively consonant, and 35 were in disagreement; for 3 compounds no Ames data were available. Consonance was therefore 62/97, i.e. 64%. (4) In the correlation of C O M P A C T with the rodent assay (1/4), 89 were positively consonant, none were negatively consonant, and 8 were in disagreement. Consonance was therefore 89 out of 97, i.e. 92%. (5) In the correlation of C O M P A C T vs. the Ames plus the rodent assay, of the 91 compounds that were ( + ) in the COMPACT, 56 were ( + ) in both Ames and the rodent assay (1/4), and of the 9 compounds that were ( - ) in C O M P A C T none was ( - ) in both Ames and the rodent assay and 6 were not tested in both Ames and the rodent assay. The consonance was 56 out of 94, i.e. 60%. (6) In the correlation of C O M P A C T plus Ames vs. the rodent assay (1/4), of the 97 compounds that were ( + ) in the rodent assay, 56 were ( + ) in both C O M P A C T and the Ames and 33 more were ( + ) in one or the other. Three were not tested in the Ames, in one of which no firm carcinogenicity data were available. The consonance was therefore 89 out of 95, i.e. 94%.
%
1. 2. 3. 4. 5. 6.
Rat vs. Mouse Rodent assay vs. Ames C O M P A C T vs. Ames C O M P A C T vs. Rodent assay C O M P A C T vs. Ames plus Rodent assay C O M P A C T plus Ames vs. Rodent assay
52 64 64 92 60 94
Consonance
% False positives given by C O M P A C T
% False negatives given by C O M P A C T
33 0
3 8
73
Figs. 1 and 2 (Lewis et al., 1986, 1987; Parke et al., 1988). The rodent carcinogenicity data are evaluated as ( + ) where 1 or more of the 4 animal studies (rat M & F; mouse M & F) was positive, and ( - ) only where none of the 4 animal studies was positive. The original Ames and rodent carcinogenicity data, and Ames plus C O M P A C T data have been combined by assigning ( + ) , ( - ) or ( + ) values by means of a notional 50 : 50 weighting between the two methods of toxicity evaluation. The total refined data set is shown in Table 4 for purposes of comparison and for inspection of the degree of correlation between each method. In the Ames test 59 chemicals were positive, 38 were negative, and 3 were not tested. In the rodent assay ( 1 / 4 ) 97 chemicals were positive and 3 were not sufficiently tested. In the C O M P A C T evaluation 91 chemicals were positive and 9 were negative. Correlations between the various combinations of Ames test, rodent carcinogenicity assay and C O M P A C T were arrived at as defined in Table 5. The rodent assay versus the Ames test gave 64% consonance, the C O M P A C T versus Ames
tors making such decisions revealed that the practice varied in different countries and different agencies, and was often dependent on a variety of other factors. It was therefore decided to follow the general practice of NCI and to apply the most stringent standard of evaluating a single positive result ( 1 / 4 or more) as an indication of positive carcinogenicity ( + ) , and assigning a negative ( - ) value only where there was no positive result in any of the 4 (rat M & F, mouse M & F) segments of the rodent assay. Results
COMPACT procedure. The full data set for 100 chemicals together with carcinogenicity data are presented in Table 3 for comparison between COMPACT, the rodent two-species life-span carcinogenicity assay and the Ames test for mutagenicity. The raw structural data were analysed and compared against those of known substrates of CYP1 and CYP2E (see Table 2). The regions of molecular parametric space defining CYP1 and CYP2E substrate specificity are presented in
TABLE 6 COMPACT DATA ON KNOWN HUMAN CARCINOGENS AND TOXIC CHEMICALS Compound
Area/depth 2
AE
COMPACT ratio
Collision diameter
Toxicity
/3-naphthylamine diethylstilboestrol dimethylaminoazobenzene
7.9 4.8 3.8
12.5 13.0 10.6
0.63 0.37 0.36
6.4 7.9 7.4
bladder carcinogen transplacental carcinogen hepatocarcinogen
paracetamol tienilic acid afla/oxin B 1
4.8 3.6 3.2
14.4 12.0 11.9
0.33 0.30 0.27
6.4 7.7 7.7
fatal hepatotoxin fatal hepatotoxin hepatocarcinogen
benoxaprofen halothane chloramphenicol
2.5 2.0 2.2
12.0 10.8 13.6
0.21 0.19 0.16
7.7 5.6 7.6
fatal hepatotoxin fatal hepatotoxin acute neonatal toxicity
thalidomide a ethanol 4-aminobiphenyl
2.0 1.8 8.3
13.6 22.4 12.9
0.15 0.08 0.64
7.4 4.5 6.8
teratogen hepatocarcinogen bladder carcinogen
4-nitrobiphenyl benzidine vinyl chloride
8.4 7.3 2.8,
12.7 12.5 16.3
0.66 0.58 0.17
7.0 7.0 4.6
bladder carcinogen bladder carcinogen hepatocarcinogen
COMPACT ratio and collision diameter values in heavy type indicates that these chemicals will be substrates of CYP1 and CYP2E1 respectively. a The teratogenic enantiomer.
74 also gave 64% agreement, C O M P A C T versus the rodent assay gave 92%, and the highest of all was 94% for C O M P A C T plus Ames versus the rodent assay.
Determination of the sensitivity of Ames test and COMPACT procedure. On the basis of the 100 chemicals evaluated in the present study, the index of "sensitivity" was calculated as follows: Sensitivity Number of carcinogens positive in test Total number of carcinogens The total number of carcinogens on the basis of the rodent bioassay ( 1 / 4 ) is 97, but of these 2 were not tested in the Ames assay so that the total number of carcinogens evaluated in this test is 95. 57 Sensitivity for Ames test = - - × 100 = 60% 95 Sensitivity for C O M P A C T procedure 89 -
97
x
100
=
92%
Validation of COMPACT against human data. Much of the unequivocal human data on the toxicity/carcinogenicity of chemicals comes from the exposure to new drugs. Table 6 shows a list of chemicals known to be toxic to humans, and many of these are seen to be substrates of CYP1 a n d / o r CYP2E. However, there are some such as chloramphenicol which is activated via nitro reduction, and not oxidation, and tienilic acid which appears also to be activated by C Y P 2 C l l in addition to CYP1. Discussion
From recent studies of mechanisms of carcinogenicity and chemical toxicity it is apparent that there are two major pathways to malignancy, namely (i) the alkylation of D N A by electrophilic
metabolites (genotoxic), and (ii) nuclear damage by reactive oxygen species generated by futile cycling of CYP2E1 and possibly other P450 enzymes, redox cycling of quinones, peroxisomal proliferation, or transoxygenation in eicosanoid biosynthesis. Genotoxic reactive electrophiles which function as genotoxins are primarily formed through metabolism of essentially planar molecules, catalysed by the CYP1A family (Ioannides and Parke, 1990). Moreover, what also prompted emphasis on this cytochrome P-450 family is the fact that this activity is highly inducible by chemicals to which man is exposed frequently through the diet, such as the indoles in cruciferous vegetables, polycyclic aromatic hydrocarbons formed during the charcoal broiling of food, and heterocyclic amines formed during the frying and grilling of meat and fish. Even drugs such as omeprazole can induce CYP1 activity in humans at therapeutic doses (Diaz et al., 1990). Indeed, human studies support the premise that high CYP1 levels predispose to chemically-induced cancers and expression of CYP1 proteins is currently being evaluated as an index of lung cancer risk in individuals exposed to pulmonary carcinogens, such as those present in tobacco smoke (Petruzzelli et al., 1988; Bartsch et al., 1991). Furthermore, CYP1 activity appears also to correlate with increased risk of breast cancer (Pyykko et al., 1989). In human lung specimens CYP1 was only present in smokers with none of this protein being detectable in non-smokers, and recent studies revealed that smokers are at an increasing risk of developing lung peripheral adenocarcinoma if they display induced pulmonary CYP1 levels (Antilla et al., 1991). Interestingly pulmonary activity was higher in the lungs of cancer patients who had smoked within 30 days before surgery than in cancer-free patients with similar smoking" habits (Bartsch et al., 1991). These findings indicate that expression of CYP1 activity in lung, and thus the ability to metabolically-activate tobacco-derived carcinogens, provide a feasible explanation as to why only a fraction of tobacco smokers develop lung cancer. Finally, in agreement with this concept, lung cancer cell lines exhibit CYP1 m R N A levels, normally absent from normal cell lines (McLemore et al., 1989).
75 The CYP2E family has also been associated with a limited role in the activation of chemical carcinogens, such as certain nitrosamines (Yang et al., 1985).
COMPACT for CYP1 and CYP2E. As there was poor concordance between the sexes, and between the two species, in the rodent assay, when three or more segments (male rat, female rat, male mouse, female mouse) were in agreement, the only rational way forward was to accept a positive result for malignancy in only one or more segment as an overall positive result in the rodent carcinogenicity assay. This resulted in 97/100 chemicals being positive, whereas the remaining three were not sufficiently tested. If the criterion was changed to 3 / 4 segments being in agreement, then only 38/100 chemicals were rated positive (Table 4). In the Ames Salmonella mutagenicity assay, 59/100 chemical were positive, 38/100 negative, and 3 were not tested. In the C O M P A C T procedure 91/100 chemicals were positive and 9 / 1 0 0 negative. From this, it would appear that the C O M P A C T procedure can identify carcinogens/toxic chemicals not positive in the Ames test. This is not surprising since the C O M P A C T procedure identifies the generation of reactive intermediates which may manifest other forms of toxicity, e.g. hepatic necrosis in the case of paracetamol, whereas the Ames test picks up only genotoxins. On the basis of the 100 chemicals evaluated in the present study, the sensitivity for the Ames test is 60% whereas for the C O M P A C T procedure it is much higher at 92%, demonstrating the high capability of the latter procedure to identify carcinogens. Among these chemicals, the rodent bioassay ( 1 / 4 ) did not reveal any unequivocal non-carcinogens, precluding the calculation of "specificity" and "predictive value" and thus the ability of these tests to identify "non-carcinogens" and to distinguish between carcinogens and noncarcinogens. Anomalous rodent carcinogens. Of the 8 chemicals not rated as positive in the C O M P A C T procedure but rated as positive in the rodent ( 1 / 4 ) assay, trifluralin (65), captan (67), chlordane (68), heptachlor (80) and aldrin (89) (Table
3) are all "mouse carcinogens", that is are tumorigenic in one or both sexes of mice but not in either sexes of rat; they are all highly halogenated and consequently resistant to metabolism, and because of this they can initiate ROS generation by the redox cycling of any of the cytochromes P450 to which they bind as substrates. Hence, this is analogous to the generation of ROS, oxidative stress, and malignant transformation seen with CYP2E1 substrates. The greater susceptibility of rodents, especially mice, to the tumorigenicity of these chemicals (Parke and Ioannides, 1990) has long been recognised by the Joint W H O / F A O Meeting on Pesticide Residues (J.M.P.R., 1978) which recommends the study of such chemicals in several other animal species. Trifluralin, which contains the CF 3 group, and a dipropylamino group hindered by two vicinal nitro groups, is a similar case, being a positive tumorigen only in the female mouse, and likely to be resistant to metabolism. Nitrilotriacetic acid (32) is tumourigenic in both sexes of rat, but has a C O M P A C T ratio of 0.07 and a collision diameter of 6.6; it readily forms a ligand complex with iron which is tumorigenic and the mechanism probably involves the catalytic generation of reactive oxygen species by the iron (Preece et al., 1989). Butyl benzyl phthalate (96) is tumorigenic only in the female rat, and is likely to be a substrate of CYP4 and a hepatic peroxisomal proliferator, generating ROS in the liver of rats via excess production of hydrogen peroxide. 4,4'-Methylene dianiline (27), is not recognised as positive in the COMPACT procedure, having a C O M P A C T ratio of 0.14 and a collision diameter of 7.2, whereas a positive value for CYP1 is taken when the ratio is > 0.15, or the collision diameter is < 6.5, for CYP2E1. Finally, dimethylmorpholino phosphoramidate is also not recognised as positive in the C O M P A C T procedure, having a C O M P A C T ratio of 0.11 and a collision diameter of 6.6, but is equivocal in the rodent assay.
Conclusions. Ashby and Tennant's (1988) survey of the rodent carcinogenicity, Salmonella mutagenicity, and chemical structure (potential electrophilic sites) of 222 chemicals tested in rodents in the U.S. N C I / N T P programme, revealed an overall correlation between these three parame-
76
ters. The Salmonella assay was considered to be a sensitive method of detecting genotoxicity, and its use reveals two groups of carcinogens, namely, genotoxic and putatively non-genotoxic chemicals. These two groups show differences in species and tissue specificity, and only 30% of those carcinogens specific for mouse liver were found to be genotoxic. The high correlation of COMPACT with the rodent assay, namely 92%, indicates that this toxicity evaluation procedure, based on chemical structure and knowledge of metabolic activation and detoxication, is highly suitable for screening chemicals of diverse structure. Furthermore, as only the chemical structure has to be known, COMPACT can be used at the earliest stages of product development, thus avoiding costly mistakes of developing new drugs, food additives, or pesticides, that finally turn out to be carcinogenic, or seriously toxic in other ways. From the COMPACT data of a number of drugs and industrial chemicals that were originally considered to be safe, but were later found to be toxic in man, all would have given rise to alerts in the COMPACT procedure (see Table 6). Data obtained by the COMPACT procedure are relevant to all animal species that express the CYP1 and CYP2E families, assuming that their substrate specificity is very similar to the rat orthologous proteins. The cost of conducting the COMPACT procedure is < 1% of a rodent assay and can be completed in days instead of years, so is a small investment to make before undertaking new product development. Furthermore, for those chemicals that are marketed in small amounts, such as food flavours, the COMPACT procedure is ideal for safety evaluation. COMPACT studies may subsequently be confirmed by CYP enzyme induction studies in rodents and other species, which take into account pharmacokinetic aspects that can add a quantitative element to the safety evaluation procedure (Ayrton et al., 1990). Thorough validation of COMPACT is essential to instil public confidence in this new procedure, and unfortunately extensive validation against toxicity/carcinogenicity in humans is limited to the availability of toxicity data from accidental poisoning. Thus one is restricted to validation against experimental animal data, essentially
rodent toxicity and carcinogenicity studies. Validation against a further 100 chemicals studied in the U.S. N C I / N T P evaluation of toxicity/carcinogenicity, has recently been concluded and will be published in due course.
Acknowledgements The authors are most grateful to Mr. A. MacAlastair-Brown and the Humane Research Trust for their continued support over the past five years in the development of the COMPACT procedures, and to the Ministry of Agriculture, Fisheries and Food for support in the application of COMPACT procedures to the screening of food chemicals and flavours for potential chemical toxicity. Our grateful thanks also to Dr. John Ashby who initially suggested this approach for the validation of COMPACT and has given continued advice and encouragement.
References Ames, B.N. and L.S. Gold (1990) Chemical carcinogens: Too many rodent carcinogens, Science, 249, 970-971. Amoah, A.G.B., B.J. Gould and D.V. Parke (1986) Further studies on the pharmacokinetics of perhexiline maleate in humans, Xenobiotica, 16, 63-68. Antilla, S., E. Hietanen, H. Vainio, A.-M. Camus, H.V. Gelboin, S.S. Park, L. Heikkila, A. Karjalainen and H. Bartsch (1991) Smoking and peripheral type of cancer are related to high levels of pulmonary cytochrome P450IA in lung cancer patients, International Journal of Cancer, 47, 681685. Aronoff, G.R., T. Ozawa, K.A. DeSante, J.F. Nash and A.S. Ridolfo (1982) Benoxaprofen kinetics in renal impairment, Clinical Pharmacology and Therapeutics, 32, 190-194. Ashby, J. and R.W. Tennant (1988) Chemical structure. salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogenesis among 222 chemicals tested in rodents by the U.S. NC1/NTP, Mutation Research, 204, 17-115. Ayrton, A.D., S. Neville and C. Ioannides (1992) Cytosolic activation of 2-aminoanthracene: Implications in its use as diagnostic mutagen in the Ames test, Mutation Research, 265, 1-8. Bartsch, H., S. Petruzzelli, S. De Flora, E. Hietanen, A.M. Camus, M. Castegnaro, O. Geneste, A. Camoirano, R. Saracci and C. Giutini (1991) Carcinogen metabolism and DNA adducts in human lung tissues as affected by tobacco smoking or metabolic phenotype: a case-control lung study on lung cancer patients, Mutation Research, 250, 103-114. Clayson, D.B. and D.J. Clegg (1991) Classification of carcinogens: Polemics, pedantics or progress?, Regulatory Toxicology and Pharmacology, 14, 147-166.
77 Diaz, O., I. Fabre, M. Daujat, B. Saint Aubert, P. Bories, H. Michel and P. Maurel (1990) Omeprazole is an aryl hydrocarbon-like inducer of human hepatic cytochrome P450, Gastroenterology, 99, 737-747. Eason, C.T., F.W. Bonner and D.V. Parke (1990) The importance of pharmacokinetic and receptor studies in drug safety evaluation, Regulatory Toxicology and Pharmacology, 11,288-307. Ennever, F.K., T.J. Noonan and H.S. Rosenkranz (1987) The predictivity of animal bioassays and short-term genotoxicity tests for carcinogenicity and non-carcinogenicity to humans, Mutagenesis, 2, 73-78. Gori, G.B. (1991a) Are animal tests relevant in cancer risk assessment? A persistent issue becomes uncomfortable, Regulatory Toxicology and Pharmacology, 13, 225-227. Gori, G.B. (1991b) Adjudicating cancer causation: Scientific, legal and political conflicts, Regulatory Toxicology and Pharmacology, 13, 309-325. Griest, F.C., I.I. Ozols, A.S. Ridolfo and J.C. Muus (1982) The phototoxic effects of benoxaprofen and their management and prevention, European Journal of Rheumatology and Inflammation, 5, 138-147. Guengerich, F.P. (1988) Roles of cytochromes P-450 enzymes in chemical carcinogenesis and cancer chemotherapy, Cancer Research, 48, 2946-2954. Guengerich, F.P. (1992) Human cytochrome P-450 enzymes, Life Sciences, 50, 1471-1478. Halliwell, B., and J.M.C. Gutteridge (1990) Role of free radicals and catalytic metal ions in human diseases: an overview, Methods in Enzymology, 186, 1-85. Ingelman-Sundberg, M. and A.L. Hagbj6rk (1982) On the significance of cytochrome P-450-dependent hydroxyl radical-mediated oxygenation mechanism, Xenobiotica, 12, 673-686. Ioannides, C. and D.V. Parke (1987) The cytochromes P448 a unique family of enzymes involved in chemical toxicity and carcinogenesis, Biochemical Pharmacology, 36, 4197-4207. Ioannides, C. and D.V. Parke (1990) The cytochrome P4501 gene family of microsomal haemoproteins and their r61e in the metabolic activation of chemicals, Drug Metabolism ReL,iews, 22, 1-86. J.M.P.R. (1978) Etiology and pathogenesis of liver tumours. Joint Meeting on Pesticide Residues, Pesticide residues in food, 1977, FAO Plant Production and Protection Paper 10, Rev., Rome: FAO, pp. 7 and 8. Lewis, D.F.V., C. Ioannides and D.V. Parke (1986) Molecular dimensions of the substrate binding site of cytochrome P-448, Biochemical Pharmacology, 35, 2179-2185. Lewis, D.F.V., C. Ioannides and D.V. Parke (1987) Structural requirements for substrates of cytochromes P-450 and P-448, Chemico-Biological Interactions, 64, 39-60. Lewis, D.F.V., C. Ioannides and D.V. Parke (1989a) Prediction of chemical carcinogenicity from molecular and electronic structure, Toxicology Letters, 45, 1-13. Lewis, D.F.V., C. Ioannides and D.V. Parke (1989b) Molecular orbital studies of oxygen activation and mechanisms of -
-
cytochromes P-450-mediated oxidative metabolism of xenobiotics, Chemico-Biological Interactions, 263-280. Lewis, D.F.V., C. Ioannides and D.V. Parke (1990) A prospective toxicity evaluation (COMPACT) on 40 chemicals currently being tested by the National Toxicology Program, Mutagenesis, 5,433-435. McLemore, T.L., S. Adelberg, M. Czerwinki, W.C. Hubbard, S.J. Yu, R. Storeng, T.G. Wood, R.N. Hines and M.R. Bord (1989) Altered regulation of the cytochrome P450IA gene. Novel inducer-independent gene expression in pulmonary carcinoma cell lines, J. Natl. Cancer Inst., 81, 1787-1794. Neuberger, J. and R. Williams (1989) Immune mechanisms in tienilic acid associated hepatotoxicity, Gut, 30, 515-519. Parke, D.V., C. Ioannides and D.F.V. Lewis (1988) Metabolic activation of carcinogens and toxic chemicals, Human Toxicology, 7, 397-404. Parke, D.V. and C. Ioannides (1990) Role of cytochromes P-450 in mouse liver tumour production, pp. 215-230, In:
Mouse Lit,er Carcinogenesis, Mechanisms and Species Comparisons, Eds: D.E. Stevenson, R.M. McClain, J.A. Popp, T.A. Slaga, J.M. Ward and H.C. Pitot, New York: Alan R. Liss. Parke, D.V., C. Ioannides and D.F.V. Lewis (1990) The safety evaluation of drugs and chemicals by the use of Computer Optimised Molecular Parametric Analysis of Chemical Toxicity (COMPACT), ATLA, 18, 91-102. Parke, D.V., C. Ioannides and D.F.V. Lewis (1991) The role of the cytochromes P-450 in the detoxication and activation of drugs and other chemicals, Canadian Journal of Physiology and Pharmacology, 69, 537-549. Petruzzel|i, S., A.M. Camus, L. Carrozzi, L. Chelarducci, M. Rindi, G. Menconi, C.A. Angeletti, M. Ahotupa, E. Hietanen, A. Aito, R. Saracci, H. Bartsch and C. Giuntini (1988) Long-lasting effects of tobacco smoking on pulmonary drug-metabolising enzymes: a case-control study on lung cancer patients, Cancer Res., 48, 4695-4700. Pople, J.A., D.P. Santry and G.A. Segal (1965) Approximate self-consistent MO theory. 1. Invariant procedures, Journal of Chemical Physics, 43, S129-135. Preece, N.E., D.E. Hall, J.A. Howarth, L.J. King and D.V. Parke (1989) Effects of acute and sub-chronic administration of iron nitrilotriacetate in the rat, Toxicology, 59, 37-58. Pyykko, K., R. Tuimala, L. Aalto and T. Perkio (1989) Aryl hydrocarbon hydroxylase activity in human breast cancer tissue, In: I. Schuster (Ed.), Cytochrome P450 Biochemistry and Biophysics, Taylor and Francis, London, pp. 528-531. Sies, H. (1991) Role of reactive oxygen species in biological processes, Klinische Wochenschrift, 69, 965-968. Vinter, J.G., A. Davis and M.R. Saunders (1987) Strategic approaches to drug design. I. An integrated software framework for molecular modelling, Journal of ComputerAided Molecular Design, 1, 31-51. Yang, C.S., Y.Y. Tu, D.R. Koop and M.J. Coon (1985) Metabolism of nitrosamines by purified rabbit liver cytochrome P-450 isozymes, Cancer Research, 45, 1140-1145.