Quantitative correlation of mutagenic and carcinogenic potencies for heterocyclic amines from cooked foods and additional aromatic amines

Mutation Research, 271 (1992) 269-287 © 1992 Elsevier Science Publishers B.V. All rights reserved 0165-1161/92/$05.00

269

MUTENV 08827

Quantitative correlation of mutagenic and carcinogenic potencies for heterocyclic amines from cooked foods and additional aromatic amines F r e d e r i c k T. H a t c h a, M a r k G. K n i z e h, D a n H . M o o r e II b a n d J a m e s S. F e l t o n b a

Meredith, NH 03253 (USA) and o Biomedical Sciences Division, Lawrence Livermore National Laboratory, University of California, Livermore, CA 94550 (USA) (Received 7 June 1991) (Revision received 23 January 1992) (Accepted 28 January 1992)

Keywords: Linear regression analysis; Salmonella mutagenicity; Rodent tumor bioassay; Structural factors modulating potency; Animal and human carcinogens

Summary Aromatic amines have long been recognized as animal and human carcinogens. Recently heterocyclic aromatic amines (thermic amines) have been found in small amounts in cooked foods, primarily meats, and have proven to be potent mutagens and rodent carcinogens. Availability of quantitative databases for mutagenic potency in Salmonella and for carcinogenic potency in rodents has made possible a study of ten heterocyclic thermic amines and 24 aromatic amines. Potencies on mutagenic and carcinogenic scales were significantly correlated. By multiple linear regression analysis and multivariate analysis of variance, two descriptive structural factors were found to modulate the two modes of biological response. These factors were number of rings and methyl substitution at carbon atoms. The quantitative correlation between mutagenic and carcinogenic potencies and the modulating structural factors suggest a significant similarity of molecular mechanisms and support the utility of the short-term bacterial assay in evaluating hazard levels.

Cooking and other heat processing of proteinrich foods, and pyrolysis of amino acids induce the formation of heterocyclic aromatic amines that have been found to be mutagens (Hatch et al., 1988). These will be termed "thermic mutagens" or "thermic amines" in this article. We recently reported a quantitative structure-activity (QSAR) study of the known thermic mutagens and their congeners, elucidating structural factors that influence mutagenic potency and discussing

Correspondence: Frederick T. Hatch, 27 Pease Road, Meredith, NH 03253, USA.

possible organic chemical and metabolic mechanisms (Hatch et al., 1991). We wished to extend this work to include other aromatic amines because many of them are confirmed or suspected human or animal carcinogens (Garner et al., 1984). Quantitative mutagenicity data, obtained under prescribed protocols, are now available for many chemicals that have been tested for carcinogenicity (Haworth et al., 1983; Dunkel et al., 1984, 1985; Mortelmans et al., 1986; Zeiger et al., 1987, 1988; Zeiger, 1990). An extensively reviewed and codified summary of animal carcinogenesis tests has been compiled by Gold, Ames and collaborators (Gold et al., 1984, 1986, 1987,

270

1989a). The availability of these data for ten thermic amines and 24 other aromatic amines enabled us to include both mutagenic and carcinogenic potencies in this study. It is of interest that the potencies of this series of amines span nine orders of magnitude for mutagenesis and four orders for carcinogenesis. Whereas extensive comparisons of the qualitative (i.e., + or - ) results of short-term genotoxicity tests and animal cancer bioassays have been made recently (Tennant et al., 1987; Ashby and Tennant, 1988; Zeiger et al., 1990), the analysis of quantitative (potency) data utilizing the recently compiled databases has been more limited. The latter process was begun by McCann et al. (1988) and was elaborated by Piegorsch and Hoel (1988). Mutagenic potency was expressed as the initial slope of the dose-response curve (Margolin et al., 1981; Bernstein et al., 1982), i.e., revertant colonies per microgram of agent, converted to a logarithmic scale. Carcinogenic potency was expressed as the TDs0 parameter, defined by Peto et al. (1984) as the daily dose of agent administered for a standard lifespan that will halve the probability of an animal remaining tumor-free. To allow for the wide range of potencies among chemicals and for graphical expression of increasing potency in a positive direction, the TDs0 was converted to its negative logarithm ( - log10 TDs0). For 80 miscellaneous chemicals McCann et al. (1988) found the correlation between mutagenic and carcinogenic potencies to be R = 0.41 ( P < 0.001). Piegorsch and Hoel (1988) extended the list to 97 chemicals and found a correlation of R = 0.48 ( P < 0.001). Miscellaneous sets of chemicals have the advantage of sizable number to improve statistical validity, but the distinct disadvantage of multiple mechanisms of metabolism and toxicity. Classes of congeneric chemicals should allow more meaningful examination of genotoxic correlations. Most subsets of the data of Piegorsch and Hoel, separated by chemical class, did not show correlations significantly different from zero; however, a set of 42 aliphatic and aromatic amines, amides, and sulfonamides was barely significant at R = 0.33 ( P < 0.05). Unfortunately the purely aromatic members of this set were not examined separately. Nitro compounds and nitrosamines showed significant cor-

relations between mutagenic and carcinogenic potencies for small data sets. A review of this subject, incorporating the authors' own extensive studies, has been presented recently by Parodi et al. (1990); the few studies of chemical classes suffered from small class sizes, limiting statistical power. Parodi et al. (1981) reported DNA-damaging assays in vivo and bacterial mutagenicity in a small set of aromatic amines and azo compounds. Fassina et al. (1990) reported that mutagenicity in V79 ceils does not correlate with carcinogenicity for 12 aromatic amines. Related studies including chemical classes are reported by Parodi et al. (1982, 1983, 1988). For heterogeneous sets of chemicals the correlations of quantitative results for short-term tests and carcinogenic potency were generally between 0.3 and 0.6. A few higher correlations (e.g. Lutz, 1986) were suggested by Parodi et al. (1990) to reflect special circumstances. Qualitative comparisons of mutagenic and carcinogenic test data generally result in analysis of 2 × 2 tables by chi-squared testing, and calculation of concordances, sensitivity and specificity. Results have recently been reported for 114 chemicals examined in the National Toxicology Program (Zeiger et al., 1990). For example, although the Salmonella assay correctly labeled 89 percent of the Salmonella-positive chemicals (high positive predictivity), it only found 48 percent of the total carcinogens in the series (low sensitivity); and its overall concordance (correct identification of both carcinogens and noncarcinogens) was 66 percent. In general, there was not a high degree of predictivity from the short-term tests to carcinogenic outcome, even without consideration of potency; and this has caused considerable consternation among interested scientists. For the thermic and aromatic amines reported in this paper the quantitative mutagenic and carcinogenic potencies were found to be significantly correlated, at a higher level than noted above for miscellaneous chemicals. A small set of structural factors descriptive of the molecules was found to modulate the mutagenic and carcinogenic potencies. We present results utilizing analyses for correlation, simple and multiple linear regression, and multivariate analysis of variance. Different approaches to the relationships among muta-

271

genicity, carcinogenicity, and chemical structure have been reviewed by Frierson et al. (1986) and Benigni et al. (1989). Experimental procedure Chemicals studied Ten thermic heterocyclic aromatic amines, isolated from fried or broiled meat or fish, or from

an amino acid pyrolysate (Felton and Knize, 1990), one non-thermic heterocyclic amine (3aminotriazole), and 23 carbocyclic aromatic amines had the requisite potency data available. Chemicals were included regardless of their classification as positive or negative in the assays. Their abbreviations, Chemical Abstract Service numbers, chemical names, and common names are listed in Table 1. Molecular structures are shown in Chart 1.

TABLE 1 ABBREVIATIONS, CAS NUMBERS, CHEMICAL NAMES (COMMON NAMES) OF AROMATIC AND THERMIC HETEROCYCLIC AMINES TRPP1 2AAF 4AB 34MIQ

75104-43-7 53-96-3 2113-61-3 77094-11-2

3-Amino-1,4-dimethyl-5 H-pyrido[4,3-b ]indole (Trp-P-1) 2-Acetylaminofluorene 4-Aminobiphenyl 2-Amino-3,4-dimethylimidazo[4,5-f ]quinoline (MeIQ)

24ATOLUENE 3MIQ BENZIDINE 246MANILIN

95-80-7 76180-96-6 531-85-1 6334-11-8

2,4-Diaminotoluene 2-Amino-3-methylimidazo[4,5-f ]quinoline (IQ) Benzidine 2,4,6-Trimethylaniline

38MIQX GLUP1 TRPP2 1M6PHIP

77500-04-0 67730-11-4 72254-58-1 105650-23-5

2-Amino-3,8-dimethylimidazo[4,5-f ]quinoxaline (MeIQx) 2-Amino-6-methyldipyrido[ 1,2-a : 3 ',2 '-d ]imidazole (Glu -P- 1) 3-Amino-1-methyl-5 H-pyrido[4,3-b ]indole (Trp-P-2) 2-Amino-l-methyl-6-phenylimidazo[4,5-b ]pyridine (PhIP)

AAC 3ATRIAZOLE AMAC 44MNDIANIL

26148-68-5 61-82-5 68006-83-7 13552-44-8

2-Amino-9H-pyrido[2,3-b ]indole (AAC) 3-Aminotriazole (Amitrol) 2-Amino-3-methyl-9H-pyrido[2,3-b ]indole (AMAC) 4,4'-Met hylenedianiline

24XYLIDINE 245MANILIN GLUP2 2TOLUIDINE

21436-96-4 21436-97-5 67730-10-3 636-21-5

2,4-Xylidine 2,4,5-Trimethylaniline 2-Aminodipyrido[1,2 a-3' : 2 '-d]imidazole (Glu-P-2) 2-Aminotoluene

2ANISIDINE 24AANISOLE 15NAPHDIAM 24CRESIDIN

134-29-2 39156-41-7 2243-62-1 102-50-1

2(o)-Anisidine 2,4-Diaminoanisole

2NA 25CRESIDIN 26ATOLUENE 13PHENDIAM

91-59-8 120-71-8 15481-70-6 541-69-5

2-Naphthylamine 2,5(p)-Cresidine 2,6-Diaminotoluene 1,3(m)-Phenylenediamine

ANILINE 25XYLIDINE 25ATOLUENE 14PHENDIAM

142-04-1 51786-53-9 6369-59-1 624-18-0

Aniline 2,5-Xylidine 2,5-Diaminotoluene 1,4(p )-Phenylenediamine

12PHENDIAM 4ANISIDINE

615-28-1 20265-97-8

1,2(o )-Phenylenediamine 4(p)-Anisidine

1,5-Naphthalenediamine 2,4(m)-Cresidine

272

Mutagenic potency A m e s / S a l m o n e l l a assay data in frameshift strain TA1538 for the thermic amines were from our laboratory (Knize and Felton, 1986; Felton et al., 1988). Data for the other amines in strains TA98 or TA1538 were taken from published summaries sponsored by the National Institute of Environmental Health S c i e n c e s / N a t i o n a l Toxicology Program (Haworth et al., 1983; Dunkel et al., 1984, 1985; Mortelmans et al., 1986; Zeiger et al., 1987, 1988; Zeiger, 1990). The only exception was data for o-toluidine taken from Garner and Nutman (1977). Metabolic activation, which is required for mutagenic activity, was provided by addition to the assay medium of the microsomal

fraction (9000 × g supernatant--S9) from liver of rats or hamsters induced with Aroclor 1254 (Analabs, Inc., Norwalk, C"F). Mutagenic potency was determined from the slope of the linear part of the d o s e - r e s p o n s e curve (Moore and Felton, 1983). Values were recalculated to revertants per nanomole because of the substantial variation in molecular weight among the chemicals, and were expressed on a log~0 scale (hereafter Log M.P.) because of the wide range of the data. These amines do not show major differences in potency between two of the frameshift strains of Salmonella TA98 and TA1538 (Felton et al., 1988; de Meester, 1989); and thus data from either strain were used. In the

CH3

TRPP1 NH2

CH3

NH2

NH2

CH3

NH2

2TOLUIDINE

2ANISIDINE

~ CN3

~N~ v -CH3 34MIQ

GLUP2

NH2 24ATOLUENE

3MIQ OCH3

H3C HEN

CH3

CH3

NH2 BENZIDINE

CH3 246MANILIN

H3C ~ N./,,,a,~

~

NH[~

~CH3 NH2 24AANISOLE

38MIQX

NH2 ISNAPHDIAM

OCH3 24CRESIDIN

NH2

CH3

N GLUP1

H

25CRESlDIN 1M6PHIP

TRPP2 H

CH3

~---~ H

AAC

__~H H2N

NH2 t ~

NH2

NH 2 3ATRIAZOLE

H

CH3

~

NH~. CH3

NH2

H3C ANILINE

13PHENDIAM

25XYLIDfNE

AMAC

CH3

~

C 44MNDIANIL

NH2 CH3 24XYLIDINE

.~"S

CH3

P~3C" "~ CH3 245MANILIN

NH2 H2N 2SATOLUENE

NHz NH2 14PHENDIAM

OCH3 12PHENDIAM

4ANISIDINE

Chart 1. Structures of aromatic and thermic amines described in Tables 1 and 3. Chemicals are arranged in descending order of carcinogenic potency ( - log TDs0) in data set No. 1 from left to right and top to bottom. Positions of AAC and AMAC, for which data are missing in data set No. 1, are arbitrarybut approximatelycorrect.

273 TABLE 2 CARCINOGENICPOTENCY DATABASES Data Set 1. Rat: LowestValues 2. Mouse:LowestValues 3. Mouse:LiverValues

n 32 34 30

Chemicalsomitted AAC,AMAC None 1M6PHIP,2TOLUIDINE 2ANISIDINE, 4ANISIDINE

case of weak mutagens, data were selected from the strain giving the most linear initial dose-response curve.

Carcinogenic potency The TDs0 parameter of Peto et al. (1984) was used for this determination. Except for PhlP, values were taken from the database of Gold et al. (1984, 1986, 1987, 1989a). The values for PhlP were calculated from tumor incidence data for mice published by Esumi et al. (1989) and for rats by Ito et al. (1991) according to the above method. Three sets of carcinogenic potency values were evaluated in this study: (1) lowest TDs0 from a significant experiment in rats; (2) lowest TDs0 from a significant experiment in mice; and (3) TDs0 for mouse liver tumors, hepatocellular carcinomas when available. Data set No. 1 lacked tumor bioassay data for AAC and AMAC, and data set No. 3 did not show significant incidence of liver tumors for 1M6PHIP, 2TOLUIDINE, 2ANISIDINE, and 4ANISIDINE (Table 2). All TDs0 values were converted to mmole/kg body weight/day, and were expressed on a negative logt0 (reciprocal) scale so that potency would increase in a positive direction. Molecular data and the genotoxic potencies for each amine are given in Table 3. Missing data are replaced by zeros.

vation or detoxication of precursor compounds or (2) the reaction of activated intermediates with DNA. Possible modes of action considered were: (1) electronic and steric effects on reaction mechanisms; (2) positions of substituents affecting the probability of detoxicating side reactions; and (3) hydrophobic regions of a molecule that could influence enzyme binding, transmembrane transport, or DNA intercalation and binding. The more heterogeneous set of chemicals studied in this paper placed limitations on the factors considered previously. After evaluation the following structural factors were selected: (1) number of rings, which relates to hydrophobicity and possibly to the potential for intercalation into DNA; (2) methyl substitution on ring nitrogen, which differentiates a small subset of potent heterocyclic amines; (3) methyl substitution on ring carbon, which may relate to localized hydrophobicity or to blocking of possible detoxication sites; and (4) number of amino groups, which differentiates a subset of aromatic amines and may offer a choice of sites for amino group activation. These factors were tabulated according to the number of occurrences in each structure. Data for each chemical are presented in Table 4. Certain attractive factors were rejected because of high correlation with another important factor, leading to multicollinearity in multiple regression models; examples are molecular weight and number of ring nitrogen atoms, both of which correlated strongly with number of rings, which was considered of primary importance. Other factors, methoxyl substitution on ring carbon and classification of amino groups as "free" or "hindered" according to neighboring structure, were rejected for lack of significant contribution to regression models.

Statistical methods Structural factors Structural features differentiating the individual chemicals of the series were tested in regression models and for pairwise correlations. The rationale for selecting structural factors that might influence mutagenesis by heterocyclic amines produced during cooking of food has been discussed by Hatch et al. (1991). Features were considered that might relate to (1) metabolic acti-

The computer program used was Goodness-ofFit, Walonick Associates, Minneapolis, MN •55423. Calculations were performed with an IBM AT-compatible Olivetti M28 microcomputer, Olivetti USA, Somerville, NJ 08876. The correlation between mutagenic and carcinogenic potencies is of primary interest. The former assay may be quickly, inexpensively, and rigorously performed; whereas the latter is labori-

274

ous, expensive, and rarely subject to replication. Although both parameters are properly "response variables", mutagenic potency comes closer to the concept of a predictor or independent variable. Therefore, we have calculated sim-

pie linear regression for the two potencies in order to show the "slope" of their relationship and the standard error of predicting carcinogenic potency from mutagenic potency. Correlation coefficients were also calculated between the po-

TABLE 3 CHEMICAL

AND POTENCY

Agent Data Set TRPP1 2AAF 4AB 34MIQ 24ATOLUENE 3MIQ BENZIDINE 246MANILIN 38MIQX GLUP1 TRPP2 1M6PHIP

DATA FOR AROMATIC

Salt

ACETATE HCI

2HCI HCI

HCI ACETATE

Mol wt.

Type

AND THERMIC - LOGTDs0 No. 1 R a t Lowest

AMINES - LOGTDs0 No. 2 M o u s e

- LOGTDs0 No. 3 M o u s e

LOGMP Salmo-

Lowest

Liver

nella

270 223 206 212

T A A T

2.675 2.542 2.360 2.301

1.033 1.667 2.318 1.297

0.959 1.460 0.747 0.762

4.030 1.740 0.610 5.300

124 198 257 172

A T A A

2.098 2.093 2.088 1.903

1.236 1.054 1.456 0.975

0.668 0.893 1.460 0.951

- 0.890 4.500 0.396 - 1.000

213 235 256 224

T T T T

1.887 1.859 1.684 1.596

0.851 1.663 1.576 0.733

0.697 1.660 1.580 0.000

4.330 4.070 4.210 2.800

T A T A

0.000 1.356 0.000 1.336

0.832 0.535 1.215 1.203

0.733 0.535 0.412 1.080

2.080 - 1.900 1.110 0.258

AAC 3ATRIAZOLE AMAC 44MNDIANIL

ACETATE ACETATE 2HC1

242 84 256 271

24XYLIDINE 245MANILIN GLUP2 2TOLUIDINE

HC1 HCI HCI HCI

158 172 221 144

A A T A

0.973 0.926 0.815 0.791

1.105 1.450 1.265 - 0.652

0.383 1.450 1.040 0.000

- 1.160 - 0.900 2.640 - 1.800

2ANISIDINE 24AANISOLE 15NAPHDIAM 24CRESIDIN

HC1 SO4.3H20

160 236 158 138

A A A A

0.760 0.545 0.493 0.418

- 0.767 - 0.045 0.375 0.300

0.000 - 0.593 0.138 - 0.173

- 1.672 0.913 - 1.350 - 2.580

A A A A

0.366 0.257 0.226 0.201

0.915 0.490 0.983 -0.145

0.844 - 0.358 0.037 -0.691

0.330 - 1.660 - 0.496 0.150

2NA 25CRESIDIN 26ATOLUENE 13PHENDIAM

2HCI 2HCI

143 138 197 181

ANILINE 25XYLIDINE 25ATOLUENE

HCI HC1 SO4

130 158 220

A A A

0.169 0.017 - 0.025

- 1.833 - 0.308 - 0.117

- 1.830 - 0.543 - 0.117

-3.390 - 1.640 - 1.280

14PHENDIAM 12PHENDIAM 4ANISIDINE

2HCI 2HCI HCI

181 181 160

A A A

- 0.135 - 0.137 - 0.770

- 0.885 - 0.447 - 1.896

- 0.879 - 0.529 0.000

- 0.983 - 0.770 - 2.305

T, thermic amine; A, aromatic amine.

275 TABLE 4 STRUCTURAL FACTORS THERMIC AMINES

FOR

AROMATIC

AND

Agent

Rings

N-Methyls

C-Methyls

Aminos

TRPP1 2AAF 4AB 34MIQ

3 3 2 3

0 0 0 1

2 0 0 1

1 1 1 1

24ATOLUENE 3MIQ BENZIDINE 246MANILIN

1 3 2 1

0 1 0 0

1 0 0 3

2 1 2 1

38MIQX GLUP1 TRPP2 1M6PHIP

3 3 3 3

1 0 0 1

1 1 1 0

1 1 1 1

AAC 3ATRIAZOLE AMAC 44MNDIANIL

3 1 3 2

0 0 0 0

0 0 1 1

1 1 1 2

24XYLIDINE 245MANILIN GLUP2 2TOLUIDINE

1 1 3 1

0 0 0 0

2 3 0 1

1 1 1 1

2ANISIDINE 24AANISOLE 15NAPHDIAM 24CRESIDIN

1 1 2 1

0 0 0 0

0 0 0 1

1 2 2 1

2NA 25CRESIDIN 26ATOLUENE 13PHENDIAM

2 1 1 1

0 0 0 0

0 1 1 0

1 1 2 2

ANILINE 25XYLIDINE 25ATOLUENE

1 1 1

0 0 0

0 2 1

1 1 2

14PHENDIAM 12PHENDIAM 4ANISIDINE

1 1 1

0 0 0

0 0 0

2 2 1

tency values and the molecular weights of the chemicals or the individual structural factors (data not shown). Multiple linear regression calculations were run separately with carcinogenic or mutagenic potencies as dependent variables, after selection of the independent structural factor variables as

described above. Regression models were constructed stepwise with slightly low stringency (Fto-enter = 2 and F-to-remove = 1.9) until an optimal set of independent variables was determined; the model was then confirmed in a single step. Evaluation for multicollinearity was performed by examination of intervariable correlation tables for substantial pairwise correlations between independent variables, which would indicate obvious multicollinearity. For more subtle problems, principal components analysis with collinearity diagnostics was performed, including calculation of variance inflation factors and condition indices (Glantz and Slinker, 1990). With the reported set of independent variables there was no evidence of significant multicollinearity. As an independent procedure principal components analysis did not contribute additional information. Residuals appeared to show normal distributions, and there were no significant correlations with the independent variables or the predicted dependent variables. For this set of data the existence of two response variables (potencies), which are themselves significantly correlated, means that separate evaluations of their individual dependence on an identical set of independent variables (structural factors) are not entirely proper. Multivariate analysis of variance (MANOVA) (Gagnon et al., 1989), including both dependent variables separately and together in the calculation, was performed at the suggestion of a reviewer. The authors of the program describe its rationale as follows: " W h e n several measurements are taken on the same experimental units, they tend to be correlated; that is, the values of some of the (dependent) variables can be readily predicted by the values of the other (dependent) variables. One way to think about the correlation of multiple variables is that they do not contain as much information about the underlying process they are measuring as you might think, because they are often just different ways of looking at the same underlying causes. The technique which performs analysis of variance on more than one dependent variable and explicitly takes into account the correlation among the dependent variables is known as the multivariate analysis of variance or MANOVA."

276 10

Results

A

Relationship of carcinogenic to mutagenic potency This relationship for 32 chemicals with rat carcinogenesis data (data set No. 1, Table 2) is shown in Fig. 1. Examination of the figure indicates that pooling of the thermic and aromatic amines for simple regression is probably justified. The thermic amines show higher mutagenicity (X-axis) than the aromatic amines in all cases. And the thermic amines show carcinogenic potency in the higher portion of its range (Y-axis). Frequency distributions of mutagenic and carcinogenic potencies are shown in Fig. 2. Correlation and regression data for the full data set No. 1 and for the separate thermic and aromatic amine subsets are shown in Table 5. All three series show significant correlation coefficients for the relationship between carcinogenic and mutagenic potencies. The slopes, intercepts, and standard error of the estimates of the simple linear regression lines are shown for interest. Correlation and simple regression results for data sets Nos. 2 and 3 are also in this table. Cross-correlation between the carcinogenic potency in mice and rats (data sets Nos. 1 and 2), for the 32 amines common to both data sets, was 0.76 ( P < 0.01). On average, the TDs0s for rats were three-fold lower than for mice, i.e., rats

•

•

2.0

•

•

O

•

O OO

1.5

O

a I- 1.0

O

•A

0.5

•

•

•

•

•

o .......

•

•

| • Aromatic amines |

•

At,-, ...........

~

/ O Thermic amines

~-

-0.5 -1.0_4

I -3

i -2

i -1

i 0

I 1

i 2

I

I

I

3

4

5

,, i!, I, Log M.P.

?,

4 3 2 1 0

-0.5

0

0.5

1.0

1.5

2.0

2.5

3.0

-Log TDs0 Fig.

2.

Frequency

distributions

for

aromatic

and

thermic

amines in data set No. 1. Panel A: frequency distribution for logarithm of mutagenic potency. Panel B: frequency distribution for negative logarithm of TDs0. Hatched bars, aromatic

amines; filled bars, thermic amines.

were three-fold more sensitive. There was considerable variation in the differences for individual chemicals. Likewise the cross-correlation between potency values for mouse liver and mouse lowest values (data sets Nos. 2 and 3) was 0.90 ( P < 0.001), reflecting the fact that in many cases the liver was the most sensitive target organ, so that the same values appeared in both data sets for these chemicals.

3.0 2.5

o" a.

6

Log MP.

Fig. 1. Scatter plot for eight thermic heterocyclic amines and 24 aromatic amines in data set No. 1. The logarithm of mutagenic potency in the Ames/Salmonella test is plotted on the X-axis and -log TDs0 (carcinogenic potency) in rats on the Y-axis. See Experimental section for definitions of potency scales. Correlation coefficientfor these data is R = 0.66.

Relationship of potencies to compound molecular weight For data set No. 1 (rat lowest values) the regression slope coefficient of Log M.P. on molecular weight was b = 0.036 ( P < 0.001). For - l o g TDs0 (rat lowest values) on molecular weight, b = 0.0088 ( P = 0.014). The positive

277 TABLE 5 R E L A T I O N S H I P O F C A R C I N O G E N I C T O M U T A G E N I C POTENCY: C O R R E L A T I O N A N D SIMPLE L I N E A R R E G R E S SION O F - L o g TDs0 O N Log M.P. Chemicals

n

R

Prob.

Slope, b

Intercept

S.E.E.

0.664 0.512 0.720

< 0.001 < 0.01 ~ 0.02

0.257 0.377 0.450

0.968 1.120 0.070

0.712 0.778 0.411

0.591

< 0.001

0.247

0.472

0.809

0.596

< 0.001

0.213

0.308

0.690

Data set No. 1: rat lowest values

Total series Aromatic amines Thermic amines

32 24 8

Data set No. 2: mouse lowest values

Total series

34

Data set No. 3: mouse liver values

Total series

30

Abbreviations: n, n u m b e r of chemicals; R, correlation coefficient; Prob., probability for correlation coefficient; S.E.E., standard

error of estimate of - Log TD50 from regression.

Modulation of mutagenic potency by structural factors Multiple linear regression of Log M.P. as de-

slopes indicate that larger molecules tend to be more potent for both mutagenesis and carcinogenesis. However, molecular weight was not used as an independent variable for two reasons: (1) it is not, strictly speaking, a structural feature, and (2) a high correlation between molecular weight and number of rings, which is an important structural feature, would introduce undesirable multicollinearity into the regression models.

pendent variable on the four structural factors as independent variables (number of rings, methyl substitution on nitrogen, methyl substitution on carbon, and number of amino groups) is summarized in Table 6. The coefficient of multiple determination R 2= 86.5% for data set No. 1 indi-

TABLE 6 M U L T I P L E L I N E A R R E G R E S S I O N : Log M.P. ON S T R U C T U R A L F A C T O R S (A) Overall results for Log M.P. on structural factors Data set

n

R 2 (%)

S.E.E.

D.F.

F-ratio

Prob.

Ind. Var.

No. 1 Rat lowest No. 2 Mouse lowest No. 3 Mouse liver

32 34 30

86.5 85.0 81.5

0.952 0.978 1.05

4/27 4/29 2/27

43.7 41.1 59.5

0.000 0.000 0.000

All 4 All 4 Rings and N-methyls

(B) Structural factors for data set No. 1: rat lowest values Predictor

Coefficient

Std. error

F-ratio

Probability

Rings N-methyls C-methyls Aminos Constant

2.36 1.42 0.445 0.835 - 5.32

0.242 0.615 0.205 0.406

95.5 5.30 4.73 4.23

< 0.000 0.029 0.039 0.050

Regression equation: Log M.P. = - 5.32 + 2.36 * Rings + 1.42 * N-methyls + 0.445 * C-methyls + 0.835 * Aminos Abbreviations: n, n u m b e r of chemicals; R 2, coefficient of determination; S.E.E., standard error of estimate of Log M.P. from

regression; D.F., degrees of freedom; Prob., probability from F-ratio; Ind. Var., significant independent variables.

278 TABLE 7 MULTIPLE L I N E A R REGRESSION: - L o g TD5o ON S T R U C T U R A L FACTORS (A) Overall results for - Log TDs0 on structural factors Data Set

n

R 2 (%)

S.E.E.

D.F.

F-ratio

Prob.

Ind. Vat.

No. 1 Rat Lowest No. 2 Mouse Lowest No. 3 Mouse Liver

32 34 30

52.7 49.0 51.8

0.677 0.740 0.607

2/29 3/30 2/27

16.1 9.62 14.5

0.000 0.0001 0.000

Rings and C-methyls Rings, C-methyls, Aminos Rings and C-methyls

(B) Structural factors for data set No. 1: rat lowest values Predictor

Coefficient

Std. error

F-ratio

Probability

Rings C-methyls Constant

0.751 0.290 - 0.447

0.136 0.136

30.5 4.55

< 0.000 0.042

Regression equation: - Log TDs0 = - 0.447 + 0.751 * Rings + 0.290 * C-methyls

Abbreviations: see Table 6.

cates very strong dependence of mutagenic potency on the structural factors. The high F-ratio of 43.7 confirms the very low probability of the model relationships being due to chance. The individual regression coefficients are all of positive sign and are significant at the P < = 0.05 level. Multiple regression results for the other two data sets are also given in Table 6. They are similar to data set No. 1 with slightly lower coefficients of determination and, in the case of mouse liver, only two significant structural factors.

Modulation of carcinogenic potency by structural factors Multiple linear regression of - l o g TDs0 (data set No. 1) on the same structural factors is summarized in Table 7. The coefficient of determination R e= 52.7% indicates moderate dependence of carcinogenic potency on the structural factors. The F-ratio of 16.1 confirms the model relationships, though less strongly than for Log M.P. Individual regression coefficients (again with positive slopes) are significant only for number of rings and presence of C-methyl substituents, so that methyl substitution on nitrogen and the number of amino groups appear not to have significant modulating influence on carcinogenic potency. Multiple regression results for the other two data sets are also given in Table 7. As above, they are similar to data set No. 1 with slightly

lower coefficients of determination and, in the case of mouse lowest values, three significant structural factors.

Multivariate analysis of variance Since we have shown a significant correlation between the two response variables, mutagenic potency and carcinogenic potency (R = 0.66, P < 0.001), it is appropriate to combine them in a single regression model to study their joint dependence on the independent variables (structural factors). When this was done for data set No. 1 (Table 8) multivariate analysis of variance (MANOVA) revealed that the number of rings ( P = 0.0001) and number of C-methyl groups ( P

TABLE 8 MULTIVARIATE (MANOVA)

ANALYSIS

Dependent

Independent variables

variable(s)

Rings

C-methyls

OF

VARIANCE

N-methyls

Aminos

( A ) Standardized regression coefficients Log M.P. - Log TDs0

0.867 0.713

0.164 0.282

0.197 0.014

0.163 0.023

0.0001 0.0002 0.0001

0.039 0.055 0.035

0.029 0.930 0.097

0.05(/ 0.880 0.151

( B ) P-values Log M.P. - Log TDs0 Combined

279 = 0.035) were the only significant structural factors when both dependent variables were included in the model. It should be noted in Tables 6 and 7 that these were the only two structural factors that were significant in both of the multiple regression models that included the potency variables separately. As shown by the standardized regression coefficients and P-values in Table 8, the number of rings is more heavily weighted than the number of C-methyl groups as a structural factor modulating mutagenic and carcinogenic potencies. Discussion

Aromatic and thermic heterocyclic amines Aromatic amines have been indicted as human carcinogens for nearly a century (Garner et al., 1984). Three of the present series, 4-aminobiphenyl, benzidine, and 2-naphthylamine are certified in group 1, 2-toluidine in group 2A, and 3-aminotriazole in group 2B, according to the International Agency for Research on Cancer (IARC) (1982). Ample confirmation of their carcinogenicity in animal experiments has followed over the last half century. In the last dozen years, heterocyclic aromatic amines have been identified in cooked foods and pyrolyzed amino acids (Hatch et al., 1988); these thermic amines are potent bacterial mutagens and all members tested to date are carcinogenic in rodents (Sugimura and Wakabayashi, 1990) and in one case a primate (Adamson et al., 1990). Although the amounts in food are small (ppblevel), exposure may be for most of a lifetime. Therefore, the series of chemicals reported here are potentially of considerable importance to human safety. Correlation of mutagenic and carcinogenic potencies We have focused the quantitative correlation of mutagenic and carcinogenic potencies, an approach begun by McCann et al. (1988) and by Piegorsch and Hoel (1988), onto a single class of related chemicals. Relevant data from the massive, carefully monitored databases for bacterial mutagenicity and animal carcinogenicity have been linked to study aromatic and thermic amines.

After translation of potencies into appropriate quantitative terms, we found significant correlation (R = 0.66, P < 0.001) between the carcinogenic and mutagenic potencies of a series of carbocyclic and heterocyclic aromatic amines (Table 5). Although the two types of potency are both response variables, we felt that it is of interest to perform simple linear regression. Log M.P. values were determined under a rigorous multilaboratory protocol and the technique is brief and well-understood, so that its assignment as the independent variable is justifiable. - L o g TDs0 values are determined with a difficult, lengthy, and very expensive protocol, so that it qualifies well as a response variable. There is, therefore, value in learning whether the latter potency can be estimated from the former for new chemicals. The regression equation for the full data set No. 1 derived from Table 5 is - l o g TDs0 = 0.968 + 0.257. Log M.P. For the amines in this study the standard error of the estimate of - l o g TDs0 from Log M.P. was sufficiently high (0.712) that direct prediction of carcinogenic potency from mutagenic potency in the .case of a single chemical would be inappropriate. Two standard errors on each side of the regression line would encompass nearly three orders of magnitude of carcinogenic potency. However, it is possible that some sense of hazard priority would be given by the predicted potency.

Short-term tests and rodent bioassays Much controversy and "soul-searching" has recently surrounded the question of even a qualitative correlation between short-term tests for genotoxicity and long-term rodent feeding bioassays for carcinogenicity (Tennant et al., 1987; Ashby and Tennant, 1988; Zeiger et al., 1990; Auletta and Ashby, 1988; Kier, 1988; Brockman and DeMarini, 1988; Shelby et al., 1988; Ashby, 1990). Therefore, we had not expected a substantial quantitative correlation to exist, even for a single class of chemicals. The relationship we did observe seems potentially very important. Although the Ames/Salmonella test for mutation appears to predict the qualitative result of rodent bioassays about as well as any short-term test, or combination of tests (Zeiger et al., 1990), there remains considerable controversy about its proper

280 role in regulatory matters and risk analysis. The fairly strong quantitative correlation we have demonstrated between the assays for mutagenic and carcinogenic potency supports the credibility of the short-term test as reflecting similar genotoxic mechanisms for mutagenesis and carcinogenesis for at least one important class of chemicals. This finding lends strength rather than weakness to the use of both microbial and rodent assays to estimate degrees of hazard.

Cytotoxicity, genotoxicity, and carcinogenicity Recently, Haseman and Clark (1990) reported qualitative dichotomous (i.e., + or - ) relationships among general chemical toxicity (as manifested by the maximum tolerated dose or MTD), rodent carcinogenicity, and four short-term tests for genotoxicity (including Salmonella). Concordance among all pairings was in the range of 60 to 70 percent. Thus it is possible that a considerable proportion of cytotoxicity results from the same mechanisms that confer mutagenicity and carcinogenicity on a chemical. It should be noted that all of the amines reported here fall well within the "toxic range" defined by Haseman and Clark. In a previous paper from our laboratory we showed a linear correlation between cell survival and mutagenic response (forward mutations) for 22 chemical mutagens in three rodent and two human cell culture systems. The relative increase in cytotoxicity was accompanied by a proportional increase in mutagenicity since the slope of the regression equation was 1.03 (Carver et al., 1979). At least for potent mutagens there appeared to be tight coupling between genetic injury (DNA damage) and cell lethality. Hoel et al. (1988) have studied the relationships between toxicity and tumor bioassays from two viewpoints: (1) general toxicity as manifested by reduced body weight and nontumor-based mortality, and (2) cytotoxicity as manifested by histopathologic changes judged not to be secondary to carcinogenesis. Only 25 percent of 53 positive chemicals (of 99 chemicals tested) showed macroscopic evidence of toxicity (body weight or survival) at every dose-species-sex measurement showing significant tumorigenesis. In some of these cases the toxicity was judged to be possibly

secondary to established neoplasia. For 207 positive site-specific carcinogenic effects, 31 percent showed associated target-organ histopathologic toxicity by their criteria. For only seven chemicals (13 percent of 53 positive) carcinogenesis was believed to have been possibly secondary to target organ toxicity; six of these were negative in Salmonella and one was weakly positive. Overall, Hoel et al. conclude that most positive chemicals are "primary" carcinogens (exhibit their carcinogenicity independent from toxicity); and that minority candidates for "secondary" carcinogens (based on local cytotoxicity) may be nongenotoxic or weakly so (as judged in Salmonella). The authors admit that chemically induced cell replication may have escaped their histopathologic detection and recommend further investigation. The criteria of Hoel et al. for microscopic toxicity have been criticized by Goodman and Wilson (1991). Only 41 percent of the positive carcinogens studied by Hoel et al. were qualitatively positive in Salmonella, although some of the nonmutagenic chemicals were positive in other short-term tests for genotoxicity. Thus, mutagenicity is by no means a requirement for carcinogenicity. The aromatic and heterocyclic amines that we have studied are generally genotoxic, so that there is not a clear link to the histopathologic studies of Hoel et al. Cytotoxicity that may be closely associated with mutagenicity, i.e., presumably involving damage to DNA, may not be detected or distinctly recognized by histopathology. A correlation of this type of DNA-mediated toxicity with carcinogenesis may, nevertheless, contribute to the observed variance in carcinogenic potency and to its linkage to two of the structural factors that also relate strongly to mutagenic potency.

Distinctiue behauior of aromatic and heterocyclic amines As indicated in the Results section, we combined thermic and aromatic amines in examining the relationship between mutagenic and carcinogenic potencies. The thermic amines are especially capable of inducing frameshift mutations in the appropriate Ames strains of Salmonella. This appears to be due to a specific frameshift mechanism, i.e., deletion of two adjacent bases in a run

281 of GC bases in the hisD gene, restoring the reading frame in strains TA98 and TA1538 (Fuscoe et al., 1988). As shown in Fig. 2 the thermic amines are also relatively potent on the rodent carcinogenesis scale. Despite their high potencies, the thermic amines do not seem to differ fundamentally from the aromatic amines in the relationship of carcinogenic to mutagenic potency (Fig. 1).

Interspecies cross-correlation of carcinogenic potency The strong correlation between the carcinogenic potencies of the amines in mice and rats (R = 0.76, P < 0.01) reflects, to a degree, a replication of the tumor bioassay. Crouch and Wilson (1979) reported an apparent good correlation of carcinogenic potency between mice and rats. Less consistent results were mentioned by Freedman and Zeisel (1988). The high interspecies correlation may have a tautologous aspect as discussed by Bernstein et al. (1985), since the maximum doses tested are highly correlated because of a correlation of toxicity limitations on dosage. However, Goodman and Wilson (1991) have critiqued the calculations of Bernstein et al., and believe that the interspecies correlation is at least partly real. They state "a study of the interspecies correlation factor K cannot be separated from a study of the relationship between toxicity and carcinogenic potency, or of the relationship between carcinogenic potency and activities at the cellular level (including cytotoxicity and genotoxicity)." On a qualitative basis predictivity between mice and rats approaches 75 percent in a large database, if chlorinated compounds are eliminated (Gold et al., 1989b).

Modulation of mutagenic potency by structural factors In Table 6 the coefficient of determination of R 2 = 86.5% indicates that a very large proportion of the variance in Log M.P. for data set No. 1 is explained by the four structural factors. The mechanisms of metabolic activation of aromatic amines have been reviewed by Kadlubar (1987) and Beland and Kadlubar (1990), and of thermic amines, by Kato (1986) and Kato and Yamazoe (1987). Possible organic chemical mechanisms by

which the factors, number of rings, and methyl substitution at ring N- and C-atoms, may affect mutagenic potency were discussed previously for the thermic mutagens (Hatch et al., 1991). Similar considerations may also apply for the aromatic amines. The remaining structural factor, number of amino groups, applies only to the aromatic amines and may relate to potency by providing in some cases a choice of two amino groups for activation to an ultimate mutagenic metabolite.

Modulation of carcinogenic potency by structural factors In Table 7 the coefficient of determination of R E = 52.7% indicates that more than one-half of the variance in - l o g TD50 is explained by two of the same structural factors that modulate mutagenic potency (three factors for the mouse lowest values, data set No. 2). The fit of the model for carcinogenic potency is weaker than that for mutagenic potency. The demonstrated correlation between mutagenic and carcinogenic potencies made it advisable to apply multivariate analysis of variance, which combines the two dependent variables in a single model. When this was done (Table 8) the structural factors, number of rings and number of C-methyl groups, were confirmed as important predictors in the combined model. Since carcinogenic mechanisms for aromatic amines are more complex and less well understood than mutagenic mechanisms, it is less feasible to hypothesize how the structural factors may relate to carcinogenic potency. Of course, a role in modulating initiation of cells by somatic mutation would presumably involve the same chemical mechanisms that apply for mutagenic potency. Also, as noted above, it is possible that cytotoxicity and cell killing arise in part from the same mechanisms as for mutagenicity. Cytotoxicity at levels which induce cell killing also induces cell regeneration in tissues. The increased replication of DNA in the tissue enhances the probability that unrepaired DNA lesions will cause mutational errors and, in consequence, enhances the probability of tumor initiation (Ames and Gold, 1990, 1991; Cohen and Ellwein, 1990). Cell regeneration might also increase the rate of clonal expansion of initiated cells, thus accelerating the appearance of detectable tumors. This conse-

282 quence of cytotoxicity would presumably increase carcinogenic potency above the level that would be observed in its absence. It might also obscure the relationship of carcinogenic potency to some of the structural factors that were found to modulate mutagenic potency, i.e., the structural basis and dose response for tumor progression may differ from those influences for initiation.

Implications of thermic amines for human health The following characteristics of aromatic and thermic amines suggest a high probability that they are carcinogenic in humans. (1) All have one or more primary amino groups, which confer "structural alerts" according to proposals of Ashby (1985) and Ashby et al. (1989). (2) Several members of the aromatic amines reported here are already classified by IARC as definite or probable human carcinogens (IARC, 1982). (3) Rodent tumor bioassays of the thermic amines reported here almost universally show activity in both sexes of two species at multiple organ sites (Ohgaki et al., 1986); and this is similar to the behavior of known human carcinogens (Wilbourn et al., 1986). (4) Short-term tests on thermic amines generally reveal conclusive evidence of gcnotoxicity (Hatch, 1986). Genotoxicity is a property of most of the known human carcinogens (Shelby, 1988). The foregoing characteristics are consistent with the criteria for human carcinogenic risk given by Weisburger (1985). Assessment of human hazard requires knowledge of the probability that a chemical would be carcinogenic at some dose. Also necessary are realistic evaluations of the magnitude of human exposure (Alexander et al., 1989; Felton and Knize, 1990; Sugimura and Wakabayashi, 1990), of the internal pharmacokinetics and mechanism of toxification after exposure (Carman et al., 1988; Turteltaub et al., 1989; Alexander and Wallin, 1991), of the shape of the dose-response curve at low doses (Hoel et al., 1983; Swenberg et al., 1987), and of the appropriate translation of potency from rodent to human. Preliminary efforts at risk assessment for the thermic amines have been published by Alexander et al. (1989) and Gaylor and Kadlubar (1991), reaching different conclusions.

Future research needs We suggest here a number of areas where further research would contribute to a better understanding of the degree of human risk posed by aromatic amines and, in particular, the thermic amines found in cooked food. A crucial need is for more extensive data on human dietary intake of thermic amines. Even if we accept the thesis that the thermic amines are probably carcinogenic in humans and a conservative linear extrapolation downward from the doses administered in rodent bioassays, we must be able to estimate realistic human ingestion levels and their proportionate distribution among the population. Only after such data become available can an acceptable estimate be made of potential cancer risk. Several laboratories are engaged in the laborintensive process of analyzing additional foods, cooked under various conditions, for the known thermic amines. A reasonable amount of such data are already available (Alexander et al., 1989; Felton and Knize, 1990; Sugimura and Wakabayashi, 1990). However, accurate data on the daily intake of the foods, primarily meats, that contain the thermic amines and on the cooking practices of the population are in short supply. In particular, the degree of "doneness", which has a very large influence on mutagen (thermic amine) content, is seldom recorded in dietary surveys. Recent studies have begun to address this issue (Schiffman et al., 1989; Schiffman and Felton, 1990; Gerhardsson de Verdier et al., 1991). Dose-response relationships with DNA adducts, rather than tumors, as the endpoint can be extended into the range between the high exposure doses required for tumor bioassays and realistic environmental exposure levels (Gupta and Earley, 1988; Turteltaub et al., 1990). The latter reference deals with a new method for measurement of DNA binding of chemicals with an enormous dynamic range, reaching realistic environmental exposure levels. Specifically, with the technique of accelerator-mass spectrometry the binding of MeIQx to D N A was found to be linear over a dose range of 10S-fold, thus ruling out saturation of repair processes as the dose increases. Application of these more sensitive

283 methods to a variety of carcinogens will be valuable. A correlation among DNA binding, mutagenicity, and carcinogenicity has been reported for two of the amines in the present series (Furlong et al., 1987). Covalent binding of 2,4-diaminotoluene in rat hepatocytes was about fourfold higher than that of 2,6-diaminotoluene. The carcinogenic potency of 2,4-diaminotoluene in the database of Gold et al. was nearly two orders of magnitude higher in rats than 2,6-diaminotoluene, with a smaller difference in mice (Table 3). The latter compound was classified as noncarcinogenic by the National Cancer Institute bioassay program (1980). A matrix of information should be developed to integrate dose-response data on the DNA binding of chemicals, mutagenicity, cytotoxicity and cell proliferation, and tumor incidence. Then we shall have an improved understanding of the significance of rodent bioassays and improved reliability of modeling the extrapolation from high to low exposure doses. Finally, extension of correlations between mutagenic and carcinogenic potencies, and their modulation by structural factors, to other important chemical classes (e.g., polycyclic aromatic hydrocarbons, nitroaromatics) is now feasible and should be of interest. Ford and Scribner (1990) have studied reactivity of alkanediazonium ions with DNA bases by molecular orbital calculations. De Compadre et al. (1990) and Debnath et al. (1992a,b) have determined relationships between Salmonella mutagenicity and combined calculations of hydrophobicity and energies of molecular orbitals for aromatic amines and nitro compounds. Shusterman (1991) has reported similar calculations for several chemical classes, including a preliminary study of some thermic and nonthermic heterocyclic amines. Conclusions

(1) About one-half of the known thermic amines produced during the cooking of food (all those with tumor bioassays completed) lie near the upper end of the mutagenic and carcinogenic potency ranges for aromatic amines. This emphasizes the need for careful study of these chemi-

cals. The thermic amines are clearly genotoxic, exhibit broad carcinogenesis in multiple target organs of both sexes of two rodent species, and are associated by chemical class with known human carcinogens. A main objective of further research must be to evaluate whether these chemicals pose a significant human risk, taking into account their presence in foods in minute quantities (individually at 1-ppb), and exposures which cover nearly an entire lifetime but are strongly dependent on dietary food choices and cooking practices. (2) A significant quantitative correlation has been demonstrated between the mutagenic and carcinogenic potencies of a series of aromatic and heterocyclic aromatic amines. Mutagenic potency was derived from histidine reversion assays in the frameshift-sensitive strains of Salmonella. Carcinogenic potency was derived from TDs0 values reviewed and compiled by Gold, Ames and collaborators. Calculations were made on a logarithmic scale because of the wide potency ranges exhibited by these chemicals and were corrected for variations in molecular weight. (3) Structural features that were considered to have potential for influencing the metabolic activiation or detoxication pathways of the amines, or their interaction with DNA were evaluated. The structural factors were shown by multiple linear regression analysis and multivariate analysis of variance to serve well as independent variables, capable of modulating mutagenic and carcinogenic potencies. We believe it is important that similar structural factors modulate both biological effects. Although the nature of the structural factors does not define their metabolic or organic chemical mechanisms for contribUting to mutation or oncogenesis, their similar effects on the two modes of response suggest a degree of common mechanism underlying the respective assay conditions. The highly significant fit of the mutagenicity model is consistent with the rather well understood mechanisms of metabolism and DNA binding of aromatic amines. The weaker fit of the carcinogenicity model is consistent with the widely accepted role of somatic mutation in tumor initiation, i.e., the "somatic mutation theory of cancer" (Boveri, 1914; Barrett et al., 1981; Moolgavkar,

284

1986; Moolgavkar and Knudson, 1981; for a contrary view, Lijinsky, 1989), plus some additional less understood mechanisms. The additional mechanisms may involve as yet unidentified structural factors to account for the wide range of carcinogenic potencies exhibited by these amines. (4) The observed correlation between the mutagenic and carcinogenic potencies and the partially common set of modulating structural factors argue in favor of the utility of the short-term mutagenicity test in evaluating hazard levels. This utility applies both in the sense of rapid, inexpensive screening for hazardous chemicals and of enhancing the interpretation of tumor bioassay data. Acknowledgments We thank Richard Wooten for producing the illustrations. This work was performed under the auspices of the U.S. Department of Energy by the Lawrence Livermore National Laboratory under contract number W-7405-ENG-48 and was supported by the National Institute of Environmental Health Sciences/National Toxicology Program under lAG NIEHS 222Y01-ES-10063. References Adamson, R.H., U.P. Thorgeirsson, E.G. Snyderwine, S.S. Thorgeirsson, J. Reeves, D.W. Dalgard, S. Takayama and T. Sugimura (1990) Carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in nonhuman primates: Induction of tumors in three macaques, Japan. J. Cancer Res. 81, 10-14. Alexander, J., G. Becher and L. Busk (1989) Cooked food mutagens--A general overview, Var Foda 42, Suppl. 2, 9-59. Alexander, J., and H. Wallin (1991) Metabolic fate of heterocyclic amines from cooked food, in: H. Hayatsu (Ed.), Mutagens in Food: Detection and Prevention, CRC Press, Boca Raton, pp. 143-156. Ames, B.N., and L.S. Gold (1990) Too many rodent carcinogens: mitogenesis increases mutagenesis, Science 249, 970-971. Ames, B.N., and L.S. Gold (1991) Cancer prevention strategies greatly exaggerate risks, Chem. Eng. News Jan. 7, 1991, 28-32. Ashby, J. (1985) Fundamental structural alerts to potential carcinogenicity or noncarcinogenicity, Environ. Mutagen. 7, 919-921. Ashby, J. (1990) Are short-term tests useful for predicting carcinogenicity? A panel discussion revisited, Environ. Molec. Mutagen. 16, 324-327.

Ashby, J., and R.W. Tennant (1988) Chemical structure, Salmonella mutagenicity and extent of carcinogenicity as indicators of genotoxic carcinogens among 222 chemicals tested in rodents by the U.S. NCI/NTP, Mutation Res. 204, 17-1~15. Ashby, J., R.W. Tennant, E. Zeiger and S. Stasiewicz (1989) Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 42 chemicals tested for carcinogenicity by the U.S. National Toxicology Program, Mutation Res. 223, 73-103. Auletta, A., and J. Ashby (1988) Workshop on the relationship between short-term test information and carcinogenicity, Williamsburg, Virginia, January 20-23, 1987, Environ. Molec. Mutagen. 11, 135-145. Barrett, J.C., B.D. Crawford and P.O.P. Ts'o (1981) The role of somatic mutation in a multistage model of carcinogenesis, in: N. Mishra, V. Dunkel and M. Mehlman (Eds.), Mammalian Cell Transformation by Chemical Carcinogens, Adv. Modern Environ. Toxicol. 1,467-501. Beland, F.A., and F.F. Kadlubar (1990) Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons, Handbook Exptl. Pharmacol. 94/I, 267-325. Benigni, R., C. Andreoli and A. Giuliani (1989) Interrelationships among carcinogenicity, mutagenicity, acute toxicity, and chemical structure in a genotoxicity data base, J. Toxicol. Environ. Health 27, 1-20. Bernstein, L., J. Kaldor, J. McCann and M.C. Pike (1982) An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test, Mutation Res. 97, 267281. Bernstein, L., L.S. Gold, B.N. Ames, M.C. Pike and D.G. Hoel (1985) Some tautologous aspects of the comparison of carcinogenic potency in rats and mice, Fundamental and Applied Toxicology 5, 79-86. Boveri, T. (1914) Zur Frage der Entstehung maligner Tumoren, Gustav Fischer, Jena; and (1929) Williams and Wilkins, Baltimore. Brockman, H.E., and D.M. DeMarini (1988) Utility of shortterm tests for genetic toxicity in the aftermath of NTP's analysis of 73 chemicals, Environ. Molec. Mutagen. 11, 421-435. Carman, R.J., R.L. Van Tasselt, D.G.I. Kingston, M. Bashir and T.D. Wilkins (1988) Conversion of IQ, a dietary pyrolysis carcinogen to a direct-acting mutagen by normal intestinal bacteria of humans, Mutation Res. 206, 335-342. Carver, J.H., F.T. Hatch and E.W. Branscomb (1979) Estimating maximum limits to mutagenic potency from cytotoxic potency, Nature 179, 154-156. Cohen, S.M., and L.B. Ellwein (1990) Cell proliferation in carcinogenesis, Science 249, 1007-1011. Crouch, E., and R. Wilson (1979) Interspecies comparison of carcinogenic potency, J. Toxicol. Environ. Health 5, 10951118. Debnath, A.K., G. Debnath, A.J. Shusterman and C. Hansch (1992a) A QSAR investigation of the role of hydrophobicity in regulating mutagenicity in the Ames test, 1. Mutagenicity of aromatic and heteroaromatic amines in Salmonella typhimurium TA98 and TA100, Environ. Molec. Mutagen., 19, 37-52.

285 Debnath, A.K., R.L.L. de Compadre, A.J. Shusterman and C. Hansch (1992b) Quantitative structure-activity relationship investigation of the role of hydrophobicity in regulating mutagenicity in the Ames test: 2. Mutagenicity of aromatic and heteroaromatic nitro compounds in Salmonella typhimurium TA100, Environ. Molec. Mutagen., 19, 53-70. de Compadre, R.L.L., A.K. Debnath, A.J. Shusterman, and C. Hansch (1990) LUMO energies and hydrophobicity as determinants of mutagenicity by nitroaromatic compounds in Salmonella typhimurium, Environ. Molec. Mutagen. 15, 44-55. de Meester, C. (1989) Assessment of bacterial mutagenesis of heterocyclic amines found in heat-processed food, Mutation Res. 221, 235-262. Dunkel, V.C., E. Zeiger, D. Brusick, E. McCoy, D. McGregor, K. Mortelmans, H.S. Rosenkranz and V.F. Simmon (1984) Reproducibility of microbial mutagenicity assays: I. Tests with Salmonella typhimurium and Escherichia coli using a standardized protocol, Environ. Mutagen. 6, Suppl. 2, 1-254. Dunkel, V.C., E. Zeiger, D. Brusick, E. McCoy, D. McGregor, K. Mortelmans, H.S. Rosenkranz and V.F. Simmon (1985) Reproducibility of microbial mutagenicity assays: II. Testing of carcinogens and noncarcinogens in Salmonella typhimurium and Escherichia coli, Environ. Mutagen. 7, Suppl. 5, 1-248. Esumi, H., H. Ohgaki, E. Kohzen, S. Takayama and T. Sugimura (1989) Induction of lymphoma in CDF 1 mice by the food mutagen, 2-amino-l-methyl-6-phenylimidazo[4,5b]pyridine, Japan. J. Cancer Res. 80, 1176-1178. Fassina, G., A. Abbondandolo, M. Mariani, M. Taningher and S. Parodi (1990) Mutagenicity in V79 cells does not correlate with carcinogenicity in small rodents for 12 aromatic amines, J. Toxicol. Environ. Health 29, 109-130. Felton, J.S., and M.G. Knize (1990) Heterocyclic-amine mutagens/carcinogens in foods, Handbook Exptl. Pharmacol. 94/1, 471-502. Felton, J.S., M.G. Knize, N.H. Shen, R. Wu and G. Becher (1988) Mutagenic heterocyclic imidazoamines in cooked foods, in: C.M. King, L.J. Romano, and D. Schuetzle (Eds.), Carcinogenic and Mutagenic Responses to Aromatic Amines and Nitroarenes, Elsevier, Amsterdam, pp. 73-85. Ford, G.P., and J.D. Scribner (1990) Prediction of nucleoside-carcinogen reactivity. Alkylation of adenine, cytosine, guanine, and thymine and their deoxynucleosides by alkanediazonium ions, Chem. Res. Toxicol. 3, 219-230. Freedman, D.A., and H. Zeisel (1988) From mouse to man: The quantitative assessment of cancer risks, Statistical Science 3, 3-56. Frierson, M.R., G. Klopman and H.S. Rosenkranz (1986) Structure-activity relationships (SARs) among mutagens and carcinogens: A review, Environ. Molec. Mutagen. 8, 283-327. Furlong, B.B., R.P. Weaver and J.A. Goldstein (1987) Covalent binding to DNA and mutagenicity of 2,4-diaminotoluene metabolites produced by isolated hepatocytes and 9000 g supernatant from Fischer 344 rats, Carcinogenesis 8, 247-251.

Fuscoe, J.C., R. Wu, N.H. Shen, S.K. Healy and J.S. Felton (1988) Base-change analysis of Salmonella hisD gene revertant alleles, Mutation Res. 201, 241-251. Gagnon, J., J.M. Roth, M. Carroll, R. Hofmann, K.A. Haycock, J. Plamondon, D.S. Feldman Jr. and J. Simpson (1989) Super-ANOVA, Accessible General Linear Modeling, Abacus Concepts, Inc., Berkeley, CA, pp. 194-195. Garner, C., and C.A. Nutman (1977) Testing of some azo dyes and their reduction products for mutagenicity using Salmonella typhimurium TA1538, Mutation Res. 44, 9-19. Garner, R.C., C.N. Martin and D.B. Clayson (1984) Carcinogenic aromatic amines and related compounds, in: C.E. Searle (Ed.), Chemical Carcinogens, 2nd edn., Amer. Chem. Soc., Washington, pp. 175-301. Gaylor, D.W., and F.F. Kadlubar (1991) Quantitative cancer risk assessment of heterocyclic amines in cooked foods, in: H. Hayatsu (Ed.), Mutagens in Food: Detection and Prevention, CRC Press, Boca Raton, pp. 229-236. Gerhardsson de Verdict, M., U. Hagman, R.K. Peters, G. Steineck and E. Overik (1991) Meat, cooking methods and colorectal cancer: A case-referent study in Stockholm, Int. J. Cancer 49, 520-525. Glantz, S.A., and B.K. Slinker (1990) Primer of Applied Regression and Analysis of Variance, McGraw-Hill, New York, pp. 189-193, 216-226. Gold, L.S., C.B. Sawyer, R. Magaw, G.M. Backman, M. de Veciana, R. Levinson, N.K. Hooper, W.R. Havender, L. Bernstein, R. Peto, M.C. Pike and B.N. Ames (1984) A carcinogenic potency database of the standardized results of animal bioassays, Environ. Health Perspect. 58, 9-319. Gold, L.S., M. de Veciana, G.M. Backman, R. Magaw, P. Lopipero, M. Smith, M. Blumenthal, R. Levinson, J. Gerson, L. Bernstein and B.N. Ames (1986) Chronological supplement to the carcinogenic potency database: Standardized results of animal bioassays published through December 1982, Environ. Health Perspect. 67, 161-200. Gold, L.S., T.H. Slone, G.M. Backman, R. Magaw, M. Da Costa, P. Lopipero, M. Blumenthal and B.N. Ames (1987) Second chronological supplement to the carcinogenic potency database: Standardized results of animal bioassays . published through December 1984 and by the National Toxicology Program through May 1986, Environ. Health Perspect. 74, 237-329. Gold, L.S., T.H. Slone, G.M. Backman, S. Eisenberg, M. Da Costa, M. Wong, N.B. Manley, L. Rohrbach and B.N. Ames (1989a) Third chronological supplement to the carcinogenic potency database: Standardized results of animal bioassays published through December 1986 and by the National Toxicology Program through June 1987, Environ. Health Perspect. 84, 215-285. Gold, L.S., L. Bernstein, R. Magaw and T.H. Slone (1989b) Interspecies extrapolation in carcinogenesis: Prediction between rats and mice, Environ. Health Perspect. 81, 211-219. Goodman, G., and R. Wilson (1991) Predicting the carcinogenicity of chemicals in humans from rodent bioassay data, Environ. Health Perspect. 94, 195-218. Gupta, R.C., and K. Earley (1988) 32p-adduct assay: comparative recoveries of structurally diverse DNA adducts in the

286 various enhancement procedures, Carcinogenesis 9, 16871693. Haseman, J.K., and A-M. Clark (1990) Carcinogenicity results for 114 laboratory animal studies used to assess the predictivity of four in vitro genetic toxicity assays for rodent carcinogenicity, Environ. Molec. Mutagen. 16, Suppl. 18, 15-31. Hatch, F.T. (1986) A current genotoxicity database for heterocyclic thermic food mutagens. I. Genetically relevant endpoints, Environ. Health Perspect. 67, 93-103. Hatch, F.T., J.S. Felton and M.G. Knize (1988) Mutagens formed in foods during cooking, ISI Atlas of Science: Pharmacology 222-227. Hatch, F.T., M.G. Knize and J.S. Felton (1991) Quantitative structure-activity relationships of heterocyclic amine mutagens formed during the cooking of food, Environ. Molec. Mutagen. 17, 4-19. Haworth, S., T. Lawlor, K. Mortelmans, W. Speck and E. Zeiger (1983) Salmonella mutagenicity test results for 250 chemicals, Environ. Mutagen. 5, Suppl. 1, 3-142. Hoel, D.G., N.L. Kaplan and M.W. Anderson (1983) Implication of nonlinear kinetics on risk estimation in carcinogenesis, Science 219, 1032-1037. Hoel, D.G., J.K. Haseman, M.D. Hogan, J. Huff and E.E. McConnell (1988) The impact of toxicity on carcinogenicity studies: implications for risk assessment, Carcinogenesis 9, 2045-2052. International Agency for Research on Cancer (1982) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Chemicals, Industrial Processes and Industries Associated with Cancer in Humans, IARC, Lyon, Suppl. 4, 14-16. Ito, N., R. Hasegawa, M. Sano, S. Tamano, H. Esumi, S. Takayama and T. Sugimura (1991) A new colon and mammary carcinogen in cooked food, 2-amino-l-methyl-6phenylimidazo-[4,5-b]pyridine (PhlP), Carcinogenesis, 12, 1503-1506. Kadlubar, F.F. (1987) Metabolism and DNA binding of carcinogenic aromatic amines, ISI Atlas of Science: Pharmacology, 129-132. Kato, R. (1986) Metabolic activation of mutagenic heterocyclic aromatic amines from protein pyrolysates, CRC Crit. Rev. Toxicol. 16, 307-350. Kato, R., and Y. Yamazoe (1987) Metabolic activation and covalent binding to nucleic acids of carcinogenic heterocyclic amines from cooked foods and amino acid pyrolysates, Japan. J. Cancer Res. 78, 297-3 l 1. Kier, L.D. (1988) Comments and perspective on the EPA workshop on "The relationship between short-term test information and carcinogenicity", Environ. Molec. Mutagen. 11, 147-157. Knize, M.G., and J.S. Felton (1986) The synthesis of the cooked-beef mutagen 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine and its 3-methyl isomer, Heterocycles 24, 1815-1819. Lijinsky, W. (1989) A view of the relation between carcinogenesis and mutagenesis, Environ. Molec. Mutagen. 14, Suppl. 16, 78-84. Lutz, W.K. (1986) Quantitative evaluation of DNA binding data for risk estimation and for classification of direct and

indirect carcinogens, J. Cancer Res. Clin. Oncol. 112. 85-91. Margolin, B.H., N. Kaplan and E. Zeiger (1981) Statistical analysis of the Ames Salmonella/microsome test, Proc. Natl. Acad. Sci., USA 76, 3779-3783. McCann, J., L.S. Gold, L. Horn, R. McGill, T.E. Graedel and J. Kaldor (1988) Statistical analysis of Salmonella test data and comparison to results of animal cancer tests, Mutation Res. 205, 183-195. Moolgavkar, S.H. (1986) Carcinogenesis modeling: From molecular biology to epidemiology, Ann. Rev. Public Health 7, 151-169. Moolgavkar, S.H., and A.G. Knudson (1981) Mutation and cancer: A model for human carcinogenesis, J. Nat. Cancer Inst. 66, 1037-1052. Moore, D.H., and J.S. Felton (1983) A microcomputer program for analyzing Ames test data, Mutation Res. 119, 95-102. Mortelmans, K., S. Haworth, T. Lawlor, W. Speck, B. Tainer and E. Zeiger (1986) Salmonella mutagenicity tests: 1I. Results from the testing of 270 chemicals, Environ. Mutagen. 8, Suppl. 7, 1-119. National Cancer Institute (1980) Bioassay of 2,6-toluenediamine dihydrochloride for possible carcinogenicity, Natl. Cancer Inst. Carcinog. Tech. Rep. Ser. vol. 195. Ohgaki, H., H. Hasegawa, T. Kato, M. Suenaga, M. Ubukata, S. Sato, 8. Takayama and T. Sugimura (1986) Carcinogenicity in mice and rats of heterocyclie amines in cooked foods, Environ. Health Perspect. 67, 129-134. Parodi, S., M. Taningher, P. Russo, M. Pala, M. Tamaro and C. Monti-Bragadin (1981) DNA-damaging activity in vivo and bacterial mutagenicity of sixteen aromatic amines and azo derivatives, as related quantitatively to their carcinogenicity, Carcinogenesis 2, 1317-1326. Parodi, S., M. Taningher, P. Boero and L. Santi (1982) Quantitative correlations amongst alkaline DNA fragmentation, DNA covalent bonding, mutagenicity in the Ames test and carcinogenicity for 21 compounds, Mutation Res. 93. 1-24. Parodi, S., M. Taningher, P. Russo, M. Pala, D. Vecchio, G. Fassina and L. Santi (1983) Quantitative predictivity of the transformation in vitro assay compared with the Ames test, J. Toxicol. Environ. Health 12, 483-510. Parodi, S., M. Taningher and L. Santi (1988) Utilization of the quantitative component of positive and negative results of short-term tests, Mutation Res. 205, 283-294. Parodi, S., M. Taningher, P. Romano, S. Grilli and L. Santi (1990) Mutagenic and carcinogenic potency indices and their correlation, Teratogenesis, Carcinogenesis, and Mutagenesis 10, 177-197. Peto, R., M.C. Pike, L. Bernstein, L.S. Gold and B.N. Ames (1984) The TDs0: A proposed general convention for the numerical description of the carcinogenic potency of chemicals in chronic-exposure animal experiments, Environ. Health Perspect. 58, 1-8. Piegorsch, W.W., and D.G. Hoel (1988) Exploring relationships between mutagenic and carcinogenic potencies, Mutation Res. 196, 161-175. Schiffman, M.H., A.W. Andrews, R.L. Van Tassell, L. Smith, J. Daniel, A. Robinson, R.N. Hoover, J. Rosenthal, R. Weil, P.P. Nair, S. Schwartz, H. Pettigrew, G. Batist, R.

287 Shaw and T.D. Wilkins (1989) Case-control study of colon cancer and fecal mutagenicity, Cancer Res. 49, 3420. Schiffman, M.H., and J.S. Felton (1990) Re: Fried foods and the risk of colon cancer, Am. J. Epidemiol. 131,376-378. Shelby, M.D. (1988) The genetic toxicity of human carcinogens and its implications, Mutation Res. 204, 3-15. Shelby, M.D., E. Zeiger and R.W. Tennant (1988) Commentary on the status of short-term tests for chemical carcinogens, Environ. Molec. Mutagen. 11, 437-441. Shusterman, A.J. (1991) Predicting mutagenicity: Use a technique that's been useful in predicting efficacy, Chemtech, October, 624-627. Sugimura, T., and K. Wakabayashi (1990) Mutagens and carcinogens in food, in: M.W. Pariza, H-U. Aeschbacher, J.S. Felton, and S. Sato (Eds.), Mutagens and Carcinogens in the Diet, Wiley-Liss, New York, pp. 1-18. Swenberg, J.A., F.C. Richardson, J.A. Boucheron, F.H. Deal, S.A. Belinsky, M. Charbonneau and B.G. Short (1987) High-to-low dose extrapolation: Critical determinants involved in the dose response of carcinogenic substances, Environ. Health Perspect. 76, 57-63. Tennant, R.W., B.H. Margolin, M.D. Shelby, E. Zeiger, J.K. Haseman, J. Spalding, W. Caspary, M. Resnick, S. Stasiewicz, B. Anderson and R. Minor (1987) Prediction of chemical carcinogenicity in rodents from in vitro genetic toxicity assays, Science 236, 933-941. Turteltaub, K.W., M.G. Knize, S.K. Healy, J.D. Tucker and J.S. Felton (1989) The metabolic disposition of 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine in the induced mouse, Fd. Chem. Tox. 27, 667-673.

Turteltaub, K.W., J.S. Felton, B.L. Gledhill, J.S. Vogel, J.R. Southon, M.W. Caffee, R.C. Finkel, D.E. Nelson, I.D. Proctor and J.C. Davis (1990) Accelerator mass spectrometry in biomedical dosimetry: Relationship between lowlevel exposure and covalent binding of heterocyclic amine carcinogens to DNA, Proc. Natl. Acad. Sci. USA 87, 5288-5292. Weisburger, J.H. (1985) Definition of a carcinogen as a potential human carcinogenic risk, Japan. J. Cancer Res. 76, 1244-1246. Wilbourn, J., L. Haroun, E. Heseltine, J. Kaldor, C. Partensky and H. Vainio (1986) Response of experimental animals to human carcinogens: an analysis based upon the IARC Monographs programme, Carcinogenesis 7, 1853-1863. Zeiger, E. (1990) Mutagenicity of 42 chemicals in Salmonella, Environ. Molec. Mutagen. 16, Suppl. 18, 32-54. Zeiger, E., B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans and W. Speck (1987) Salmonella mutagenicity tests: III. Results from the testing of 255 chemicals, Environ. Mutagen. 9, Suppl. 9, 1-110. Zeiger, E., B. Anderson, S. Haworth, T. Lawlor and K. Mortelmans (1988) Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals, Environ. Molec. Mutagen. 11, Suppl. 12, 1-158. Zeiger, E., J.K. Haseman, M.D. Shelby, B.H. Margolin and R.W. Tennant (1990) Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: confirmation of earlier results with 41 additional chemicals, Environ. Molec. Mutagen. 16, Suppl. 18, 1-14.