Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines

Mutation Research 498 (2001) 99–105

Studies on the antimutagenesis of Phyllanthus orbicularis: mechanisms involved against aromatic amines Mirle Ferrer a , Angel Sánchez-Lamar b , Jorge Lu´ıs Fuentes c , Jordi Barbé a , Montserrat Llagostera a,∗ b

a Dpto de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Edifici Cn, 08193 Bellaterra, Barcelona, Spain Laboratorio de Genética Toxicológica, Facultad de Biolog´ıa, Universidad de La Habana, Calle 25 No. 455, Ciudad de La Habana, Cuba c Centro de Estudios Aplicados al desarrollo Nuclear (CEADEN), Calle 30 No. 502 e/5ta y 7ma., P.O. Box 6122, Miramar, Playa, Ciudad de la Habana, Cuba

Received 1 May 2001; received in revised form 5 July 2001; accepted 5 July 2001

Abstract Phyllanthus orbicularis is a medicinal plant, endemic to Cuba, whose aqueous extract has proven antiviral properties. This plant extract is being studied for treatment of viral diseases in animals and humans. Antimutagenic activities of this plant aqueous extract have been investigated as an additional and possible valuable property. Antimutagenesis was assayed against the mutagenic activity of m-phenylenediamine (m-PDA), 2-aminofluorene (2-AF), 1-aminopyrene (1-AP), 2-aminoanthracene (2-AA) and 9-aminophenantrene (9-AP) in Salmonella typhimurium (S. typhimurium) YG1024, in different co-treatment approaches. This plant extract produced a significant decrease of the mutagenesis mediated by these aromatic amines (AA) in the following order: m-PDA > 2-AA > 2-AF > 9-AP > 1-AP. Interactions with S9 enzymes and transformation of promutagenic amines and their mutagenic metabolites by chemical reactions to non-mutagenic compounds are proposed as possible mechanisms of antimutagenesis. Mutagenesis mediated by m-PDA was almost completely abolished when S9 mixture was co-incubated with the plant extract during 40 min, previous to the addition of the m-PDA and bacterial cells to the assay. Similar results were found with 2-AA and 1-AP, but the reduction of the mutation rate was not so dramatic. In contrast, the most significant antimutagenic effect against 2-AF and 9-AP was seen when these chemicals were co-incubated with the plant extract, before addition of the S9 mixture and bacterial cells to the assay. Therefore, inhibition or competition for S9 enzymes seems to be the main antimutagenic mechanism of this plant extract against m-PDA, 2-AA and 1-AP, whilst a chemical modification of 2-AF and 9-AP into non-promutagenic derivatives is likely to be the main mechanism of antimutagenesis against both compounds. © 2001 Elsevier Science B.V. All rights reserved. Keywords: P. orbicularis; Antimutagenesis; Aromatic amines; S9 enzymes; Chemical inactivation of promutagens

1. Introduction It has recently been proposed that mutations in somatic cells can also be involved in the pathogenesis ∗

Corresponding author. Tel.: +34-3-5812615; fax: +34-3-5812387. E-mail address: [email protected] (M. Llagostera).

of some chronic degenerative diseases such as heart diseases, in addition to carcinogenic processes [1,2]. Chemoprevention of mutation-related diseases is an area of increasing research with the final objective of a rational implementation of chemoprevention measures to improve human health. In order to achieve this purpose, the search of chemopreventive agents, the assessment of their efficacy and safety and the

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knowledge of the involved mechanisms are essential [3–5]. In fact, over the last several years there has been an increase of the number of studies reporting medicinal plants and dietary components as an excellent source of chemopreventive agents. Phyllanthus is a widespread genus in most tropical and sub-tropical countries, and their extracts or infusions have long been used in folk medicine as diuretic, for intestinal infections, and for other disorders [6–8]. Different species of Phyllanthus show inhibitory activity against some animal and human viruses [8–11]. Furthermore, some species of this genus inhibit the Epstein–Barr virus DNA polymerase [12] and the reverse transcriptase of human immunodeficiency virus-1 [13,14]. In addition, antigenotoxic and/or antitumoral properties of some species of Phyllanthus have been reported [15–20]. Among the different species included in this genus, Phyllanthus orbicularis HBK is an endemic plant of Cuba, the aqueous extract of which has different antiviral activities [21,22] and currently it is being evaluated in preclinical trials for its application in the treatment of certain viral diseases. Besides this activity, we have recently reported that this extract has also antimutagenic properties because it reduced the number of chromosome aberrations, induced by hydrogen peroxide, in Chinese hamster ovary (CHO) cell line [23]. On the basis of these indications, we decided to assess the antimutagenic properties of the aqueous extract of P. orbicularis on the mutagenesis induced by different promutagenic aromatic amines (AAs). This family of chemicals is a known class of environmentally hazardous compounds, widely distributed in our environment, which includes a wide number of mutagenic and promutagenic compounds, some of them being potent carcinogens. In mammals, the first step of activation of promutagenic AAs to mutagenic metabolite(s) by hepatic microsomal enzymes [cytochrome P450-dependent monooxygenases (cyt-P450) and flavin-containing monooxygenases (FMO)] involves an N-oxidation giving rise to N-hydroxylamine derivatives, which are further metabolised by mammalian N-acetyltransferases (NATs) to ultimate mutagenic metabolites [24]. Also, an NADPH-dependent arylamine oxidase from hepatic cytosol is known to activate AAs, probably by N-hydroxylation [25]. Besides these mechanisms of activation, promutagenic AAs can also be activated in

mammalian extrahepatic tissues through peroxidative transformations, catalyzed by prostaglandin H synthase, myeloperoxidase or lactoperoxidase, to form reactive compounds that damage DNA [24]. Plant enzymes (cyt-P450, FMO and peroxidases) can also activate this class of compounds, giving rise to mutagenic metabolites [26–29]. To conduct the proposed antimutagenic studies, the Salmonella plate incorporation assay was used, because it is versatile, flexible and useful for the assessment of the antimutagenic properties of complex mixtures, for the identification of active components and for the study of mechanisms of antimutagenesis [30]. Among the wide number of bacterial strains available for this assay, strain YG1024, isogenic to TA98, is especially sensitive to AAs because it overproduces the acetyl-CoA:N-hydroxylarylamine O-acetyltransferase (OAT). This bacterial enzyme, like mammalian NATs, is involved in the activation of AAs, catalysing an O-acetylation of N-hydroxylamine derivatives which are formed from AAs by cyt-P450 and FMO activities of the S9 fraction, used in the Ames assay as exogenous activation system [31]. In this work, this assay system has been used to assess the antimutagenic properties of an aqueous extract of P. orbicularis against different AAs and to study the possible mechanisms involved in antimutagenesis.

2. Material and methods 2.1. Chemicals Glucose-6-phosphate, 2-aminofluorene (2-AF) and 1-aminopyrene (1-AP) were from Sigma while m-phenylenediamine (m-PDA), 2-aminoanthracene (2-AA) and 9-aminophenanthrene (9-AP) were from Aldrich and NADP was from Boehringer Mannheim. 2.2. Aqueous extract of P. orbicularis P. orbicularis aqueous extract (POE) employed on this work was obtained from leaves and stems of the plant, collected from Cuba, and lyophilised as reported [21].

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Table 1 Co-treatment proceduresa Approach

Possible mechanisms of antimutagenesis

Outline of the procedure

A B C

Combined effect of all the possible mechanisms Effect of the POE over the metabolites from promutagen Interaction of the POE with the promutagen

D

Interaction of the POE with S9 enzymes

S9 mixture + promutagen + POE + bacteria and plate S9 mixture + promutagen (20 min), add POE (20 min), add bacteria and plate Promutagen + POE (20 min), add S9 mixture (20 min), add bacteria and plate S9 mixture + POE (20 min), add promutagen (20 min), add bacteria and plate

a All incubations were performed at 37◦ C with shaking. Control for each approach was performed following the procedure indicated, but without the addition of POE.

2.3. Ames assay and methodological approaches to study the mechanisms of antimutagenesis The Ames test was performed following the method of plate incorporation as previously described, using three plates per dose [32]. Following the outlines proposed, some modifications were introduced in order to study the antimutagenic mechanisms of POE [30]. Co-treatment approaches used in our work are presented in Table 1. All experiments were conducted using phenobarbital/5,6 benzoflavone-induced rat-liver S9 from Moltox at a concentration in the activation mixture of 0.4% (v/v). Salmonella typhimurium strain YG1024 was kindly provided by Dr. T. Nohmi. 2.4. Determination of the antimutagenic effect Antimutagenesis was determined as the percentage of remaining mutagenesis (%RM). This value was

calculated from results of a minimal of two independent Ames assays as follows: %RM = 100 × (number of induced revertants per plate in the treatment with both mutagen and POE/number of induced revertants per plate with mutagen and without POE). Number of induced revertants was calculated subtracting the number of spontaneous revertants obtained in the negative control of each experiment.

3. Results In order to study the antimutagenic properties of the POE against different AAs (1-AP, 2-AA, 2-AF, 9-AP and m-PDA), their mutagenicity in the YG1024 strain was firstly determined by the classical method of plate incorporation of Ames (data not shown). On the basis of these results, the dose of the AA that gave a number of revertants per plate of between 500–1000 was

Table 2 Optimal antimutagenic effect of POE against AAsa Aromatic amine (AA) (␮g per plate)

POE (␮g per plate)

Number of revertants per plate

1-AP (12.5) 2-AA (10.0) 9-AP (2.5) 2-AF (5.0) m-PDA (250)

0 100 0 100 0 100 0 200 0 100

669.3 458.7 585.3 276.7 927.3 518.2 856.7 400.0 947.8 289.7

a

± ± ± ± ± ± ± ± ± ±

33.5 13.7 29.3 9.6 42.0 9.9 33.1 44.2 0.5 2.1

%RM 100 67.5 100 43.7 100 54.6 100 44.5 100 27.9

± 2.6 ± 1.5 ± 1.2 ± 3.5 ± 3.0

Standard deviations from at least two independent experiments are shown. The mean value of the number ofYG1024 spontaneous revertants per plate of these experiences was 36.33 ± 2.05.

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chosen for studies of antimutagenesis. The selected doses were 12.5 (1-AP), 10.0 (2-AA), 2.5 (9-AP), 5.0 (2-AF) and 250 (m-PDA) ␮g per plate. Following the experimental approach A, the antimutagenic effect of POE (from 50 to 400 ␮g per plate) against all of these AAs was assessed. Results found indicated that the extract produced a significant decrease of the number of induced revertants by all compounds assayed. Table 2 shows the concentration of POE (␮g per plate) that produced the highest reduction of the mutagenicity induced by each AA. It can also be seen in this Table that the optimal antimutagenic effect, expressed as %RM, decreased in the following order: m-PDA > 2-AA > 2-AF > 9-AP > 1-AP. The fact that the highest reduction of the mutagenesis was detected against m-PDA leads us to choose

Fig. 1. The %RM in experimental approach A (Table 1) at different concentrations of POE and 250 ␮g of m-PDA per plate (A) and at 100 ␮g of POE per plate and different concentrations of m-PDA (B). Standard deviations of a minimal of two independent experiments are shown (%RM = 100 × (number of induced revertants per plate in the treatment with both mutagen and POE/number of induced revertants per plate with mutagen and without POE)).

this compound as the model in elucidating the possible mechanisms of antimutagenesis of the extract. Firstly, and following the experimental approach A, the antimutagenesis against m-PDA (250 ␮g per plate) mediated by a range of doses of POE wider than those assayed before was determined. Fig. 1 shows that increasing doses of POE (from 12.5 to 100 ␮g per plate) provoked a decrease of the number of m-PDA induced revertants with a good dose-response relationship, the optimal dose of POE being 100 ␮g per plate. Concentrations of POE which were higher than 100 ␮g per plate gave rise to a decrease of the antimutagenesis mediated by POE, until reaching values of about 60% of RM (Fig. 1A). Due to the higher antimutagenesis potency was at 100 ␮g of POE per plate, the effect of this concentration against a wide range of doses of m-PDA was assayed, following the same experimental approach. Results obtained indicated that this dose of POE is able to reduce, to the same extent, the mutagenesis induced by m-PDA from 6.26 to 1000 ␮g per plate (Fig. 1B). The next step was the study of the possible interactions among m-PDA, S9 enzymes and POE, through the experimental approaches B, C and D, described in Table 1, using 250 and 100 ␮g per plate of m-PDA and POE, respectively. Data of Fig. 2 clearly indicated that the higher antimutagenic effect of POE was found in approaches C and D with a RM of about 18% and 6%, respectively. The high decrease of the mutagenesis induced by m-PDA seen in approach D was corroborated by studying the effect

Fig. 2. The %RM induced by m-PDA in experimental approaches A, B, C and D (Table 1). Standard deviations of a minimal of two independent experiments are shown.

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2-AA and 1-AP, but the kinetic was different for this last compound (Fig. 3). In contrast, the antimutagenic effect of POE against 2-AF and 9-AP was independent of the time of co-incubation of the POE and the S9 mixture. Nevertheless, a marked relationship was found between the time of co-incubation of both AAs with POE (approach C) and the %RM (Fig. 4).

4. Discussion

Fig. 3. The %RM induced by m-PDA (䊉), 2-AF (䊏), 2-AA (䉫), 1-AP (䊊) and 9-AP (䉱) at different time of co-treatment in the experimental approach D (Table 1). Standard deviations of a minimal of two independent experiments are shown.

of the time of co-incubation of the POE with the S9 mixture on m-PDA mutagenesis. Data found indicated that the mutagenesis was almost completely abolished after 40 min of co-incubation (Fig. 3). This dramatic decrease of the mutagenesis was not found when the same experiment was performed with the other AAs previously studied. However, a relationship between the time of co-incubation and the %RM was found for

Fig. 4. The %RM induced by 2-AF (䊏) and 9-AP (䉱) at different time of co-treatment in the experimental approach C (Table 1). Standard deviations of a minimal of two independent experiments are shown.

Using the Ames assay, we have demonstrated that the POE is a potent inhibitor of the mutagenesis mediated by different AAs (Table 2, which gives the results for optimal inhibition). Our results clearly show that the higher antimutagenic activity was found with 100 ␮g of POE and against m-PDA. The finding that doses of POE higher than 100 ␮g per plate produced a minor antimutagenic effect could suggest that POE contains both mutagenic and antimutagenic compounds. However, no increase of the number of revertants was found when the mutagenicity of POE was assessed in strains TA98, TA100, TA1535, TA1537, TA102 and YG1024 in a range of doses from 3.12 to 4,000 ␮g per plate (data not shown). Taking these results into account and the fact that POE is a complex mixture of many compounds, we believe that this reduction might be attributed to a chemical inactivation of POE antimutagenic compounds by other components of this plant extract. Studies about the antimutagenic mechanisms of POE against m-PDA clearly show that this plant extract contains compounds able to interact with m-PDA and with S9 enzymes involved in the activation of this AA. Thus, the mutagenesis of m-PDA was reduced by about 80% when m-PDA was incubated with POE, previous to the addition of the S9 mixture and bacterial cells. A more dramatic antimutagenic effect was observed when the S9 mixture was incubated with POE because the m-PDA mutagenesis was almost completely abolished after 40 min of co-treatment. In contrast, POE had a minor effect on mutagenic metabolites from m-PDA. Thus, the lowest antimutagenesis activity was found when m-PDA was incubated with S9 mixture, before the addition of POE and bacterial cells. Therefore, compounds of POE mainly act at two levels, reacting with m-PDA to form non-promutagenic derivatives and interacting with S9

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enzymes. Both kinds of antimutagenic activities have been reported for some whole plants, as well as for some isolated plant compounds [3,33]. The reduction of the m-PDA mutagenesis by about 70% found in approach A must be understood as the final result of several reactions with different affinities among POE antimutagenic compounds, S9 enzymes and m-PDA. It is remarkable that the antimutagenic effect of POE in co-treatment A increases in the order m-PDA > 2-AA > 2-AF > 9-AP > 1-AP. This could be due to differences in the chemical reactivity of POE compounds with these AAs, in order to generate non-promutagenic compounds. Additionally, it can also reflect different affinities of S9 enzymes for these AAs and for POE components that inactivate these enzymes or compete with AAs. Reduction of mutagenesis mediated by 2-AA and 1-AP, at different times of POE and S9 co-incubation, suggests that interactions of POE components with S9 enzymes might be one of the main mechanisms of antimutagenesis that operates against both chemicals, likewise against m-PDA. Interaction with both cyt-P450 and FMOs are possible because both kinds of enzymatic activities are involved in the activation of the AAs studied [24,29]. In contrast, data obtained with 2-AF and 9-AP strongly suggest that a chemical inactivation of both promutagenic amines by POE components must be one of the most important mechanisms of antimutagenesis. Thus, the mutagenesis mediated by both compounds remained constant and independent of the time of co-incubation of POE with S9, whilst a significant decrease of mutagenesis was seen when both AAs were co-incubated with POE. We have additional evidence indicating that POE components, responsible for chemical reactivity with AAs, are different from those that interfere with S9 enzymes. Thus, when assays were performed with POE treated at 100◦ C during 5 min, following the approach A, antimutagenesis against 2-AF remained at the same level, but it was only very slight against m-PDA (data not shown). These suggest that POE components associated with a chemical reactivity with AAs are resistant to heat treatment, and those that interact with S9 enzymes are heat sensitive. Antimutagenicity of various food-derived antioxidants and flavones and flavonols against heterocyclic amines (HCAs) has been reported [34,35]. Also, soyasaponins and soyasapogenol B repressed the genotoxic capacity of 2-acetoxyacetylaminofluorene [36]. Mechanisms

involved seem to be the inhibition of the formation of HCAs in cooked foods [34], a strong inhibition of the cyt-P450 1A family [35] and an interception of or competition with reactive molecules [36]. In the extract of P. orbicularis studied here all of these mechanisms have been detected. The strong antimutagenic activity of POE against m-PDA and 2-AF, in the conditions of assay described here, provides us with a useful bioassay to further identify the antimutagenic compounds of POE. The antimutagenic potential of this complex mixture against hydrogen peroxide and AAs is remarkable. This fact must be taken into account as an additional valuable property in the current use of P. orbicularis in traditional medicine as well as in its future use for treatment of viral diseases.

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