Fundamental and Molecular

Mechanisms of Mutagenesis
Mutation Research

350 (1996) 109- 129

Activity profiles of antimutagens: in vitro and in vivo data

Michael D. Waters il.*, H. Frank Stack b, Marcus A. Jackson ,

Herman E. Brockman , Silvio De Flora d

Accepted

3 June 1995

Abstract
In this review, retinol. chlorophyllin, and N-acetylcysteine are examined and compared with regard to their antimutagenic
activity against some promutagens and a group of direct-acting alkylating agents. The promutagens included aflatoxin B , .
certain polycyclic aromatic hydrocarbons (e.g.. benzo[ alpyrene). and certain heterocyclic amines (e.g.. food pyrolysates).
Results of antimutagenicity
testing selected from data surveyed in the published literature are displayed graphically as
activity profiles of antimutagens showing both the doses tested and the extent of inhibition or enhancement of mutagenic
activity. All three antimutagens are discussed in terms of their putative mechanisms of action in vitro and in vivo with
emphasis on the xenobiotic metabolizing enzyme systems.
Kr,wortfs: Antimutagenicity
profile: Aflatoxin B,: Benro[n]pyrene;
Chlorophyllin;
Mechanisms; N-Acetylcysteine:
Polycyclic aromatic hydrocarbon; Retinol: Retinoid

Food pyrolysatea

or heterocyclic

amine; Metabolism;

Abbreviations:
2-AA. ?_-aminoanthracene:
A CUC. amino-a-carboline:
2.AF, 2.aminofluorene:
2-AAF. 2.acetylaminofluorene:
AFB,,
aflatoxin B,; B[n]P. benzo[n]pyrene
; BA. benz[a]anthracene:
CHL. chlorophyllin: CPA, cyclophosphamide:
CsCI, cesium chloride: DEB,
dicpoxybutane;
DEN. diethylnitrosamine:
DMBA, 7.12.dimethylbenz[c~]anthracene;
DMN. dimethylnitrosamine;
ECH. epichlorohydrin;
EMS. ethyl methanesulfonate:
EtBr. ethidium bromide: Glu-P-l. 2-amino-6-methyldipyrido[l,2-tr:3,2dlimidazol;
Gh-P-2. 2-aminodipyrido[ I .2-cr:3,2-dlimidazol:
GSH. glutathione;
H .O,, hydrogen peroxide; HAS, heterocyclic
amines; H&l, mercury chloride; IQ,
2-amino-3-methylimidazo[3.5-f]quinoline;
MCA, 3-methylcholanthrene:
MeA (YC, aminomethyl-wcarboline:
MeIQ. 2.amino-3.4dimethylimidazo(4.5,fJquinoline:
MeIQx, 2.amino-3.8.dimethylimidazd4,5-f)quinoxaline:
MMC. mitomycin C: MMS. methyl methanesulfonate;
MNNG. N-methyl-Nnitro-N-nitrosoguanidine:
4NPDA, 4.nitro-o-phenylenediamine:
4NQ0. 4.nitroquinoline-N-oxide:
NAC. N-acetyl-Lcysteine: NNN. N-nitroaonornicotine:
NNK, J-f N-methyl-N-nitrosoamino~-l-~3-pyridinyl~-2-butanone;
NOP. 4.nitro-o-phenylenediamine;
OAAT. waminoazotoluene:
PAHs. polycyclic aromatic hydrocarbons; ROL. retinal; Thio-TEPA, triethylene thiophosphoramide:
TrpP-I.
3-~mino-3.4-dimethyl-SH-pyrido[4,3-h]indole:
Trp-P-2. 3.amino- I-methyl-5H-pyrido[4.3-h]indole:
Trp-P-2tNHOH).
3.hydroxyamino-lmethyl-5H-pyrido[4.3-h]indole
Corresponding
author
This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication.
Mention of trade names or commercial products does not constitute endorsement or recommendation
for use.
0027.5 107/96/$15.00
0 1996 Elaevier Science B.V. All rights reserved
SSLZI0027.5
107(95)00097-6

I IO

M.D. Wutrrs et rrl./ Mutcltim Rr.wtdt

1. Introduction
The term antimutagen
was used originally to
describe those agents that reduce the frequency 01
rate of spontaneous or induced mutation independent
of the mechanisms
involved (Novick and Szilard.
1952). This is the general meaning of the word used
in this paper. We also use the word anticarcinogen
in the same general way to describe any agent that
reduces the incidence of cancer.
The ability of man and other animal species to
resist the toxic effects of environmental
agents is
dependent on the detoxication and antioxidant systems. These two defense systems are closely related
and the cytochrome P450 superfamily
of enzyme
isoforms is a component
of both. P450 isoforms
function in the detoxication system as oxygenases or
as reductases. yielding products that can generate
oxygen radicals by redox cycling (Kappus. 1986).
P4SO isoforms function in the antioxidant system in
scavenging and neutralizing compounds that generate oxygen radicals. free radicals. and active oxygen
(Parke et al.. I99 I).
Recently. it has been recognized that certain drugs,
environmental
chemicals,
endogenous
regulatory
molecules. and dietary components
may alter the
levels and activities of P450 isozymes. Such changes
may involve the transcription of specific P4SO genes.
the degradation of specific P4SO mRNAs, the translation of these mRMAs. and/or
the degradation of
P450 through protein turnover or by suicide inhibition. Some drugs and dietary components can function as antimutagens
affecting other drugs or toxicants by: (I) altering the rates of absorption and
uptake; (2) reacting or tightly binding with the drug;
(3) competing with the drug for binding to plasma
proteins, or (4) affecting activation and detoxication
systems. Interfering with P450-dependent metabolism
appears to be the most selective mechanism by which
dietary components
exert their effects on drug
metabolism and carcinogenesis (Yang et al.. 1992).
One means to decrease the rate of mutation in
humans, and subsequently to decrease the incidence
of cancer, may be to identify effective antimutagens
and anticarcinogens
and to increase our exposure to
them (Ames. 1983; Wattenberg, 1985; Ramel et al..
1986). especially through the diet (Hayatsu et al.,
1988). In order to seriously entertain such a possibil-

350 ( IYYO) IOY-12Y

ity one should have an appreciation of the mechanism(s) whereby an agent functions as an antimutagen. To gain some perspective on mechanisms of
antimutagenicity,
three antimutagens, retinol (ROL).
chlorophyllin (CHL) and N-acetyl-L-cysteine
(NAC),
are examined and compared with regard to their
activity against: (1) aflatoxin B, (AFB, 1; (2) some
polycyclic aromatic hydrocarbons (PAHs): (3) certain heterocyclic
amines (HAS). i.e.. food pyrolysates: and (4) some miscellaneous
direct-acting
alkylating agents. The results are discussed in terms
of their putative mechanisms of action, with special
attention to their possible influence on activation and
detoxication pathways.

2. Methods
In assembling the database for the present investigation. the literature was surveyed for the availability
of data on the three antimutagens
ROL, CHL. and
NAC. Publications were selected that presented original quantitative
data for any of the genotoxicity
assays that fit the scope of the genetic activity
profiles (Waters et al.. 1988). Data were selected.
classified. and organized into data listings (Tables
l-4)
for three chemical classes of promutagens
[AFB,. PAHs (e.g., B[ n]P), and the HAS] and some
direct-acting alkylating agents (MNNG, EMS. MMS
and MNU) which were tested in combination
with
each of the antimutagens.
The data are displayed graphically as antimutagenicity profiles. These profiles are two parallel sets
of bar graphs (Fig. 1). The upper graph displays the
mutagen concentration and the range of antimutagen
concentrations.
The lower graph shows either the
maximum percent inhibition (represented by a bar
directed upward from the origin) or the maximum
percent enhancement
(represented by a downwarddirected bar) of the genotoxic response. A bar at zero
on the lower graph indicates that the difference in
the response of the mutagen when tested alone and
the response when tested in combination
with the
antimutagen was less than 20%. Codes used to represent the short-term tests have been reported previously (Waters et al.. 1988). and the subset of tests
represented in this paper is defined in the Appendix.
Two formats were adopted for the organization of

M.D. Waters et ~1. /Mutation

Table I
Antimutagenic
Mutagen

Aflatoxin B ,
AFB,
AFB ,
AFB ,

AFB,
AFB,
AFB,
AFB,
AFB ,

III

Research 350 (1996) 109%129

effects of retinal
Mutagen dose
(M or mmol/kgl

4.lE-7
5.7E-8
6.4E-8
6OE-7
6.4E-7
6.4E-7
I .6E-6
6.4E-7
2.OE-6

AFB,
Polycvclic aromatic hydrodcarbons
3.8E-6
B[ ,T]P7.3E-6
B[tr]P
8.OE-6
B[ci]P
-l.OE-1
B[ cl]P
6.9E-7
2-.AF
7.5E-7
2-.4F
1AE-6
2-.4F
2.8E-6
2-4F
4.5E-6
2-4F
I .3E-5
2-.4F
1.8E-5
2-AF
l.SE-7
2-AF
I .5E-6
2AF
4.5E-6
2-AF
I .3E-5
2-AF
I .8E-5
2-AF
4.OE-6
2.AAF
3.OE-5
DMBA
2.OE-5
DMBA
3.OE-5
DXIBA
7.8E-8
DMBA
7.8E-8
DMBA
78E-6
DMBA
3.9E-3
DMBA
7.1 E-6
MCA
9.3E-6
MCA
I .9E-5
MCA
8.4E-6
MCA
8.8E-6
BA
Heterocyclic amines
3.4E-8
Glu-P-l
2.4E-6
Glu-P-2
2.OE-7
IQ
1.9E7
MeIQ
I .9E-7
MeIQx
I .7E-7
Trp-P- 1
2.3E-8
Trp-P-2
1.OE-5
OAAT

Test
code

% Inhibit.
(% enhanc.)

Citation

Retinol
(M or mmol/kg)
Low

High

SAO
SA9
SA9
SA9
SIC
SIC
SIC
CIC
BID

98
99
71
89
33
38
33
56
91

3.7E-5
9.3E-6
2.8E-6
3.2s.6
I .7E-6
1.JE-5
2.8E-5
5.OE-5
1.OE-4

I .9E-4
3.1E-4
2.8E-5
I .OE-3
6.9E6
2.?E-4
l.lE-4
I .OE-4
5.OE-4

Bhattacharya et al.. 1987

Whong et al.. 1988
Busk and Ahlborg, 1980
Qin and Huang. 1985
Qin et al.. 1985
Huang et al., 1982
Huang et al., 1982
Qin et al., 1985
Bhattacharya et al., 1984

SA9
SA9
SIC
CIC
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SAO
URP
GCO
SIC
SIC
SIC
CIC
SAO
TCM
SIC
SAO
SIC

91
0
0
0
95
23
84
88
53
74
62
(88)
(105)
(182)
(49)
(86)
(145)
0
83
72
47
25
0
0
53
97
0
0
0

2.8E-5
3.28-6
7.OE-6
I .4E-5
3.4E-5
1.6E-4
8.7E-6
8.7E-6
2.8E-4
I .X4
.3E-4
3.2E-6
3.2E-6
5.6E-6
2.3E-6
l.lE-5
1.8E-6
3.28-6
1.OE-6
2.5E-6
2%5
2.5E-5
7.OE-6
I .JE-5
3.E-6
I .7E-7
?.OE-6
3.2E-6
7.OE-6

I .4E-4
I .OE-4
1.OE-3
1.OE-4
I .7E-4
I .6E-4
I .7E-4
I .7E-J
5.6E-4
5.6E-4
5.1E-4
1.6E-5
3.2E-5
I .7E-4
l.lE-4
l.lE-4
3.4~~4
5.3E-5
S.OE-5
2.5E-5
2.5E-5
2.5E-5
5.6E-5
I.lE-4
5.3E-5
I .7E-6
5.66-5
5.38-5
5.6E-5

Calle and Sullivan. 1982

Qin and Huang. 1985
Qin et al.. 1985
Qin et al., 1985
Baird and Birnbaum, 1979
Busk and Ahlborg, 1982a
Baird and Birnbaum. 1979
Baird and Bimbaum. 1979
Balbinder et al.. 1983
Balbinder et al., 1983
Balbinder et al.. 1983
Busk and Ahlborg. l982a
Buak and Ahlborg, l982a
Balbinder et al.. 1983
Balbinder et al.. 1983
Balbinder et al., 1983
Busk and Ahlborg, 1982a
Qin and Huang. 1985
Budroe et al., 1987
Budroe et al.. 1988
Cozzi et al.. 1990
Cozzi et al.. 1990
Qin et al.. 1985
Qin et al.. 1985
Qin and Huang. 1985
Merriman and Bertram, 1979
Qin et al.. 1985
Qin and Huang, 1985
Qin et al.. 1985

SA9
SA9
SA9
SA9
SA9
SA9
SA9
SAO

75
90
50
27
61
92
88
92

7.8E-6
3. IE-6
3.5E-5
3.5E-5
3.5E-5
3.1E-6
3.IE-6
8.OE-6

2.3E-4
2.4E-4
1.JE-4
I .JE-4
I .4E-4
2.4E-4
I .6E-4
2.4E-4

Busk et al.. 1982

Busk et al., 1982
toannides et al., 1990
loannides et al.. 1990
Ioannides et al.. 1990
Busk et al., 1982
Busk et al.. 1982
Busk and Ahlborg. 1982b

Table

I (continued)

Mutagen

Mutagen dose
CM or mmol/kg)

Nitrosamines
DEN
3.8E-4
DEN
Y.XE-2
DMN
5.3E-4
DMN
6.7E-3
Cyclophosphamide
CPA
2.7E-4
CPA
6SE-4
CPA
I .OE-3
CPA
I .OE-3
CPA
I .SE-5
CPA
I I E-5
CPA
5.4E-4
CPA
7.2E-5
Alkylating agents
MNNG
I .OE-7
EMS
I .6E-3
8. I E-4
EMS
EMS
2.OE-3
MMS
3.6E-4
Miscellaneous compounds
Melphalan
4.9E-7
Mitomycin C
Y.OE-8
Mitomycin C
1.6E-7
Adriamycin
4.6E-6
DEB
5.3E-5
4NPDA
5.38-S
Buaulfan
2.OE-4
Thio-TEPA
5.3E-5

Teat
code

% Inhibit.
(c/ enhanc.)

Citation

Retinal
(M or mmol/kg)
LOW

High

Huang.
Hung.
Huang.
Huang,

SAO
SIC
SAO
SIC

61
53
9X
60

6.7E-6
6.7E-6
I .1E-5

S.-IE-5
I. I E-4
5.4s5
I. I E-4

SA5
SA5
SIC
SIC
SIC
SIC
CIC
CBA

7X
11
ix
47
61
34
96
0

i.ZE-6
I .OE-5
1.5E-5
2.5E-5
I .lE-6
I .4E-5
I .1E-5
3.5t-6

5.3E-5
I .OE-4
2.5E-5
2.5E-5
7.OE-6
I. I E-4
I. I E-4
8.7E-5

Qm and Hung. 1985

Busk et 31.. 1983
Cozzi et al.. 1990
Cozzi et al.. 1990

SIC
URP
cc0
SIC
CBA

(X0)
0
0
0
0

7.OE-6
I .OE-6
I .OE-6
7.OE-6
35E-6

5.6E-5
5.OE-5
2.SE-5
5.6E-5
8.7E-5

Qin et al., 1985

Budroe et al.. 19X7
Budroe et al.. IYXX
Qin et al.. I985
Renner. I YX5

SHL
SIC
SA2
SA9
SA5
SA9
CBA
CBA

(35)
0
0
0
0
0
0
0

I .3E-5
?SE-6
32E-6
J.-K5
I .3E-5
5.6E-6
3.SE-6
3.5E-6

I .&E-5
I .1E-5
5.3E-5
1.4E-5
1.5E-5
5.6E-4
8.7E-5
8.7E-5

Dori-Vassiliadeh et al.. 19X5

Sirianni et 31.. 1981
Qin and Hung, 1985
Baird and Birnbaum. 1979
Rusk and Ahlborg. 1280
Balhinder et al.. 19X.3
Renner. I985
Renner. I YXS

I .4E-5

I987
1337
1987
I987

Qin et al..

IS35

HuangL et al. , 1982

Qin et al., 1985
Renncr. I985

A positive value is the percentage inhibition of the effect induced by the mutagen in the short-turn teht; a negative value ( ) i.\ the percent
enhancement of the effect: and a value of zero indicates no significant effect observed. A value of zero i\ not a precise caluc and in general
the true value is less than 20% inhibition or enhancement.

data in this report. For the three antimutagens.

the
profiles were arranged according to the chemical
classes of the mutagens tested (Fig. 2. Figs. 4 and 5).
For the mutagen plot (showing two mutagens), the
profiles were organized to exhibit contrasting interactions with the antimutagens
under consideration
(Fig. 3).

3. Results
3. I. At~titturtuget~icity pmfile ,for- retinal (ROIL
Retinoids have a well-established
role in the control of epithelial cell differentiation
(Roberts and

Sporn, 1984; Lowe. 1986: Darwiche et al., 1993:

Denning and Verma, 1994). A substantial body of
evidence has been produced indicating that retinoids
affect processes related to tumor promotion (Spom
and Roberts, 1983; Verma, 1987; De-Luca et al..
1989; Grubbs et al., 1990: Athar et al., 1991).
Retinoids also may influence the initiation step in
carcinogenesis
(McCormick et al., 1980. 1981; Zile
et al.. 1986: Decoudu et al.. 1992). It is in the
context of initiation that short-term tests for antimutagenicity have been most useful, and most of the
data presented here will relate to initiation.
We reported previously (Brockman et al., 1992)
that IS retinoids had been tested for antimutagenicity
and that ROL had been studied in combination with

M.D. Wmers rt d/Mutation

28 mutagens. The results for ROL antimutagenicity

were divided into three groups: (I ) chemicals whose
activity was inhibited: (2) chemicals whose activity
was not affected, and (3) chemicals whose activity
showed mixed results. To facilitate the presentation
of this information in a concise manner, the entire
antimutagenicity
profile for ROL has been reproduced in Fig. 2 and the data for this figure are
presented in Table 1. The antimutagenicity
profile in
Fig. 3 gives data for three retinoids (retinol, retinoic
acid, and retinol acetate) evaluated in common tests
with AFB, and B[ a]P. The corresponding
data are
listed in Table 2. Only selected portions of the ROL
data are described below. and for more details refer
to Brockman et al. (1992).
ROL inhibited the mutagenicity
of AFB, in S.
~phimurhm
in the presence of S9 activation (Busk
and Ahlborg. 1980; Qin and Huang. 1985; Bhattacharya et al.. 1987; Whong et al., 1988). It also

Table 2
Antimutagenic
Antimutagen

Aflatoxin B ,
Retinol

Retinoic acid

Retinol acetate
BenzoIalpyrene
Retinol

Retinoic acid
Retinol acetate

113

Resrurch 350 (IYYhi IOY-1ZY

inhibited the induction of sister-chromatid exchanges

(SCEs) and chromosomal
aberrations
in Chinese
hamster cells (Huang et al., 1982; Qin et al., 1985)
and covalent binding of AFB, to calf thymus DNA
(Bhattacharya et al.. 1984) in the presence of S9. In
addition, Fig. 3 and Table 2 show that retinoic acid
and retinol acetate inhibited the mutagenicity of AFB,
in vitro in the presence of S9. Such inhibitory effects
could result from reduced metabolic activation of
AFB, or from trapping of the AFB , epoxide.
ROL gave mixed results with the PAHs. Calle
and Sullivan ( I982), using
,&naphthoflavone-induced rat liver S9. demonstrated that ROL inhibited
the mutagenicity of B[cr]P in TA98. But when Aroclor- 1254-induced S9 was used. ROL did not inhibit
B[ a]P mutagenicity
(Qin and Huang, 1985) or the
induction of SCEs or chromosomal
aberrations in
Chinese hamster cells in vitro (Qin et al., 1985).
These different effects of ROL on the mutagenicity

effects of three retinoids

Mutagen dose
(M or mmol/kgl

Test
code

8 Inhibit.
(% enhanc.)

Citation

Retinoid
(M or mmol/kg)
Low

High

5.7E-8
6.4E-8
6.OE-7
6.4E-7
6.4E-7
I .6E-6
6.4E-7
S.7E-8
2.48-7
6.OE-7
6.OE-7
I .OE-4

SA9
SA9
SA9
SIC
SIC
SIC
CIC
SA9
SA9
SA9
SA9
SVA

99
71
89
33
38
33
56
54
70
64
83
46

9.3E-6
2.w6
3.2E-6
I .7E-6
I .4E-5
2.8E-5
5.OE-5
9.3E-6
7.OE-IO
3.2E-6
8.OE-6
I .3E-4

3.1E-4
2.8E-5
I .OE-3
6.9E-6
2.2E-4
l.lE-4
I .OE-4
3. IE-4
2.OE-6
I .OE-4
I .3E-1
I .OE-3

Whong et al., 1988

Busk and Ahlborg, I980
Qin and Huang. 1985
Qin et al., 1985
Huang et al., 1982
Huang et al., 1982
Qin et al.. 1985
Whong et al., 1988
Raina and Gurtoo. I985
Qin and Huang. 1985
Qin and Huang. 1985
Qin and Huang, 1985

2.8E-6
7.3E-6
S.OE-6
4.OE-4
7.38-6
7.3E-6
1.6E-3

SA9
SA9
SIC
CIC
SA9
SA9
SVA

91
0
0
0
0
0
0

28E-5
3.E-6
7.OE-6
I .4E-5
3.2E-6
8.OE-6
I .3E-3

1.4E-4
I .OE-4
I .OE-4
I .OE-4
I .OE-4

Calle and Sullivan, 1982

Qin and Huang. 1985
Qin et al., 1985
Qin et al.. 1985
Qin and Huang, I985
Qin and Huang. 1985
Qin and Huang, 1985

1.3E-4
I .OE-3

A positive value is the percentage inhibition of the effect induced by the mutagen in the short-term test: a negative value ( 1 is the percent
enhancement of the effect; and a value of zero indicates no significant effect observed. A value of zero is not a precise value and in general
the true value is less than 20% inhibition or enhancement.

114
Table 3
Antimutagenic

effects of chlorophyllin

Mutagen

Mutagen dose
(M or mmol/kg)

Tebt
code

% Inhibit. ,
(% enhanc.)

High
Atlatoxin B ,
1.3E-7
AFB,
AFB,
5.78-7
AFB,
6.78-6
4.8E-6
AFB,
2.OE-8
AFB,
AFB, -&$epoxide
3.OE-7
Polycyclic aromatic hydrocarbons
3.6E-7
B[u]P
I .SE-6
B[a]P
1.OE-6
B[ CLIP
5.2E-6
B[a]P
3.68-7
B[cr]P

B[ulP

1.1E-5

B[a]P
B[ n]P
BPDE
BPDE
B[(i]P-7,X-dial
2-AF
3-AAF
MCA
MCA
?-AA
Heterocyclic amines

l.lE-5
1.0%6
3.3E-7
S.OE-7
3.5E-6
I .2E-5
3.8E-5
3.38-5
3.38-S
I .1E-5

IQ
IQ
IQ
IQ
IQ

1.9E-8
2.3E-8
6.5E-6
5.OE-5
2.5E--l
6.78-7
I .4E-7
2.3E-7
3.7E-8
3.8E-8
6.1 E-8
I .7E-6
1.5E-4
25E-4
2.5E-4
7.7E-8

Trp-P- I
Trp-P- I
Trp-P- I
Trp-P-2
Trp-P-2
Trp-P-2
Trp-P-7
Trp-P-2
Trp-P-2
Trp-P-2
Glu-P- 1
Glu-P- I
GIU-P-l
Glu-P-2
Gh-P-2
Glu-P-2
MeIQ
MeIQ

1.5E-7
6.3E-7
3.1E-6
3.8E-6
3.3E-5
1.3E-9
6. I E-9

Citation

CHL
(M or mmol/kg)

SAO
SA9
SAF
SAO
BVD
SA9

100
100
95
73
70
91

I .2E-6
3.OE-5
8.3E-4
l.lE-4
S.OE-4
I .3E-1

3.8~.4
3.0~.3
3.3E-3
3.8E-3
?.OE-3
4.2E-4

Arimoto et al., 1993

Whong et al., 1988
Warner et al., 1991
Dashwood et al., 1991
Dashwood et al.. 1991
Dashwood et al., 199 I

SA9
SA9
SA9
SA9
SAO
SAO
SAF
G90
SAO
G9H
G9H
SA9
SA9
SAO
SAO
SAF

100
67
95
90
100
93
95
15
99
100
85
0
60
85

8.9E-5
3.6E-5
I .5E-4
5.2E-5
X.98-5
6.4E-3
8.3E-4
I .SE-6
l.lE-4
I .OE-6
5.OE-5
I .ZE-6
I .ZE-6
5.78-3
6.4E-3
I .lE-3

5.1E-3
9.lE-5
5.OE-4
2.6E-4
l.lE-3
I .9E-3
I ..iE-2
I .SE-5
I .3E-3
2.OE-5
I .SE-3
1.E-4
I .2E-4
Z.lE-3

Romert et al.. 1992

Elias et al.. 1990
Arimoto et al.. 1980a
Terwel and van der Hoeven.
Romert et al.. 1991
Lai, 1979
Warner et al.. 1991
Katoh et al.. 1983
Romert et al.. 1992
Romert et al.. 1997
Romert et al.. 1992
Arimoto et al.. 1993
Arimoto et al.. 1903
Lai et al.. 1980
Lai. 1979
Warner et al.. 1991

SA9
SAY
SA9
BID
BVD
SA9
SA9
SA9
SA9
SA9
SA9
SA9
DMM
DMM
DMM
SA9
SA9
SA9
SA9
SA9
SAY
SA9
SA9

96
100
91
80
58
95
95
100
95
100
92
98
81
48
100
100
97
95
95
100
86

3,s5
3.8E-7
3.9E-3
I .5E-4
3.OE-3
3.7E-5
1.9E-5
I .1E-6
7.3E-5
3.8E-7
I .9E-5
3.9E-4
3.OE-3
1.OE-3
1.OE-3
3.8E-7
3.8E-S
3.7E-5
5.5E-5
I .2E-6
I .9E-5
I .7E-6
I .9E-5

3.XE-4
I .ZE-4
3.9E-4
I .5E-4
3.OE-4
7.1E-5
I .9E-4
3.8E-4
I.IE-4

100
100

100
87

1.9E-2
6.7E-3

1.3E-1
1.9E-4
3.9E-3
8.OE-3
8.OE-3
8.OE-3

1.2E-4
3.8E-4
I.lE-4
I.lE-4
3.8E-4
1.9E-4
3.8E-4
I .9E-1

Dashwood and Guo. lYY3

Arimoto et al.. 1993
Dashwood et al., 199 I
Dashwood and Guo. 1992
Daahwood. 1992
Arimoto et al.. 1980b
Dabhwood and Guo. 1993
Arimoto et al.. 1993
Arimoto et al., 1980b
Arimoto et al., 1993
Dashwood and Guo, 1993
Dashwood et al.. 1991
Negishi et al.. 1989
Negishi et al.. 1989
Negishi et al.. 1989
Arimoto et al., 1993
Ddshwood and Guo. 1993
Arimoto et al.. 19XOb
Arimoto et al., I980b
Arimoto et al., 1993
Dashwood and Guo. 1993
Arimoto et al., 1993
Dashwood and Guo. 1993

I985

M.D.

Waters et al. /Mutation

115

Research 350 (1996) 109-129

Table 3 (continued)
Mutagen

Heterocyclic amines
MeIQx
MeIQx
AaC
Me AaC
Activated heterocyclic

IQ

Mutagen dose
(M or mmol/kg)

Test
code

0 Inhibit.
(8 enhanc.)

CHL
(M or mmol/kg)
Low

High

1.2E-6

Citation

3.8E-9
5.4E-8
3.OE-5
9.3E-5
amines

SA9
SA9
SA9
SA9

100
100
95
95

3.8E-5
5.5E-5
7.4E-5

3.8E-4
3.8E-4
l.lE-4
I .XE-4

Arimoto et al..
Dashwood and
Arimoto et al..
Arimoto et al.,

7.4E-7

SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9
SA9

95
95
95
95
95
95
95
95
100

3.7E-6
7.4E-7
l.lE-5
3.7E-7
7.4E-5
3.3E-5
9.3E-5
3.7E-6
3.7E-7

3.7E-6
7.4E-7
l.lE-5
3.7E-7
7.4E-5
3.3E-5
9.3E-5
3.7E-6
3.lE-5

Arimoto
Arimoto
Arimoto
Arimoto
Arimoto
Arimoto
Arimoto
Arimoto
Negishi

and Hayatsu.
and Hayatsu.
and Hayatsu.
and Hayatsu.
and Hayatsu,
and Hayatsu.
and Hayatsu.
and Hayatsu,
et al.. 1989

SAO
SAO
SAO
SAO
G9H
G9H

(235)
100
(771)
100
(30)
50

5.48-5
2.78-4
5.4E-5
l.lE-3
4.OE-5
I .5E-4

l.lE-4
2.lE-3
5.4E-5
3.2E-3
4.OE-5
3.OE-4

Romert
Romert
Romert
Romert
Romert
Romert

et
et
et
et
et
et

SAO
SA9
HMM
HMM

77
85
90
0

7.3E-5
7.E-5
6.OE-5
6.OE-5

7.?E-5
7.2E-5
6.OE-5
6.OE-5

Kimm
Kimm
Barale
Barale

SAO
SAO
SAF
SAO
SAO
G9H
G9H

73
68
96
70
0
49
0

7.2E-5
7.2E-5
8.3E-4
7.2E-5
l.lE-4
2.OE-5
I .OE-3

7.2E-5
I .4E-4
1.3E-2
7.2E-5
6.58-3
I .OE-4
?.OE-2

Kimm et al.. 1982

Kimm and Park. 1982
Warner et al.. 1991
Kimm et al.. 1982
Romert et al., 1992
Romert et al.. 1992
Romert et al.. 1992

SAO
SAS
SAS
SAS
SCG
SCR
SCG
SCR
ECK
ECK
SA9
SA9

100
100
IO0
100
73
71
92
95
100
100
61
86

I .2E-6
I .2E-6
I .2E-6
I .2E-6
5.OE-3
5.OE-3
5.OE-3
5.OE-3
4.OE-4
4.OE-4
7.8E-5
I .9E-5

I .2E-4
3.88-4
3.8E-5
3.86-4
3.OE-2
3.OE-2
2.OE-2
2.OE-2
S.OE-4
4.OE-4
3.IE-4
3.OE-4

Arimoto et al., 1993

Arimoto et al.. 1993
Arimoto et al.. 1993
Arimoto et al., 1993
Bronzetti et al.. 1990
Bronzetti et al., 1990
Bronzetti et al.. 1990
Bronretti et al.. 1990
Clarke and Shankel. 1989
Clarke and Shankel, 1989
Gentile and Gentile. 1991
Gentile and Gentile, I99 I

MeIQ
5.6E-8
I .5E-7
MeIQx
Trp-P- I
I .8E-7
3.7E-7
Glu-P- I
Glu-P-2
I .8E-6
7.-IE-5
AaC
I .5E-4
MeAaC
TV-P-~ (NHOH)
I .8E-8
Nitrosamines
I .8E-3
NNK
NNK
I .8E-3
NNN
I .4E-3
NNN
I .4E-3
I .OE-2
DMN
DMN
I .OE-2
Nitrosated agents ( +NaNO, 1
?.OE-3
Methylguanadine
Methylguanadine
2.OE-3
Methylurea
2.7E-4
,.2E-5
Aminopyrene
Alkylating agents
2-K5
MNNG
2.3E-5
MNNG
2.lE-6
MNNG
MNU
3.5E-4
I .7E-4
MNU
4.OE-4
MNU
EMS
5.OE-3
Miscellaneous compounds
4-NQO
3.8E-7
ICR- I70
3.8E-8
Quinacrine
I .7E-4
Aminoacridine
I .5E-4
Ethidium Br (EtBr)
5.OE-4
Ethidium Br (EtBr)
5.OE-4
Styrene Ox.
XOE-3
Styrene Ox.
2.OE-3
Caffeine
.6E-3
Nitro-furazone
5.OE-6
7.4E-6
NOP
NOP
7.4E-6

et
et
et
et

al.,
al..
al..
al.,
al..
al..

al.,
al.,
al..
al.,

1993
Guo, 1993
l980b
l980b
1989
1989
1989
1989
1989
1989
1989
1989

1992
1992
1992
1992
1992
1992
1982
1982
1983
1983

Mutagen dose
(M or mmol/kg)

Miscellaneous compounds
NOP
HgCl 2
Thio-tepa
Chromium (VI) oxide
CSCI
Chlordane

7.4E-6
I. I E-5
6.9E-5
2.OE-4
7.4E-4
2.4s5

Trbt
code

SAY
CBA
CBA
CBA
CBA
CBA

% Inhibit. (
(U enhanc.)

93
87
85
93
46
0

CHL
(M or mmol/kg)
LoM

Hich

5.OE-5
3.OE-6
I .OE-3
3.OE-6
3.OE-6
3.OE-6

3.6E-4
3.OE-6
5.OE-4
3.OE-6
3.OE-6
6.OE-6

Citation

Gentile
Gho.\h
Rrnner.
Sarkar
Ghoah
Sarkar

and Gentile, IY91

et al.. I99 I a
1990
et al.. 1993
et al.. 199lb
et al.. IY93

A positive value is the percentage inhibition of the effect induced by the mutagen in the short-term test: a negative \aluc t ) i\ the percent
enhancement of the effect; and a value of zero indicates no significant effect observed. A value of zero ih not B precise value and in general
the true value is less than 20% inhibition or enhancement.

of B[rr]P. may be due to differences in the relative

levels of the cytochrome P450 isoforms induced by
P-naphthoflavone
and Aroclor-1254 as will be discussed later.
Baird and Bimbaum (1979) reported that ROL
inhibited the mutagenicity of 2-AF in TA98 in the
presence of suboptimal S9 concentrations.
Busk and
Ahlborg ( 1982al subsequently showed that ROL inhibited the mutagenicity
of 2-AF in TA98 in a
dose-related manner but that at superoptimal S9 concentrations ROL enhanced the mutagenicity
of this
compound in the same system, except at the highest
ROL dose tested. Balbinder et al. (1983) made similar observations. showing that high S9 concentrations
enhanced mutagenicity
while lower levels did not.
Busk and Ahlborg (1982a) also showed that ROL
enhanced the mutagenicity of 2-AAF in TA98 in the
presence of high S9 concentrations.
This study is
inconclusive,
however. because the mutagen was
tested at a marginally active dose in the presence of
ROL. thereby precluding the possibility of observing
an inhibitory effect.
Inhibitory effects of ROL were obtained in 4 of 6
studies with DMBA and in 2 of 3 studies with MCA.
whereas the mutagenicity
of BA was not affected.
Additional testing is necessary to determine whether
these mixed results represent real differences among
the short-term tests used and/or the concentration
ratios of ROL to mutagen.
The mutagenicity
of 7 HAS formed during the
cooking of meat (TrpP- 1, Trp-P-2. Cm-P- 1. Glu-P-2,
IQ. MeIQ and MeIQxl were inhibited by ROL in
TA98 in the presence of S9 (Busk et al.. 1982:

Ioannides et al.. 1990). Using IQ. Ioannides et al.

(1990) excluded the possibilities that ROL (1) scavenged microsome-generated
reactive intermediates,
because no dose-dependent
effects were seen when
ROL was added to the incubation
mixture after
completion of microsomal metabolism, and (2) interacted with bacterial DNA, thereby protecting it from
genotoxic metabolites, because no difference in the
mutagenic response was evident when the bacteria
were exposed to ROL prior to the mutagenicity
assay. They concluded that ROL is a non-selective in
vitro inhibitor of the hepatic cytochrome P450-dependent mixed function oxidase (MFO) system.
ROL had no effect on the induction of SCEs in
Chinese hamster V79 cells by MNNG in the absence
of S9, but it enhanced SCE induction by MNNG in
the presence of S9 (Qin et al.. 19851. It was ineffective in inhibiting the genotoxicity of EMS in vitro.
and of MMS and seven other direct-acting mutagens,
representing a variety of chemical classes, in vitro
and in vivo.

CHL, which has a porphyrin structure, is a manmade salt of chlorophyll containing a chelated metal
ion. either sodium or copper. CHL is antimutagenic
to a wide variety of individual mutagens. including
AFB,. PAHs. HAS, direct-acting
compounds,
and
complex mixtures. e.g., cigarette smoke condensate.
The antimutagenicity
profile for CHL is shown in
Fig. 4, and the corresponding
data are presented in
Table 3. CHL is a more effective antimutagen than

M.D. Waters et (11./ Mutatim

Table 4
Antimutapenic
Mutagen

effects of N-acetylcyseine
Mutagen dose
(M or mmol/kg)

Atlatoxin B,
4.7E-7
AFB,
I .6E-6
AFB,
I .6E-6
AFB,
2.OE-6
AFB,
I .hE-6
AFB,
I .6E-6
AFB,
4.7E-7
AFB,
2.OE-6
AFB,
Polycyclic aromatic hydrocarbons
3.OE-6
B[ tr]P
3.OE-6
B[o]P
-l.OE-6
B[ tr]P
4.OE-6
B[ LI]P
9.96-5
B[ tr]P
9.9E-5
B[ u]P
I .3E-5
?-AF
I .4E-5
2.AF
I .3E-5
?-AF
9.OE-5
?-AAF
Heterocyclic amine
I .3E-7
Trp-P-2
I .3E-7
TipP-7
Cyclophosphamide
3.8E-3
CPA
1.78-3
CPA
3.8E-3
CPA
CPA
7.7E-3
Alkylating agents
5.OE-6
MNNG
6.OE-6
MNNG
MNNG
3.OE-6
6.OE-6
MNNG
I .SE-5
MNNG
MNNG
5.OE-6
5.OE-6
MNNG
4.OE-J
MNU
Miscellaneous compounds
ZOE-5
Adriamycin
I .3E-2
Cig. Smoke
1.3E-2
Cig. Smohe
I .OE-6
4NQ0
7.6E-6
JNQO
2.7E-3
ECH
7.4E-3
H,O,
Na Dichrom.
5.7E-5

117

Resrarclr 350 C/Y961 IOY-129

(NAC)
Test
code

Inhibit.
(7r enhanc.)

Citation

NAC doses
(M or mmol/hgl
Low

High

SAO
SAO
SAO
SAO
SAO
SAO
SA9
BID

60
70
86
100
(2751
(65)
53
0

3.78-5
3.IE-2
3.1E-3
3.OE-2
3.8E-3
7.7E-3
3.7E-5
I .OE-J

I .9E-3
3.lE-2
3.lE-3
3.OE-2
l.5E-7
7.7E-3
I .9E-4
5.OE-4

Shetty et al., 1989

De Flora et al.. 1985
De Flora et al.. l984a
De Flora et al., l983a
De Flora et al., l984a
De Flora et al., 1985
Shetty et al.. 1989
Bhattacharya et al.. 1984

SA8
SAO
SAO
SAO
MVR
BVD
SA9
SA9
SA9
BVD

(10)
83
100
(33)
7
100
80
96
(23)
76

2.2E-4
3.1E-2
3.OE-2
I .9E-3
6.1 E-3
6.1E-3
3.IE-2
3. IE-2
7.7E-3
1.5E-4

l.lE-3
3. IE-3
3.OE- I
I .5E-2
6. I E-3
6. IE-3
3.1 E-3
3. IE-2
7.7E-3
3.5E-4

Wilpart et al., 1985

De Flora et al., l984a
De Flora et al., 1984,
De Flora et al., 1984a
De Flora et al., I99 I a
De Flora et al.. 199la
De Flora et al., 1985
De Flora et al.. 1984a
De Flora et al., 1985
Izzotti et al., 1994

SA9
SA9

21
(125)

3.1E-2
3.8E-3

3.lE-2
I .5E-2

De Flora et al.. 1984a

De Flora et al., l984a

SA5
SA5
SAS
SA5

71
80
(331
(60)

3.IE-2
I .9E-3
3.8E-3
7.7E-3

3.lE-2
3. IE-2
I .5E-2
7.7E-3

De
De
De
De

SAO
SAO
SA3
SA3
SA3
G9H
G9H
G9H

75
(83)
90
58
60
90
(160)
0

I .6E-5
5.OE-3
1.5E-4
2.5E-4
2.5E-4
I .OE--l
I .OE-4
I .OE-2

I .3E-4
5.OE-3
7.5E-3
5.0~.4
5.OE-4
1.OE-3
1.OE-3
1.OE-2

Camoirano et al.. 1988

Camoirano et al., 1988
Wilpart et al., 1985q
Wilpart et al.. 1985
Wilpart et al., 1985
Romert and Jenssen. 1987
Romert and Jenssen. I987
Romert and Jenssen. 1987

SA9
MVR
BVD
SAO
SAO
SA5
SA2
SA2

86
85
84
100
94
50
50
82

I .OE-2
I .2E-2
I .2E-2
I .7E-4
-l.XE-4
-1.8E-4
.i.SE-3
2.4E-4

1.OE-2
l.ZE-3
I .2E-2
1.7E-3
3.lE-2
3.1E-2
3.1 E-2
3.1E-2

De Flora
Balansky
Izzotti et
De Flora
De Flora
De Flora
De Flora
De Flora

Flora
Flora
Flora
Flora

et
et
et
et

al..
al.,
al..
al.,

l984a
I985
I984a
1985

et al.. I99 I a
et al., 1992
al.. 1992
et al.. 1994
et al.. l984a
et al., 1984a
et al.. I984a
et al., 1984a

A positive value is the percentage inhibition of the effect induced by the mutagen in the short-term test: a negative value ( ) is the percent
enhancement of the effect: and a value of zero indicates no significant effect observed. A value of zero is not a precise value and in general
the true value is less than 20% inhibition or enhancement.

Log Dose
(M or mmollkg)
-1

-31

IL

-51 -

-7 LA
lnhibiion

100

Maximum % inhibimn
of mutagenic aawiiy

53

No significant change in activity

0
Maximum % enhancement
of mutagenic aaiiiiy

(50)
(% Enhance.)

Test system code word

Antimutagens
(or Mutagens1

Fig. I. Schematic diagram of an antimuta,oenicity profile. Profiles

are organized

to di>play

either

the antimutqenic

acricity

ot

various antimutagens in combination with a single mutagen or the

activity of a single antimutagen with various mutagen>. The upper
bar graph display\

the mutagen concentration

antimutagen concentration
the maximum

and the range of

tested, The lower graph show\ either

percent inhibition

represented by a bar directed

upward from the origin or the maximum percent enhancement of

the genotoxic response represented by a downward-directed
As illustrated in the lower craph, ;Lbar acres

bar.

the origin indicates

that no significant effect was detected (designated a

negative

data in the text). Test code\ are defined in the Appendix.

ROL in vitro (Ong et al., 1989): it consistently

inhibited the mutagenic activity of AFB, and its
epoxide. the PAHs, and the HAS in the Ames
SalmoneIIa/microsome
assay. Furthermore, the antimutagenic capacity of CHL in vitro has been confirmed in vivo: CHL inhibited the covalent binding
of AFB, to trout liver DNA (Dashwood et al.. 1991)
and of IQ to rat liver DNA (Dashwood.
1992). It
also reduced the clastogenic effects of cesium and
methyl mercury chloride in mice (Ghosh et al., 199 1a.
b) and thio-tepa in Chinese hamsters (Renner, 1990).
In contrast to ROL (and retinoids in general).
several studies (Lai, 1979; Arimoto et al., 1980a:
Terwel and van der Hoeven. 1985: Elias et al.. 1990:
Warner et al.. 1991; Romert et al., 1992) showed that
CHL was highly effective in inhibiting the mutagenicity of B[N]P. This antimutagenicity
was also
found with BPDE in the Salmonella/microsome
assay. and with B[a]P-7,X-diol and BPDE in the V79
assay (Romert et al.. 1992). indicating that the mechanism underlying this effect does not involve the
metabolizing
system but rather arises from the for-

Log Dose
(Mor mmot/kg)

Retinal Dose Range

Mutagen Dose

Promutagens
I

i
AFBl

b.

Fig. 2. Antimutqenicity

I
tI

PolycycliiAromatic
Hydrocarbons

I Hetfw3- / N_
I cydic
I_
j Amines
i

CPA

profile of retinal. Hatched bars represent in vitro test> and solid bars represent in viva tehts.

mation of a chemical complex . CHL also inhibited

the activity of 2-AA, MCA. and 2-AAF but had no
effect on 2-AF in Salmonella (Lai, 1979; Lai et al..
1980; Warner et al., 199 I ; Arimoto et al., 1993).
The mutagenic activity of the HAS (AaC. MeAaC.
Glu-P-l,
Gh-P-2,
IQ, Tip-P-l,
and Tip-P-21 was
inhibited by CHL in the Salmonella/microsome
assay with TA98 in the presence of S9 (Arimoto et al.,
I980a. 1993; Dashwood et al.. 199 1; Dashwood and
Guo, 1993). CHL also inhibited the activity of some
activated HAS [AaC. MeAaC, Glu-P- 1, Glu-P-2. IQ,
MeIQ. MeIQx, TrpP- I, and Trp-P-2(NHOH)]. These
activated forms were obtained by incubating the HAS
alone with S9 and extracting the activated products
with acetone (Arimoto and Hayatsu, 1989). These
results further indicate that CHL inhibits the mutagenicity of promutagens through complex formation.
CHL also inhibited
somatic
cell mutations
in
Drosophila (Negishi et al.. 1989) and covalent binding of IQ to calf thymus DNA in vitro (Dashwood
and Guo, 1992).
Romert et al. (1992) reported that CHL inhibited
the mutagenicity of three nitrosamines (NNN, NNK

Log Dose
(M or mmol/kg)

Aflatoxin Bl

% Inhib.

Fig. 3. Antimutagenic

and DMN) at high molar excess but enhanced the

mutagenicities at lower relative concentrations. Interestingly, biliverdin, which also has a porphyrin structure but without the central metal ion, was unable to
potentiate
the mutagenicity
of NNK
in the
Salmonella/microsome
assay. These results, therefore, indicate that the chelated metal ion is necessary
for the enhancing effect and suggest that low doses
of CHL may facilitate electron transport due to its
redox cycling capacity. This suggestion is supported
by the finding that glucose 6-phosphate can be replaced by CHL in the NADPH-generating
system.
CHL inhibited the activity of MNNG (Kimm and
Park, 1982: Kimm et al.. 1982; Warner et al., 1991)
and
gave
mixed
results
for MNU
in the
Salmonella/microsome
assay. Kimm et al. (1982)
reported that CHL inhibited the activity of MNU in
TAIOO, whereas Romert et al. (1992) found no
effect. CHL weakly inhibited MNU activity but had
no effect on EMS in the V79 system (Romert et al.,
1992). These results indicate that complex formation
may not occur with monofunctional
alkylating agents.
Rather. CHL, like the aminothiols to be discussed

effect of three retinoids against aflatoxin B, and benzo[a]pyrene.

represent in viva tests.

Antimutagen Dose Range

Mutagen Dose

Benzo[a]pyrene

Hatched bars represent in vitro tests and solid bars

later, may enhance the formation of reactive species

from compounds like MNNG and MNU outside the
cell where they would immediately react with water
and not reach the target DNA (Romert et al.. 1992).
3.3. At~titnutaget~icit~ profile ,for NAC
Aminothiols,
both natural [e.g., reduced glutathione (GSH)] and synthetic [e.g.. N-acetylcysteine
(NAC)]. are known to antagonize a broad range of
mutagens and carcinogens (De Flora et al.. 1989).
The antimutagenicity
profile
of NAC is shown in
Fig. 5. and the corresponding
data are presented in
Table 4.
In the Ames Salmonella/microsome
assay. NAC.
tested in varying amounts, enhanced the mutagenicity of several promutagens (i.e., AFB, . B[ NIP), 2-AF.
TrpP-2,
and CPA) at the intermediate
doses in

TA98. TA 100. TAlS35 and TAl5Y8 in the presence

of S9 from Aroclor-pretreated
rats (De Flora et al..
1984a. 1985: Wilpart et al.. 1985). This effect requires the presence of the thiol during metabolic
activation in a preincubation
step. However, at a
higher sublethal dose, NAC almost completely inhibited the mutagenicity of these same compounds. This
inhibitory effect was also observed by adding NAC
after the metabolic activation of promutagens
had
been achieved (De Flora et al., 1984a).
Studies on AFB,. 2-AF and CPA in the presence
of liver preparations from ratx, either untreated or
induced with phenobarbital,
3-methylcholanthrene.
or Aroclor 1254, confirmed the inhibitory properties
of high doses of NAC. whereas the enhancement of
mutagenicity at intermediate doses occurred mainly
in the presence of suitably induced liver preparations
(De Flora et al.. 1985). Similar effects have been

Log Dose
(M or mmol/kg)

CHL Dose Range

Mutagen Dose

Heterocyclic Amines

% Inhib.
100

(50)
(% Enh.)

FiCT
C 1. Antimutagenicity

profile of chlorophyllin. Hatched harh represent in 1 itro test\ and solid bars rrprccnt

in viko trots

M.D.

Waters et al. /Mututim

reported for GSH with AFB, (Booth et al., 1981).

PAHs (Glatt and Oesch. 1977; Malaveille
et al.,
1980). and Trp-P-2 (De Waziers and Decloitre. 1984).
These results suggest that low doses of aminothiols
potentiate activity of promutagens in vitro by affecting metabolic activation. whereas high doses inhibit
mutagenicity by binding electrophilic metabolites.
NAC inhibited the activity of MNNG (Wilpart et
al., 1985; Camoirano et al., 1988) but had no effect
on MNU in the Chinese hamster V-79 assay (Romert
and Jenssen. 1987). Also. it has been shown to
inhibit the mutagenicity
of nine other direct acting
compounds
in the Salmonella
reversion test (De
Flora et al., 1984a, 1987, 1991a. 1994).
The site of reaction appears to be a critical factor
affecting the activity of NAC against alkylating
agents. Thus. MNNG mutagenicity
was eliminated
when the reaction with GSH or NAC occurred outside the cells. However, MNNG mutagenicity in S.
(vphinnfri~lrn (Camoirano
et al.. 1988) and DNAdamaging activity in E. coli (De Flora et al., 1989)
were enhanced when the intracellular thiol concen-

trations were increased by pretreating bacteria with

either NAC or GSH. The opposite effect was observed by pretreating bacteria with dimethyl maleate
which depletes GSH (Camoirano et al.. 1988). Similar results were obtained in the Chinese hamster
V-79 mutation assay (Romert and Jenssen, 1987).
Cells pre-treated with NAC or GSH gave an increased response in mutagenic activity when the
thiol was removed prior to treatment with MNNG
but showed decreased activity when treatment with
the thiol continued throughout the incubation period.
These findings are consistent with the hypothesis
that MNNG may react with thiols to form a carbon
ion, which inside the cells could methylate critical
molecules (such as DNA) but outside the cells would
react with water to form methanol (Margison and
OConnor, 1979).
In vivo, NAC inhibited the induction of micronuclei in pulmonary alveolar macrophages of rats exposed to B[a]P by intratracheal instillation on three
consecutive days (De Flora et al., 1991 b) or cigarette
smoke for l-40 days (Balansky et al.. 1992) 5 h

Log Dose
(M or mmol/kg)

1.

-3

I
-5

r - -----

-7 -

, I :
!
_ iI ---j

NAC Dose Range

Mutagen Dose

1
j1

I..

--

121

Reseurch 350 (19961 109-129

---

II

I/
I --

Promutagens
I

% Inhib.

Fig. 5. Antimutagenicity

AFBl

PAHS

profile of N-acetylcyateine.

TW
P-2

Hatched bars represent in vitro tests and solid bars represent in viva tests.

Retinal*
CHL
NAC

t = Inhibition
- = No Effect
4 = Enhancement

Includes retinal acetate for AFBI and B[a]P

Fig. 6. Comparative summary of in vitro effects of retinol. chlorophyllin and N-acetylcysteine against atlatoxin B,. benzo[cr]pyrene
and the heterocyclic amines. Circled result.\ were confirmed

in

viva

after receiving NAC by gavage. Similarly, NAC

inhibited DNA adduct formation in rats exposed to
B[ a]P (De Flora et al.. 199 1b), 2-AAF (Izzotti et al..
1994). or cigarette smoke (Izzotti et al.. 1992). In
these studies, animals were treated with NAC 5 h
prior to exposure to the test compounds and 5 h after
exposure.
Overall, while studies on the antimutagenicity
of
ROL (retinoids), CHL and NAC in vivo are very
limited. the data confirm the in vitro results summarized in Fig. 6. Enhancement
of mutagenic activity
by ROL, CHL and NAC was not observed in vivo
and may represent an artifact of in vitro testing
methodology.

4. Discussion
3. I. Retid

ROL inhibited the activity of chemicals whose

mutagenicity required activation by one or more of
the cytochrome P450 isoforms (i.e., AFB,, PAHs
and HAS), whereas it did not inhibit the activity of
direct-acting
mutagens. These and other data (e.g.,
Ioannides et al., 1990) indicate that the major mechanism for the antimutagenic
activity of ROL in in
vitro tests. and probably in some in vivo tests, is to
inhibit the activity of the cytochrome P450 isoforms.
Although Ioannides et al. ( 1990) have concluded that
ROL has poor selectivity
in inhibiting
the cytochrome P450 isoforms, the antimutagenicity
profile
of ROL (Fig. 2) and of three retinoids against AFB,

and B[ a]P (Fig. 3) argue for some degree of selectivity of retinoids for the P4501 isoforms.
ROL, retinoic acid, and retinol acetate displayed a
preponderance of negative results for antimutagenicity against B[ a]P (Fig. 3). a compound that is metabolized in vitro by cytochromes P450IA 1 and P450IIB
as described below. In contrast, these retinoids consistently inhibited the activity of AFB,. which is
metabolized
by P450IA2 and other isoforms. As
suggested previously (Brockman et al.. 1992). the
retinoids, which also are metabolized
by the cytochrome P450IA2 isoforms (Leo and Lieber. 1985:
Roberts et al., 1992). may competitively
inhibit the
P4SOIA2-mediated
activation of AFB, thereby inhibiting its mutagenic activity. This inhibitory effect
would not be expected with B[a]P because it is
activated by different isoforms. Also. the HAS were
antagonized by ROL and are known to be metabolically activated
by P450IA2
and IA1 isoforms
(Guengerich.
1988). These results indicate that competitive inhibition of cytochrome P450 activity by
ROL may also involve the IA1 isoforms. This suggestion is further substantiated
by the results presented from the studies with B[u]P and ROL.
The only instance in which the activity of B[a]P
was inhibited by ROL in vitro was when /3-naphthoflavone-induced
S9 was used in the activation
system. ROL had no effect on B[a]P activity in the
presence of Aroclor 1254-induced
S9. The mutagenicity of S9 activated B[u]P in vitro is due to the
secondary metabolite,
B[ tt]P-7,8-dial-9. IO-epoxide.
which is mediated at the sterically hindered carbon 7
and 8 positions by P450IAl (Gozukara et al.. 1982:
Jefcoate et al.. 1983). and to the primary metabolites.
B[ cr]P-4.5-oxide and &phenol.
which are oxidized
at the nonhindered
carbon 4 and 5 positions by
P4SOIIB (Parke et al., 199 1). The 7.8-dial-9, IOepoxide is at least five times as mutagenic as the
4.5oxide (Wislocki et al.. 1976b; Malaveille et al.,
1977: Wood et al., 1977) which is about four times
more mutagenic than the 6-phenol in TA98 (Wislocki
et al.. 1976a).
P-Naphthoflavone
induces
primarily
P450IA1
isoforms. whereas Aroclor 1254 uniformly induces
the cytotochrome P45Os. Therefore, in the presence
of P-naphthoflavone-induced
S9. an increase in mutagenic activity would be expected due to the increased production
of the 7,8-diol-9, IO-epoxide.

M.D. Waters rt nl./Mutntion

However. B[ a]P activity was inhibited by ROL when

P-naphthoflavone-induced
S9 was used, suggesting
that ROL may be competing with B[alP for the
P450IAl isoform. Metabolism at the 45 position by
P450IIB would be unaltered. Aroclor 1254 induced
S9, on the other hand, would elevate metabolism at
the 4.5 (P450IIB) and 7,8 (P450IAl) positions. Reduction in activity by competition of ROL and B[ a]P
for the P450IAl cytochromes could, therefore, be
counteracted by increased production of B[ a]P-4,5oxide and -6-phenol resulting from the induction of
P450IIB cytochromes. In other words, inhibition of
the mutagenicity of B[a]P by ROL could be caused
by changes in the amount of activation due to competitive inhibition or by changing the ratio of primary to secondary metabolites.
The data are convincing
that the major mechanism for the antimutagenicity
of ROL in vitro is the
inhibition of the activation of promutagens to electrophilic mutagens by the cytochrome P45Os, particularly the P4501 isoforms. The antimutagenicity
of
retinoids in vivo (and in some in vitro tests) is
undoubtedly subject to additional mechanisms, such
as stimulation
of glutathione
S-transferase activity
(McCarthy et al.. 1987). The same is true for the
mechanisms of the anticarcinogenicity
of ROL (De
Flora and Ramel. 1988; Hartman and Shankel, 1990).
As mentioned previously,
there is evidence in
vivo that the retinoids may influence the initiation
stage of chemical carcinogenesis (McCormick et al..
1980, 198 1) and also exhibit anticarcinogenic
effects
through inhibition of the promotion stage, by virtue
of a role in the differentiation
of epithelial tissues
(Sporn and Roberts, 1983). Indeed, vitamin A was
effective in preventing
chemically-induced
cancer
even when it was administered to animals following
completion of the treatment with the carcinogenic
initiator (Grubbs et al., 1977). On the other hand,
vitamin A deficiency had no major effect on the
hepatic mixed function oxidases and glutathione Stransferases, two of the most important enzyme systems in the activation and deactivation of chemical
carcinogens (Rozman et al.. 1987; Ayalogu et al.,
1988).
4.2. Chlorophyllir~
The mechanisms by which CHL and other porphyrins exert their antimutagenic
activities are not

Research 350 (19961 109-I-79

123

entirely clear at present. Scavenging of radicals (Sat0

et al.. 1984; Ong et al., 1986) or suppression of
metabolic activation (Kimm et al., 1982; Terwel and
van der Hoeven, 1985; Ong et al., 1986) have been
suggested as two possible mechanisms.
However,
Arimoto et al. (1980b), Arimoto and Hayatsu (1989).
Negishi et al. (1989), and more recently Guo et al.
(1994) have clearly shown that CHL and some other
porphyrins inactivate HAS by complex formation.
Most of the results presented here support the
hypothesis that CHL acts through complex formation
with promutagens or ultimate mutagens. The antimutagenicity of CHL with respect to B[a]P and its
metabolites can best be explained by such complex
formation.
The reduced mutagenicity
of BPDE.
which does not require metabolic activation. obviously does not involve suppression of bioactivation.
Similar
arguments
apply
for AFB,
epoxide
(Dashwood et al., 1991) and the activated HAS
(Arimoto and Hayatsu, 1989).
The potentiating
effect of CHL on the nitrosamines, however, does appear to involve interaction with bioactivating enzymes. Low doses of CHL
enhance mutagenicity,
but higher doses inhibit the
effect. Romert et al. (I 992) suggested that low doses
of CHL may interact with the electron transport
system involved in activation due to its redox cycling capacity. Results showing that CHL can replace glucose 6-phosphate in the NADPH-generating
system further support this idea. The potentiating
capacity of CHL at low concentrations also could be
overwhelmed at higher concentrations by its interaction with activating enzymes and/or by simple complex formation. Similar effects have been demonstrated for endogenous compounds that function at
low concentrations
as preoxidants to generate toxic
radicals, and at higher concentrations as antioxidants
that detoxify the radicals (Ehrenberg et al., 1989).
The ability of CHL to counteract the mutagenicity
induced by monofunctional
alkylating agents through
complex formation was not clearly demonstrated.
The mutagenicity
of EMS was not influenced by
CHL and MNU showed conflicting results. In the
case of MNNG, a methylating homolog of MNU,
CHL inhibited mutagenicity. However, Romert et al.
(1992) indicate that CHL may act similar to amines
and thiols to facilitate the activation of MNNG extracellularly so that the electrophilic
species formed

react with water and do not reach the target DNA. If

such interaction occurred intracellularly
one would
expect an enhancement
of mutagenicity.
as was
demonstrated
with NAC and MNNG (Romert and
Jenssen. 1987; Camoirano et al.. 1988; De Flora et
al., 1989).
As regards carcinogenicity.
Guo et al. (1994)
reported that CHL inhibited the carcinogenicity
of
IQ in rat liver. colon, Zymbals gland, and small
intestine, but enhanced carcinogenicity
in the skin.
The enhancement of skin tumors could be related to
CHL photoreactivity
and production of active oxygen species.
4.3. N-Ace~lcysteir~e

(NAC)

The antimutagenic activity of NAC with promutagens and direct-acting

agents has been shown to
involve multiple mechanisms both intracellularly and
extracellularly.
These mechanisms include its ability
to act as a blocking agent: a reducing agent: a
nucleophile,
trapping electrophilic
molecules;
an
oxygen scavenger: and a precursor of intracellular
glutathione (De Flora et al., 1989). The activity of
NAC as a blocking agent was demonstrated by experiments with the alkylating agent MNNG. NAC
inhibited the mutagenic activity extracellularly
by
blocking its transport into the cell. However, when
MNNG was allowed to be taken up by the cell prior
to treatment with NAC, an electrophilic product was
formed which increased
the mutagenic
activity
(Camoirano et al., 1988).
Extracellular NAC and GSH are equally effective
in eliminating
MNNG mutagenicity
in neutral and
acidic environments (Camoirano et al.. 1988). This is
an important prerequisite for inhibitors working in
the stomach (Wattenberg et al., 1987: De Flora and
Ramel. 1988). It should be noted that enteric bacteria. which have high intracellular GSH concentration
(in the millimolar range). can export GSH extracellularly. thus providing an important defense mechanism in the gut against toxic agents that, like MNNG,
are active in the micromolar
range (Owens and
Hartman. 1986).
Further evidence for the ability of NAC to act as a
blocking agent was provided by the finding that liver
and lung preparations
from rats treated intraperitoneally with NAC had an enhanced capacity to

detoxify direct-acting
mutagens (De Flora et al.,
1985). The concentrations
or the spectral properties
of total cytochromes P450 in liver or lung microsomes were unaffected by administration of NAC to
rats, either intraperitoneally
(De Flora et al.. 1985) or
in the diet (De Flora et al., 1991a). However, a
significant induction of arylhydrocarbon
hydroxylase
activity was produced by dietary NAC both in control rats and in rats receiving four consecutive cycles
of treatment with 2-AAF (De Flora et al.. 199la).
The ability of NAC to act as a reducing agent was
demonstrated with the direct acting mutagens hydrogen peroxide and sodium dichromate. Hydrogen peroxide was nonenzymatically
reduced forming a nonreactive species. NAC disulfide (Moldeus et al..
1986). The hexavalent chromium salt. which is reduced by GSH to a stable trivalent form (De Flora et
al.. 1984b). appears to be similarly affected by NAC.
NAC, like most known inhibitors of mutagenesis
or carcinogenesis
(Ramel et al., 1986). acts as a
stimulator as well as an inhibitor. depending on the
experimental
conditions.
It was demonstrated
that
preincubation
of promutagens (AfB,. B[ a]P, ?-AF,
and Trp-P-2) with intermediate doses of NAC and a
metabolic activation system increased the mutagenic
activity of these agents, while higher sublethal doses
inhibited this activity. When NAC was added following the preincubation
phase, the mutagenicity
was
inhibited regardless of the dose (De Flora et al..
1984a. b. 1985). These findings illustrate NACs
ability to act as a nucleophile trapping electrophilic
molecules formed during metabolism of promutagens. The results also suggest that thiols can modulate the biotransformation
of mutagens but do not
seem to inhibit metabolic activation or influence
microsomal pathways (De Flora et al.. 1989).
Studies by De Flora et al. (l984a) also provided
evidence that NAC stimulates cytosolic enzyme activities involved in the hexose monophosphate shunt
and in the GSH cycle. Under in vitro conditions, i.e..
by supplementing
rat liver S9 fractions with varying
amounts of NAC. GSSG reductase was the only one
of the enzymes monitored that was significantly
stimulated by NAC. suggesting an enhanced rate of
GSH regeneration (De Flora et al.. 1984a).
The detoxication of mutagens has been associated
with the occurrence of thresholds in in vivo genotoxicity and carcinogenesis
(De Flora, 1978, 1984.

M.D. Wuters et al. /Mutation

1985). Available in vitro and in vivo data suggest

that GSH is responsible
for these thresholds (De
Flora et al., 1989). Thus, NAC could play a major
role in the detoxication process by increasing GSH
levels either by direct mechanisms or as a precursor
to intracellular GSH.
With regard to anticarcinogenicity,
De Flora et al.
(1986) demonstrated that NAC decreased the induction of lung tumors in Swiss albino mice exposed to
urethane. NAC and GSH delayed the development of
y-glutamyl transpeptidase-positive
foci in the liver of
rats treated with 2-AAF and prevented 2-AAF-induced sebaceous squamocellular
carcinomas of the
Zymbals gland (Cesarone et al., 1987). NAC also
exerted protective effects in a model of rat colon
carcinogenesis (Wilpart et al., 1986).
hz conclusion, most of the studies reviewed herein
were carried out using liver microsomes from rats.
These results may provide some understanding of the
mechanisms by which dietary antimutagens and drugs
affect xenobiotic metabolism. However, caution must
be applied when extrapolating
the information obtained from hepatic tissues to nonhepatic tissues and
from animals to humans. As noted previously the in
vivo antimutagenicity
data tend to confirm the in
vitro results. Furthermore, the antimutagenic
activities have been confirmed by anticarcinogenicity
studies. With the understanding
of influences of antimutagens/anticarcinogens
on human
xenobiotic
metabolism as a goal, researchers are faced with the
following challenges:
(1) to further elucidate the
detailed mechanisms by which antimutagens
affect
xenobiotic-metabolizing
enzymes; (2) to understand
the basis for the tissue and species specificities of
xenobiotic-metabolizing
enzymes;
(3) to further
characterize the catalytic properties of human xenobiotic metabolizing
enzymes;
and (4) to pursue
well-planned
human studies concerning
the nutritional
impact
of antimutagens
on xenobiotic
metabolism and toxicity.

Appendix

A.I. Test Code Definitions

Test
BID

Definition
Binding (covalent)

to DNA in vitro

Research 350 (1996) 109-129

BVD
CBA
CIC
DMM
ECK
G9H
G90
GCO
HMM
MVR
SAO
SA2
SA3
SA5

125

Binding (covalent) to DNA, animal cells in

vivo
Chromosomal aberrations, animal bone marrow cells in vivo
Chromosomal
aberrations, Chinese hamster
cells in vitro
Drosophila melanogaster, somatic mutation
Escherichia coli K12, forward or reverse
mutation
Gene mutation, Chinese hamster V-79 cells
in vitro, HPRT
Gene mutation, Chinese hamster V-79 cells
in vitro, ouabain
Gene mutation, Chinese hamster ovary cells
in vitro
Host mediated assay. microbial cells in animal hosts
Micronucleus test, rats in vivo
Salmonella vphimurium TA 100, reverse mutation
Salmonella typhimurium TA 102, reverse mutation
Salmonella
typhimurium
TA 1530, reverse
mutation
Salmonella typhimurium TA1535, reverse

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