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

350 ( IYYO) IOY-12Y

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-

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

Research 350 (1996) 109%129

III

effects of retinal Mutagen dose (M or mmol/kgl Test code % Inhibit. (% enhanc.) Retinol (M or mmol/kg) Low High Citation

Aflatoxin B , AFB,
AFB , AFB ,

AFB, AFB, AFB, AFB,

AFB ,

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

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

SAO SA9 SA9 SA9 SIC SIC SIC CIC BID 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 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SAO

98 99 71 89 33 38 33 56 91 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 75 90 50 27 61 92 88 92

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

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 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 2.3E-4 2.4E-4 1.JE-4 I .JE-4 I .4E-4 2.4E-4 I .6E-4 2.4E-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 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 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 dose CM or mmol/kg) Teat code % Inhibit. (c/ enhanc.) Retinal (M or mmol/kg) LOW High Citation

Mutagen

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

SAO SIC SAO SIC SA5 SA5 SIC SIC SIC SIC CIC CBA SIC URP cc0 SIC CBA SHL SIC SA2 SA9 SA5 SA9 CBA CBA

61 53 9X 60 7X 11 ix 47 61 34 96 0 (X0) 0 0 0 0 (35) 0 0 0 0 0 0 0

6.7E-6

I .4E-5
6.7E-6 I .1E-5 i.ZE-6 I .OE-5 1.5E-5 2.5E-5 I .lE-6 I .4E-5 I .1E-5 3.5t-6 7.OE-6 I .OE-6 I .OE-6 7.OE-6 35E-6 I .3E-5 ?SE-6 32E-6 J.-K5 I .3E-5 5.6E-6 3.SE-6 3.5E-6

S.-IE-5 I. I E-4 5.4s5 I. I E-4 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 5.6E-5 5.OE-5 2.SE-5 5.6E-5 8.7E-5 I .&E-5 I .1E-5 5.3E-5 1.4E-5 1.5E-5 5.6E-4 8.7E-5 8.7E-5

Huang. Hung. Huang. Huang,

I987 1337 1987 I987

Qm and Hung. 1985 Busk et 31.. 1983 Cozzi et al.. 1990 Cozzi et al.. 1990
Qin et al..

IS35

HuangL et al. , 1982 Qin et al., 1985 Renncr. I985 Qin et al., 1985 Budroe et al.. 19X7 Budroe et al.. IYXX Qin et al.. I985 Renner. I YX5 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

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

Resrurch 350 (IYYhi IOY-1ZY

113

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

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

Table 2 Antimutagenic Antimutagen

effects of three retinoids Mutagen dose (M or mmol/kgl Test code 8 Inhibit. (% enhanc.) Retinoid (M or mmol/kg) Low High Citation

Aflatoxin B , Retinol

Retinoic acid

Retinol acetate BenzoIalpyrene Retinol

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

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

99 71 89 33 38 33 56 54 70 64 83 46 91 0 0 0 0 0 0

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

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

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 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

Retinoic acid Retinol acetate

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 Mutagen

effects of chlorophyllin Mutagen dose (M or mmol/kg) Tebt code % Inhibit. , (% enhanc.) CHL (M or mmol/kg) High Citation

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 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 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

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

SAO SA9 SAF SAO BVD SA9 SA9 SA9 SA9 SA9 SAO SAO SAF G90 SAO G9H G9H SA9 SA9 SAO SAO SAF SA9 SAY SA9 BID BVD SA9 SA9 SA9 SA9 SA9 SA9 SA9 DMM DMM DMM SA9 SA9 SA9 SA9 SA9 SAY SA9 SA9

100 100 95 73 70 91 100 67 95 90 100 93 95 15 99 100 85 0 60 85

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

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

I985

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

1.1E-5
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 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

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

1.9E-2
6.7E-3 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

IQ IQ IQ IQ IQ
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

1.3E-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

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

100
87

M.D.

Waters et al. /Mutation

Research 350 (1996) 109-129

115

Table 3 (continued) Mutagen Mutagen dose (M or mmol/kg) Test code 0 Inhibit. (8 enhanc.) CHL (M or mmol/kg) Low Heterocyclic amines MeIQx MeIQx AaC Me AaC Activated heterocyclic High Arimoto et al.. Dashwood and Arimoto et al.. Arimoto et al., Arimoto Arimoto Arimoto Arimoto Arimoto Arimoto Arimoto Arimoto Negishi Romert Romert Romert Romert Romert Romert Kimm Kimm Barale Barale 1993 Guo, 1993 l980b l980b 1989 1989 1989 1989 1989 1989 1989 1989 Citation

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

SA9 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SA9 SAO SAO SAO SAO G9H G9H SAO SA9 HMM HMM SAO SAO SAF SAO SAO G9H G9H SAO SAS SAS SAS SCG SCR SCG SCR ECK ECK SA9 SA9

100 100 95 95 95 95 95 95 95 95 95 95 100 (235) 100 (771) 100 (30) 50 77 85 90 0 73 68 96 70 0 49 0 100 100 IO0 100 73 71 92 95 100 100 61 86

1.2E-6
3.8E-5 5.5E-5 7.4E-5 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 5.48-5 2.78-4 5.4E-5 l.lE-3 4.OE-5 I .5E-4 7.3E-5 7.E-5 6.OE-5 6.OE-5 7.2E-5 7.2E-5 8.3E-4 7.2E-5 l.lE-4 2.OE-5 I .OE-3 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

3.8E-4 3.8E-4 l.lE-4 I .XE-4 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 l.lE-4 2.lE-3 5.4E-5 3.2E-3 4.OE-5 3.OE-4 7.?E-5 7.2E-5 6.OE-5 6.OE-5 7.2E-5 I .4E-4 1.3E-2 7.2E-5 6.58-3 I .OE-4 ?.OE-2 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

IQ

7.4E-7

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

and Hayatsu. and Hayatsu. and Hayatsu. and Hayatsu. and Hayatsu, and Hayatsu. and Hayatsu. and Hayatsu, et al.. 1989 et et et et et et et et et et al., al.. al.. al., al.. al.. 1992 1992 1992 1992 1992 1992 1982 1982 1983 1983

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

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 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

Mutagen dose (M or mmol/kg)

Trbt code

% Inhibit. ( (U enhanc.)

CHL (M or mmol/kg) LoM Hich

Citation

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

SAY CBA CBA CBA CBA CBA

93 87 85 93 46 0

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

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

Resrarclr 350 C/Y961 IOY-129

117

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

(NAC) Test code Inhibit. (7r enhanc.) NAC doses (M or mmol/hgl Low High Citation

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

SAO SAO SAO SAO SAO SAO SA9 BID SA8 SAO SAO SAO MVR BVD SA9 SA9 SA9 BVD SA9 SA9 SA5 SA5 SAS SA5 SAO SAO SA3 SA3 SA3 G9H G9H G9H SA9 MVR BVD SAO SAO SA5 SA2 SA2

60 70 86 100 (2751 (65) 53 0 (10) 83 100 (33) 7 100 80 96 (23) 76 21 (125) 71 80 (331 (60) 75 (83) 90 58 60 90 (160) 0 86 85 84 100 94 50 50 82

3.78-5 3.IE-2 3.1E-3 3.OE-2 3.8E-3 7.7E-3 3.7E-5 I .OE-J 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 3.1E-2 3.8E-3 3.IE-2 I .9E-3 3.8E-3 7.7E-3 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 .OE-2 I .2E-2 I .2E-2 I .7E-4 -l.XE-4 -1.8E-4 .i.SE-3 2.4E-4

I .9E-3 3.lE-2 3.lE-3 3.OE-2 l.5E-7 7.7E-3 I .9E-4 5.OE-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 3.lE-2 I .5E-2 3.lE-2 3. IE-2 I .5E-2 7.7E-3 I .3E-4 5.OE-3 7.5E-3 5.0~.4 5.OE-4 1.OE-3 1.OE-3 1.OE-2 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

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 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 De Flora et al.. 1984a De Flora et al., l984a De De De De Flora Flora Flora Flora et et et et al.. al., al.. al., l984a I985 I984a 1985

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

I +

-51 %
-7 LA lnhibiion 100 53 0 (50) (% Enhance.)

IL
either the antimutqenic

Maximum % inhibimn of mutagenic aawiiy No significant change in activity Maximum % enhancement of mutagenic aaiiiiy Test system code word Antimutagens (or Mutagens1

Fig. I. Schematic diagram of an antimuta,oenicity profile. Profiles are organized to di>play 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 maximum the mutagen concentration and the range of antimutagen concentration tested, The lower graph show\ either represented by a bar directed bar.

percent inhibition

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 that no significant effect was detected (designated a the origin indicates 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)

1 -

Retinal Dose Range Mutagen Dose

Promutagens
I

i AFBl

b.

I I t

PolycycliiAromatic Hydrocarbons

I Hetfw3- / N_ I cydic I_ j Amines i

CPA

Fig. 2. Antimutqenicity

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

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

Log Dose (M or mmol/kg)

1 -

Antimutagen Dose Range Mutagen Dose

% Inhib.

Aflatoxin Bl

Benzo[a]pyrene

Fig. 3. Antimutagenic

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

Hatched bars represent in vitro tests and solid bars

represent in viva tests.

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)

1 -

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

Reseurch 350 (19961 109-129

121

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) -

NAC Dose Range

Mutagen Dose

1.

. / I..

-3 I
-5 -7 -

1 j --II / I / I --

r - -----

, I : ! I ---_ i j

--

Promutagens
I

% Inhib.

AFBl

PAHS

TW
P-2

Fig. 5. Antimutagenicity

profile of N-acetylcyateine.

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 viva in

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

Research 350 (19961 109-I-79

123

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

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.

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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.

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

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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Appendix

A.I. Test Code Definitions Test BID Definition Binding (covalent)

to DNA in vitro

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