Mechanism of antimutagenic action of (+)-catechin against the plant-activated aromatic amine 4-nitro-o-phenylenediamine

Environmental Mutagenesis

ELSEVIER

Mutation Research 361 (1996) 81-87

Mechanism of antimutagenic action of( +)-catechin against the plant-activated aromatic amine 4-nitro-o-phenylenediamine Stephanie J. Toering, Glenda J. Gentile, James M. Gentile * Biology Department, Hope College, Holland, M149423, USA

Received 1 February 1996; revised 20 April 1996;accepted 15 May 1996

Abstract

Aromatic amines are activated into mutagens by both animal and plant systems. For plant-activated aromatic amines an important step in this process involves peroxidase enzymes. 4-nitro-o-phenylenediamine (NOP) is a well known direct-acting mutagen that can be enhanced in mutagenic potency by intact plant cells and also by isolated peroxidase enzymes. This activation process is inhibited by several different chemical agents including potassium cyanide (KCn), a known peroxidase inhibitor, and (+)-catechin. In our laboratory both KCn and (+)-catechin inhibited peroxidase-mediated NOP activation into a Salmonella mutagen. However, while KCn demonstrated strong peroxidase enzyme inhibition (as measured biocbemically), (+)-catechin showed only minimal inhibition of peroxidase. Experiments comparing NOP direct and plant-activated mutagenic activity to different Salmonella strains (in the presence and absence of (+)-catechin) suggest that ( + )-catechin may inhibit the mutagenic process by limiting O-acetyltransferase (OAT) activity in Salmonella. OAT activity in Salmonella is a required process for mutations to be induced following treatment with NOP and other aromatic amines. Keywords: (+)-Catechin; Peroxidase; Plant activation; O-Acetyltransferase;Salmonella; Aromatic amine

1. Introduction

Plant activation of promutagens into mutagens was first recognized as an important aspect of genetic toxicology research about 20 years ago (Plewa and Gentile, 1975, 1976). The importance of understanding this process is realized when the number of chemicals to which plants are exposed is considered. Aniline and its derivatives warrant attention because they are degradation products of numerous pesticides (Lamoureux and Frear, 1979). Aromatic amines such as 2-aminofluorene (2-AF) are also activated by

* Corresponding author. Tel.: 1 (616) 395-7714; Fax: 1 (616) 395 7923; e-mail: [email protected]

plant cells (Plewa et al., 1983). Activation of 2-AF and 4-nitro-o- phenylenediamine (NOP) has been demonstrated using both plant cell cultures (Plewa et al., 1983; Gentile et al., 1986) and isolated plant peroxidase (Gentile et al., 1985). A model of plant activation of aromatic amines has been suggested by Plewa (1993). This model includes the oxidation of an aromatic amine by plant peroxidase, conjugation to a macromolecule, transport to the bacterial cell, and acetylation/deacetylation by acetyl CoA:N-hydroxyarylamine O-acetyl transferase (OAT) which produces a nitrenium ion capable of DNA damage. Numerous chemicals, including acetaminophen, 7,8-benzoflavone, diethyldithiocarbamate, methimazole, and metapyrone, have been investigated for

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s.J. Toering et al. /Mutation Research 361 (1996) 81 87

their ability to inhibit the plant activation process (Plewa, 1993; Wilson et al., 1994). For our study we focused on potassium cyanide and (+)-catachin. Both have been previously demonstrated to be effective inhibitors of enzymatic process and both have been previously studied in experiments involving plant activation (Table 1). The purpose of our research was to clarify the mode of action of ( + ) catechin in the inhibition of plant activation of the aromatic amine 4-nitro-o-phenylenediamine (NOP). These data support a better understanding of the mechanisms of activation of aromatic amines by plant systems as well as further our understanding of mechanisms of antimutagens involved in blocking this process (Gentile and Gentile, 1991).

and S. ~phimurium strain TA98 was provided by Dr. B.N. Ames (Berkeley, CA). All strains maintained as a frozen stock at - 8 0 ° C . Confirmation of the genetic integrity of the strains was made with each experiment according to the methods of Zeiger et al. (1981). 2.3. Salmonella plate incorporation assay

The standard plate incorporation method described by Maron and Ames (1983) was performed except that HRP was used in some experiments as the enzymatic activating component. Revertant colonies were counted after a 72 h 37°C incubation period.

2. Materials and methods

2.4. Plant c e l l / m i c r o b e coincubation assay

2.1. Chemicals

The plant cell/microbe coincubation assay was performed as previously described (Plewa et al., 1983, Plewa et al., 1988; Wagner et al., 1989). Nicotiana tabacum, cell line TX1, was used for all the coincubation assays. TX1 was obtained from Dr. J. Widholm, University of IL (Urbana, IL), and maintained in MX medium, a liquid culture medium described by Murashige and Skoog (1962).

4-Nitro-o-phenylenediamine was purchased from Aldrich Chemicals Milwaukee, WI). DMSO, ( + ) catechin, phenol, horseradish peroxidase, and 4aminoantipyrine were obtained from Sigma Chemicals (St. Louis, MO). 30% hydrogen peroxide was purchased from J.T. Baker Chemicals (Phillipsburg, N J). Potassium cyanide was purchased from Fisher Chemicals (Fair Lawn, NJ). ICR- 191 was obtained from Polysciences (Warrington, PA). 2.2. Bacteria Salmonella typhimurium strains YG1024 and 1,8DNP6 were provided by Dr. M. Plewa (Urbana)

2.5. Peroxidase assay

The activity of HRP was measured using the standard protocol methods described in The Worthington Manual (1988). When an inhibitor is used, 0.1 ml of the inhibitor is added to the cuvette prior to

Table 1 Modes of inhibition suggested for potassium cyanide and (+)-catechin Inhibitor Mode of action Ref. Steele et al. (1985) ( + )-Catechin • Interfereswith P-450 bioactivation and interacts with the proximate mutagen Nagabhushan et al. (1988); Nagabhushan and Bhide (1988) • Reduces binding of benzo[a]pyrene metabolites to DNA; scavenges nitrite, reducing the formation of nitroso compounds Frankel et al. (1995); Slater and Scott (1981) • Antioxidant;scavenges free radicals Wise et al. (1983); Wilson et al. (1994) • Inhibitionof peroxidase activity Potassium cyanide Dennis and Kennedy(1986) • Inhibits monooxygenase-type peroxidase reactions

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S.J. Toering et a l . / Mutation Research 361 (1996) 81-87 1,~0-

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Fig. l. Percentinhibitionof SalmonellastrainTA98mutationsfollowingHRP activationof NOP in the presenceof (+)-catechin(•) and KCn (D).

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Fig. 2. Percentinhibitionof SalmonellastrainTA98mutationsfollowingTXI activationof NOP in the presenceof (+)-catechin(0) and KCn (D).

S.J. Toering et al./Mutation Research 361 (1996) 81 87

84

the addition of the enzyme. 10 mM ( + )-catechin and 37.5 mM KCn were used. The concentrations of hydrogen peroxide used to construct the double reciprocal plot were 0.0002, 0.0005, 0.0008, 0.0011, 0.0014 and 0.0017 M.

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3. Results and discussion

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Our data demonstrate the ability of (+)-catechin and KCn to inhibit the HRP activation and TXI activation of NOP (Figs. 1 and 2). In both the intact plant cell and isolated HRP activation systems, ( + ) catechin is the stronger inhibitor, reducing the activated mutagenicity below levels of direct-acting mutagenicity. Results from the peroxidase activity assay (Fig. 3) confirm that KCn is a strong peroxidase inhibitor. Some inhibition of peroxidase is observed with (+)-catechin (Fig. 3). The level of inhibition found, however, could not account for the high levels of (+)-catechin-associated inhibition of NOP mutagenicity demonstrated in Salmonella. These data thus suggested that a different mode of action must account for the antimutagenic activity of ( + ) catechin in this system.

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Fig. 3. Enzymatic activity of HRP in the presence and absence o f ( + ) - c a t e c h i n and K C n .

Consideration of the published model for plant activation of aromatic amines led us to question if (+)-catechin could have an involvement in the Salmonella-associated OAT steps of the activation process. The direct-acting mutagenicity of NOP is

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Fig. 4. Mutagenicity of NOP to Salmonella strains T A 9 8 ( O ) , Y G 1 0 2 4 ( [] ) a n d 1 , 8 D N P 6 ( • ).

85

S.Z Toering et aL / Mutation Research 361 (1996) 81-87 5000 T

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Fig. 5. Mutagenicity of HRP-activated NOP in Salmonella strains TA98 ( • ) , YG1024 ((3) and 1,8DNP~, ([]).

increased in the OAT-enhanced Salmonella strain YGI024, and is not seen in the OAT-deficient strain 1,8DNP 6 (Fig. 4). Activation of NOP by HRP greatly increases the mutagenicity demonstrated in YG1024,

but has no effect on 1,8DNP 0 (Fig. 5). The lack of mutagenicity in 1,8D6NP confirms OAT involvement in the activation process for NOP. Similar results were demonstrated with another aromatic

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Fig. 6. lnhibtion of HRP activated of NOP in Salmonella strain YG 1024 in the presence ( • ) and absence ( [] ) of ( + )-catechin.

86

S.J. Toering et a l . / Mutation Research 361 (1996) 81-87 3O0

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Fig. 7. Mutagenicity of ICR- 191 to Salmonella strain TA98 in the presence ( • ) and absence ( [] ) of ( + )-catechin.

amine, 2-aminofluorine (2AF) (Wagner et al., 1994). The presence of (+)-catechin decreased the level of activated-NOP mutagenicity in YGI024 to the level of direct-acting mutagenicity (Fig. 6). This was also demonstrated in a pilot experiment with 2AF (Gentile, unpublished observation). To further investigate effects of (+)-catechin we tested its effectiveness as an antimutagen in combination with the acridine half-mustard ICR-191, an direct-acting intercalating agent previously demonstrated to be mutagenic in TA98 (DeMarini et al., 1984) and a chemical that does not require Salmonella-associated OAT activity to be an effective mutagen. The presence of (+)-catechin had no effect on the mutagenicity of ICR-191 (Fig. 7). This overall set of data suggest that one mechanism by which ( + )-catechin inhibits the direct-acting and plant activated mutagenicity of NOP in Salmonella is by inhibiting OAT enzyme activity. Other mechanisms of (+)-catechin inhibition, such as antioxidant activity, must also be considered for NOP and other aromatic amine mutagenicity. Future experiments in our laboratory will focus on gaining a better understanding of specificity of (+)-catechin for OAT activity as well as its role as an antimutagen with aromatic amines using other test conditions and genetic indicator organisms.

Acknowledgements This research supported by a grant from the Howard Hughes Medical Research Institute and by a grant from the National Science Foundation (NSFBIR 9322220).

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