Mutation Research, 282 (19925241-246

241

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-7992/92/$05.00

MUTLET 0686

Decreased electrophilicity of chemicals carcinogenic only at the maximum tolerated dose Herbert S. Rosenkranz and Gilles Klopman b a

a Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA and b Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA

(Received 14 February 1992) (Accepted 7 April 1992)

Keywords: Electrophilicity,decreased; Maximum tolerated dose; MTD; LUMO energy; DNA reactivity

Summary While there was no significant difference between the actual or predicted mutagenicity and clastogenicity of a group of chemicals carcinogenic only at the maximum tolerated dose (MTD) and a group of chemicals carcinogenic below the MTD, as a group, the chemicals carcinogenic below the MTD exhibited a significantly decreased L U M O (Lowest Unoccupied Molecular Orbital) energy, indicative of increased electrophilicity (i.e. D N A reactivity). These findings suggest that chemicals carcinogenic only at the M T D either require increased doses of "weak" electrophiles to be carcinogenic or that they may act by a "non-genotoxic" mechanism.

The mechanism of action and potential health hazards of chemicals carcinogenic to rodents only at the maximum tolerated dose (MTD) are currently the subject of extensive debate (Marx, 1990; Weinstein, 1991; Ames and Gold, 1990). It has been suggested that such chemicals act through a "non-genotoxic" mechanism that might include stimulation of mitogenesis or systemic toxicity (Cohen and Elwein, 1990, 1991). In contrast to "non-genotoxic" carcinogens, "genotoxic" carcinogens, as a group, are carcinogenic to both rats and mice at multiple sites in both gende.rs (Ashby and Tennant, 1989; Gold et al., 1989), Correspondence: Dr. Herbert S. Rosenkranz, Department of Environmental and Occupational Health, Graduate School of Public Health, University of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, USA.

they are more potent carcinogens (Rosenkranz and Ennever, 1991) and, moreover, the vast majority of human carcinogens are genotoxic (Ennever et al., 1987; Shelby 1988; Bartsch and Malaveille, 1989). It has, therefore, been suggested, that "non-genotoxic" carcinogens pose less of a threat to humans than "genotoxic" ones (Williams, 1987; Ashby and Morrod, 1991). In that context, however, an analysis of a subset of chemicals tested under the aegis of the U.S. National Toxicology Program (NTP) has suggested that chemicals carcinogenic only at the M T D are not enriched with respect to "nongenotoxic" carcinogens when compared to chemicals active below the M T D (Hoel et al., 1988). In view of this controversy~ we have investigated additional properties of the chemicals carcinogenic below as well as only at the MTD.

242

Methods

Data bases The present analyses were restricted to chemicals tested for carcinogefiicity by the NTP. The results of the carcinogenicity and Salmonella mutagenicity assays were taken from Ashby and Tennant (1991), as were the "structure alerts" for DNA reactivity. The results of the assays for the induction of sister-chromatid exchanges and chromosomal aberrations were abstracted from published reports (Galloway et al., 1985, 1987; Gulati et al., 1989; Loveday et al., 1989; Tennant et ah, 1987). The designation of chemicals as carcinogenic only at the MTD or below the MTD was taken from Hoel et al. (1988). For the present analyses a chemical carcinogenic "only at the MTD" is defined as one that is carcinogenic only at the high dose but not at one-half the high dose and a chemical carcinogenic "below the MTD" is defined as one that is carcinogenic at both the high dose and one-half that dose (Hoel et al., 1988). CASE predictions The CASE method has been described on a number of occasions (Klopman et al., 1990; Rosenkranz and Klopman, 1989, 1990a, b). CASE selects its own descriptors from a learning set composed of active as well as inactive molecules. These descriptors are readily recognized as continuous structural fragments embedded in the molecule. The descriptors consist of either activating (biophore) or deactivating (biophobe) fragments. Each biophobe and biophore is characterized by its distribution among active and inactive

molecules and the associated p value. Once biophores and biophobes have been identified, unknown molecules may be analyzed. Upon submission of such a molecule, the CASE program will generate all possible fragments ranging from 2 to 10 "heavy" atoms (i.e. C, O, N, S, P and halogens) accompanied by their hydrogens and these will be compared to the previously identified biophores and biophobes. On the basis of the presence a n d / o r absence of these descriptors, CASE predicts activity or lack thereof. In order to generate the structural determinants associated with the induction of unscheduled DNA synthesis and the in vivo micronuclei, the compilations of Williams et al. (1989) and of Mavournin et al. (1990) were used. These were used to predict the properties of the two groups of carcinogens.

Quantum-mechanical calculations The quantum-mechanical calculations were performed by an ad hoc subroutine in the CASE program (Klopman et al., 1990). The methodology has been optimized to provide CNDO-like results (Houser and Klopman, 1988) using a combination of Del Re and Hiickel methodologies. It allows rapid calculations to be made of charge densities of large numbers of compounds without minimization of atomic coordinates. The purpose of these calculations is to provide fast access to reasonable estimates of physical chemical properties of large numbers of molecules. The Lowest Unoccupied Molecular Orbitals (LUMO) were obtained from these quantum-mechanical calculations. It should be noted that decreased LUMO energies indicate increased electrophilicity.

Notes to Table 1: Abbreviations: CA, carcinogenicity in rodents; Salm, mutagenicity in Salmonella; Chrom. aberr., chromosomal aberration; SCE, sister-chromatid exchange; Mnt, in vivo micronucleus test; UDS, unscheduled D N A synthesis. " + ", " • " and " - " denote positive, marginal and negative responses, respectively. * Data derived from NTP bioassays. The assignment of the chemicals to various groups based upon spectrum of carcinogenic responses is taken from Ashby and T e n n a n t (1988), Ashby et al. (1989) and T e n n a n t and Ashby (1991). * * C A S E predictions of ability to induce micronuclei and UDS. A, Agents carcinogenic to rats and mice at one or more sites. B, Agents carcinogenic only to the rat or m o u s e at two or more sites. C, Agents carcinogenic to the rat or mouse at a single site in both sexes. D, Agents carcinogenic at only a single site in a single sex of a single species.

243

TABLE

1

GENOTOXICITY

OF

GROUPS

Chemical

OF

CHEMICALS

CA

*

Salm

CARCINOGENIC

*

AT

Chrom. aberr.

SCE

THE

*

MTD

AND

Str.

*

alert

BELOW

Mnt

* *

THE

MTD

UDS

* *

LUMO

*

Carcinogenic only at the MTD 1,2-Dichloropropane

C

+

+

+

+

-

-

- 2.18

1,2-Propylene o x i d e

A

+

+

+

+

+

-

0.61

1,4-Dichlorobenzene

A

.

+

-

1.00

2,6-Dichloro-p-phenylenediamine

C

+

+

+

+

+

+

0.81

2-Biphenylamine

D

+

+

-

+

+

-

1.18

Allyl isothiocyanate

D

+

+

+

+

-

-

- 0,77

Allyl isovalerate

A

-

+

-

+

-

0,91

Butyl

D

.

+



1.05

C

+

-

+

+

+



0.00

Chlorodibromomethane

D

+

-

+

+

-

-

1.01

Isophorone

D

-

-

+

-

-

-

0.61

Melamine

D

-

-



-

-

-

1.01

Monuron

B

-

+

+

-

+

+

1.00

Trichloroethylene

C

-

-

+

-

+

+

1.22

Ziram

D

+

+

-

-

+

+

1.09

1,1,2,2-Tetrachloroethane

C

-

-

+

-

-

-

0.91

1,2-Dibromo-3-chloropropane

A

+

+

+

+

-

+

- 1.40

1,2-Dibromoethane

A

+

+

+

+

-

-

- 0.64

+

-

-

-

0.91

+

-

- 1.13

benzyl

• HCI

phthalate

C.I. solvent

yellow

14

.

.

.

+ .

.

.

Carcinogenic below the MTD

B

-

2,3,7,8-Tetrachlorodibenzo-p-dioxin

11-Aminoundecanoic

acid

A

.

3-Chloro-2-methylpropene

A

+

+

+

+

-

-

i. 17

A

+

+

+

+

+

+

0.55

A

+

+

+

-

- 1.82

A

-

-

+

-

+

-

1.00

B

.

+



0.91

B

+

+

+

+

+

-

- 1.21

A

+

-

-

+

+

-

0.12

C

+

+

+

+

+

-

0.09

A

+

-

+

+

+

-

0.34

A

-

-

+

-

-

-

0.91

-

+

+



0.29

-

4,4'-Methylenedianiline.

2HCI

.

.

.

4-Vinyl-l-cyclohexene diepoxide Benzene Benzyl

acetate

Bis(2-chloroC.I. Basic

1-methylethyl)ether

Red

9, HCI

C.I. Disperse

Blue

C.I. Disperse

Yellow

Chlorendic D and

1 3

acid

C Red

9

.

.

.

B

+

C

.

C

-

+



-

+

Di(2-ethylhexyl)phthalate

A

+

-

+

-

+

-

0.29

Dichloromethane

A

+

+

-

-

- 1.00

A

+

1.02

Decabromodi0henyl

oxide

Di(2-ethylhexyl)adipate

Diglycidyl

resorcinol

Dimethylvinyl Ethyl NC

ether

chloride

acrylate Blue

1

Pentachloroethane

.

0.83

+

+

+

+



0.91

A

+

-

+

+

+

-

1.23

-

+

+

+

+

-

0.44

A

+

+

+

+

+

+

0.00

+

-

+

-

- 1.58

+

-

0.77

+



0.54

C

.

Tetrachloroethylene

A

.

Zearalenone

B

-

biphenyl

.

A

A

Polybrominated

.

.

. .

. .

+

. +

.

. -

1.28

244

Results and discussion

In an earlier study we reported that as a group, carcinogens exhibited decreased M T D values (i.e. increased systemic toxicity) when compared to non-carcinogens (Rosenkranz, 1992). Moreover, we also found an association between mutagenicity and increased toxicity suggesting that factors such as D N A reactivity (e.g. electrophilicity) may be associated causally with increased toxicity. Additionally, we found that as a group, chemicals carcinogenic only at the M T D exhibited decreased toxicities and decreased carcinogenic potencies (Rosenkranz, 1992). In order to gain a further understanding of the relationship between carcinogenicity and mutagenicity, we determined the relationship between the energy of the Lowest Unoccupied Molecular Orbital (LUMO), a quantitative measure of the electrophilicity, and we showed that (a) mutagens had lower L U M O energies (i.e. greater electrophilicity) than non-mutagens and (b) that carcinogens, as a group, had lower L U M O energies than noncarcinogens (Rosenkranz and Klopman, 1992). Further comparisons of mutagenic and non-mutagenic carcinogens and mutagenic and non-mutagenic non-carcinogens, showed that mutagenicity, rather than carcinogenicity, was the primary determinant of decreased L U M O energy (Rosenkranz and Klopman, 1992). Although TDs0 values and M T D values are available for chemicals tested in the NTP rodent cancer bioassay, unfortunately chemicals carcinogenic only at the MTD are not readily identifiable from an examination of the NTP data. However, Hoel et al. (1988) in analyzing a subset of the NTP rodent bioassay data identified a group of chemicals carcinogenic only at the MTD and another one carcinogenic below the MTD. These authors pointed out that there was no significant difference in the proportion of mutagens in each of these groups. Our own analysis of the results of Salmonella mutagenicity, sister-chromatid exchanges, chromosomal aberrations and "structural alerts" for genotoxicity as well as the predicted potentials for inducing micronuclei and unscheduled DNA synthesis also indicated that there were no significant differences among the genotoxic potentials of these chemicals (Tables 1

TABLE 2 SUMMARY OF M U T A G E N I C I T Y A N D CLASTOGENICITY OF CHEMICALS C A R C I N O G E N I C ONLY AT MTD AND BELOW MTD At MTD only Below MTD positives a positives a Ratio Percent Ratio Percent Salmonella "Structural alerts" Chromosomal aberrations Sister-chromatid exchanges

8/15 7/15 8/15 10/15

53 47 53 67

Unscheduled D N A synthesis b 4/15 27 Micronuclei (in vivo) b 10/15 67

15/28 14/28 11/21 15/22

54 50 52 68

4 / 2 8 14 18/28 64

~ There were no statistically significant differences between the two groups. b Predictions based on CASE program.

and 2). However, analysis of the L U M O energies indicated a trend, chemicals carcinogenic only at the MTD, as a group, exhibited higher L U M O energies (i.e. decreased electrophilicity) than the chemicals which are carcinogenic below the M T D (Table 3). Moreover, it is of interest that this trend is a result, primarily of the mutagens in the group, i.e. 0.066 for chemicals carcinogenic below the MTD versus 0.22 at the M T D ( p value _< 0.05). Thus, even though there is no qualitative difference between the overall genotoxicity of chemicals carcinogenic only at the MTD and those carcinogenic below the MTD (Tables 1 and 2), the intrinsic electrophilicity, and ultimately DNA reactivity, of the chemicals carcinogenic below the MTD is greater. The present findings are consistent with the possibilities (1) that some chemicals carcinogenic TABLE 3 LUMO E N E R G I E S ASSOCIATED W I T H D I F F E R E N T G R O U P S OF C A R C I N O G E N S All N

Salm + LUMO N

All NTPcarcinogens 150 0.39 All CA in subgroup 43 0.33 CA at MTD only 15 0.57 CA below MTD 28 0.21 ~

86 23 8 15

Salm -

LUMO N

LUMO

0.20 0.03 0.22 0.07 a

0.67 0.68 0.97 0.52 ~

62 20 7 13

a All of the differences between LUMO energies of the chemicals carcinogenic below the MTD and carcinogenic only at the MTD are significant at p _< 0.05.

245

only at the MTD act by a "genotoxic mechanism" but given their lower DNA reactivity (a) they have lower toxicity and hence the animals tolerate higher doses and (b) higher doses are indeed required to induce "sufficient" DNA damage to initiate the carcinogenic event, and (2) that even though chemicals carcinogenic only at the MTD may possess some electrophilic potential (but possibly insufficient to cause a critical amount of DNA damage), they act primarily by a non-genotoxic mechanism. The latter possibility is consistent with the finding that some carcinogens mutagenic in Salmonella or possessing "structural alerts" may display a spectrum of carcinogenicity (single species at a single site) characteristic of "non-genotoxic" carcinogens (Ashby and Tennant, 1988; Ashby et al., 1989; Tennant and Ashby, 1991).

Acknowledgements This investigation was supported by the National Institute of Environmental Health Sciences (ES04659) and The U.S. Environmental Protection Agency (R818275).

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Communicated by J.M. Gentile

Decreased electrophilicity of chemicals carcinogenic only at the maximum tolerated dose.

While there was no significant difference between the actual or predicted mutagenicity and clastogenicity of a group of chemicals carcinogenic only at...
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