Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites

Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites

Mutation Research 536 (2003) 103–115 Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites Eliz...

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Mutation Research 536 (2003) 103–115

Modulation of the mutagenicity of heterocyclic amines by organophosphate insecticides and their metabolites Elizabeth D. Wagner∗ , Matthew S. Marengo, Michael J. Plewa Department of Crop Sciences, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, 366 NSRC MC-635, 1101 West Peabody Drive, Urbana, IL 61801, USA Received 2 October 2002; received in revised form 13 January 2003; accepted 15 January 2003 This paper is dedicated to Sonia Carringer for her many years of excellent service to the University of Illinois Campus Honors Program

Abstract People are commonly exposed to organophosphorus ester (OP) insecticides through the treatment of pets, homes, lawns, gardens, workplaces and in commercial agriculture. Aromatic amines are another chemical class with wide human exposure particularly dietary heterocyclic aromatic amines (HAAs). Previously, we reported that specific aromatic amines and ethyl paraoxon (the metabolite of the insecticide ethyl parathion) induced enhanced mutagenic responses in Salmonella typhimurium. In the present study, we demonstrated that the mutagenicity of 2-acetoxyacetylaminofluorene (2AAAF) and the heterocyclic dietary carcinogen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) was enhanced in the presence of the OP insecticides, ethyl parathion or methyl parathion or a metabolite (methyl paraoxon). The mutagenicity of 2-amino-3-methylimidazo-(4,5-f )quinoline (IQ) was increased by methyl parathion and methyl paraoxon but not by ethyl parathion. This mutagenic synergy was expressed in S. typhimurium strain YG1024. Mammalian microsomal activation was required for PhIP and IQ to express mutagenic synergy. Synergistic responses are rarely incorporated in risk assessment models, yet such responses are important in establishing accurate toxicological characteristics of agents. Under real world conditions where people are exposed to a multitude of agents, the results of this study raise a concern about the environmental and public health impacts of OP insecticides. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Mutagenic synergy; Ethyl parathion; Methyl parathion; Methyl paraoxon; 2AAAF; PhIP; IQ; S. typhimurium

1. Introduction During the past four decades, there has been nearly universal exposure to organophosphorus ester (OP) insecticides that are used to treat pets, homes, lawns, gardens, and workplaces as well as in commercial agri∗ Corresponding author. Tel.: +1-217-244-9869; fax: +1-217-333-8046. E-mail address: [email protected] (E.D. Wagner).

culture [1]. During 2000 in the US, over 17.8 × 106 kg of three OP insecticides (methyl parathion, chlorpyrifos and malathion) were applied to six major crops on 5.1 × 106 ha [2]. Kutz et al. [3] reported that approximately 7.5% of the general population had urinary residues consistent with recent organophosphorus ester exposure. In a recent study, children exhibited higher metabolite levels of OP insecticides than in previous studies measuring adult levels [4].

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Aromatic amines are another class of chemicals with wide human exposure. Aromatic amines are employed as intermediates in textile dying, wood processing, and in the manufacture of pharmaceuticals, pesticides and plastics. A subset of this class is the heterocyclic aromatic amines (HAAs) that are generated when meat or fish are cooked. These very potent carcinogens are highly implicated in the induction of colon and breast cancer [5–7]. Exposure to HAAs is primarily through the diet, however, several HAAs have been identified in river water used as a source of drinking water [8]. We demonstrated that the mutagenicity of aromatic amines was enhanced in the presence of ethyl paraoxon in Salmonella typhimurium [9–11]. Ethyl paraoxon (diethyl-p-nitrophenylphosphate) is the non-mutagenic active metabolite of the OP insecticide ethyl parathion. The mutagenic synergy was observed with a diverse variety of aromatic amines including m-phenylenediamine (mPDA), benzidine, the heterocyclic dietary carcinogens 2-amino3-methylimidazo-(4,5-f)quinoline (IQ) and 2-amino1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) as well as with 2-acetoxyacetylaminofluorene (2AAAF). Mutagenic synergy is the interaction of two or more agents that causes an increased response greater than the sum of their individual effects. The demonstration of mutagenic synergy between the metabolite of an OP insecticide and a wide range of environmental and dietary amines demonstrates the genotoxic risk posed by this specific combination of agents and may underestimate the risk posed by exposure to both OP insecticides and aromatic amines in general. Although ethyl parathion was banned in the US, its close analogue, methyl parathion is a restricted use pesticide. More than 7×105 kg of methyl parathion were legally applied to 4 × 105 ha in 2000 [2]. Additionally over the last decade thousands of homes and businesses have been illegally treated with methyl parathion with major occurrences in Mississippi, Ohio and New York [12–14]. The purpose of this study was two-fold: (i) to investigate other OP insecticides or oxon metabolites to determine if the mutagenic synergy is specific to ethyl paraoxon or is a general phenomenon; and (ii) to determine the toxicity of each OP insecticide in a recently developed microplate assay [15]. The agents chosen for this study were ethyl parathion, methyl parathion and methyl paraoxon.

2. Materials and methods 2.1. Chemicals and cells General laboratory reagents were purchased from Fisher Scientific Co. (Itasca, IL, USA) and Sigma Chemical Co. (St. Louis, MO, USA). S. typhimurium strain YG1024 (hisD3052; rfa; uvrB-bio; ampr ; tetr ; pKM101, pYG219) was originally obtained from Dr. T. Nohmi, National Institute of Health Sciences (Tokyo, Japan) and was stored as frozen cultures in 10% DMSO: 90% Luria–Bonner (LB) broth at −80 ◦ C. We used this strain because it contains a multicopy plasmid that carries OAT (the gene that encodes O-acetyltransferase). YG1024 is highly sensitive to aromatic and heterocyclic amines. Aroclor 1254-induced rat hepatic S9 fraction was purchased from Molecular Toxicology Inc. (Boone, NC, USA). Table 1 lists the sources of the mutagens and insecticides with their CAS numbers. The insecticides were of 98–99.5% purity level. Concentrated solutions of 2AAAF, IQ and PhIP were prepared in dimethyl sulfoxide (DMSO). Concentrated stock solutions of methyl parathion, ethyl parathion and methyl paraoxon were dissolved in DMSO followed by the addition of a surfactant, Alkamuls EL-620 (Rhodia Inc., Cranbury, NJ, USA) for a final concentration of 60% DMSO: 40% Alkamuls. 2.2. Salmonella microplate cytotoxicity assay The microplate cytotoxicity assay was modified from a previously published method [15,16]. The log-phase S. typhimurium YG1024 cells, previously frozen (−80 ◦ C) in LB plus 10% DMSO, were thawed and grown in 5 ml LB at 37 ◦ C for 2 h while shaking. The cell titer was adjusted to an optical density (OD) of 0.030 at 595 nm. Treatment with each insecticide or metabolite was conducted in a 96-well microplate. In general, dilutions of the concentrated stock solutions of insecticide or metabolite were prepared in sterile distilled water followed by dilutions in 100 mM potassium phosphate buffer (PPB), pH 7.4 and used immediately. Each treatment well contained 30 ␮l of the titered YG1024 cells, ␮l amounts of the test agent, and PPB for a total volume of 100 ␮l. In experiments with mammalian activation, the S9 fraction was prepared according to the methods of Maron

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Table 1 Description of chemical agents Chemical agent

Methyl parathion

CAS number

298-00-0

Source

ChemService Inc., West Chester PA, USA

Methyl paraoxon

950-35-6

ChemService Inc., West Chester PA, USA

Ethyl parathion

56-38-2

ChemService Inc., West Chester PA, USA

Ethyl paraoxon

311-5-5

2-Acetoxyacetylaminofluorene (2AAAF)

6098-44-8

2-Amino-1-methyl-6-phenyl-imidazo(4,5-b)pyridine (PhIP) 2-Amino-3-methylimidazo(4,5-f)quinoline (IQ)

105650-23-5 76180-96-6

Sigma Chemical Co., St. Louis, MO, USA Chemsyn Science Co., Lenexa, KS, USA Toronto Research Chem. Inc., North York, Ont., Canada Toronto Research Chem. Inc., North York, Ont., Canada

±S9

%C1/2 valuea mM

mM-h



1.16

3.20

+

0.94

2.59



1.37

3.77

+

1.61

4.42



8.07

22.20

+

8.00

22.01



2.45

6.74

+

0.92

2.53

NA

NA

NA

NA

NA

NA

NA

NA

NA

a The %C1/2 value is the concentration of the test agent that corresponded to 50% of the growth of the negative control. The %C1/2 value was determined by regression analysis.

and Ames [17]; the final concentration of S9 was 5% (v/v) per well. The cells were treated for 1 h at 37 ◦ C shaking at 200 rpm. The number of S. typhimurium cells per microplate well was 1.2 × 106 cells. At the end of the treatment time 100 ␮l of 2× LB medium were added to each microplate well and the initial OD of each well was measured using a Bio-Rad model 550 microplate reader at 595 nm. This value served as the blank for each individual well. The microplate was then incubated at 37 ◦ C while shaking for an additional 210 min. The final OD of each well was determined at 595 nm. The OD data were stored on a computer spreadsheet file. For each well, the blank OD value (time 0 reading) was subtracted from the OD reading of that specific well after 210 min of incubation. A concurrent negative control consisting of S. typhimurium without the test chemical was included with each microplate. The blank-corrected OD data for the negative control was set at 100% viability for each specific microplate. The blank-corrected data for each test agent was converted to a percentage of the negative control. Combinations of a constant concentration of mutagen or promutagen with increasing

concentrations of insecticide were examined. The concentrations of the test compounds were expressed as mM (or ␮M) and as mM-h. The concentrations expressed as mM-h included the exposure concentration of the test compounds in the reaction wells plus the 210 min in the wells at one-half of the original concentration (due to the addition of 2× LB to the microplate well). The mM-h measurement was required to integrate the S. typhimurium cytotoxicity and mutagenicity data [15]. 2.3. Salmonella preincubation mutagenicity assay S. typhimurium strain YG1024 was grown from a single colony isolate in 100 ml LB medium supplemented with ampicillin (50 ␮g/ml) and tetracycline (3.1 ␮g/ml) at 37 ◦ C with shaking (200 rpm). The bacteria were harvested by centrifugation, washed twice with PPB and the titer was adjusted to 2 × 1010 cells/ml. Treatments were conducted in three dram glass vials with screw caps. Each reaction mixture consisted of ␮l amounts of insecticide, 2 × 109 cells and PPB in a total volume of 1 ml. A non-toxic

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concentration range for each insecticide was determined from the microplate cytotoxicity assay. In the mutagenic synergy experiments, a single concentration of each mutagen or promutagen was selected that induced approximately 200–400 YG1024 revertants/5 × 108 cells plated. This constant concentration of each aromatic amine was titrated against a wide concentration range of each OP insecticide or OP metabolite. The protocol with hepatic S9 activation followed that described above. Each reaction vial was incubated for 1 h at 37 ◦ C while shaking. Triplicate 250 ␮l aliquots from the reaction vials (5 × 108 cells) were added to 2 ml of molten Vogel Bonner (VB) top agar supplemented with 550 ␮M histidine + biotin. The top agar was poured onto VB minimal medium plates, incubated at 37 ◦ C for 72 h and revertant his+ colonies were scored. 2.4. Statistics In general most mutagenicity experiments were repeated three times with three VB plates per reaction vial. The data were analyzed using the statistical and graphical functions of Table Curve 4.03, SigmaStat 2.03 and SigmaPlot 4.01 (SPSS Inc., Chicago, IL). To determine significant differences in the mutagenicity experiments, an analysis of variance was conducted. The null hypothesis was rejected if a significant difference (P ≤ 0.05) was obtained and if the power of the test exceeded 0.8 with α = 0.05. 3. Results 3.1. Cytotoxicity of insecticides A series of insecticides and their metabolites were examined for bacterial cytotoxicity. A concurrent negative control consisted of cells not treated. The blank-corrected OD of the negative control consisted of the 210 min reading minus the 0 min reading; this value was set at 100%. The blank-corrected data for each test agent concentration was normalized as a percentage of its corresponding negative control. These data were plotted as a function of the test agent concentration versus the percentage of the negative control. Solvent controls that included DMSO plus Alkamuls that were equivalent to the highest OP insecticide concentration tested did not induce cytotoxicity (data not

shown). The concentration–response curves of methyl and ethyl parathion with or without S9 activation are presented in Fig. 1A. The concentration–response curves of methyl and ethyl paraoxon with or without S9 activation are presented in Fig. 1B. To rank order these agents we calculated the %C1/2 values by regression analysis that corresponded to 50% of the growth of the negative control. The cytotoxicity of the OP insecticides or metabolites expressed an approximately nine-fold range from a %C1/2 value of 2.53 mM-h for ethyl paraoxon with S9 activation to 22.2 mM-h for ethyl parathion without S9 activation (Table 1). The rank order of cytotoxicity (decreasing toxicity) without S9 activation was: methyl parathion > methyl paraoxon > ethyl paraoxon ethyl parathion. With S9 activation the rank order of cytotoxicity was: ethyl paraoxon ≈ methyl parathion > methyl paraoxon ethyl parathion. 3.2. Mutagenicity of OP insecticides Amounts of the solvent mixture of DMSO plus Alkamuls that were equivalent to that used with the highest OP insecticide concentration tested were not mutagenic in strain YG1024 with or without S9 activation (data not shown). Without S9 activation, methyl parathion, methyl paraoxon or ethyl parathion were not mutagenic (Table 2). Ethyl parathion was not mutagenic with mammalian S9 activation. Methyl parathion with S9 activation was mutagenic at concentrations of 500 ␮M and above; 500 ␮M and 1 mM methyl parathion induced 142.8 and 237.8 YG1024 revertants/5 × 108 cells plated, respectively. With S9 activation methyl paraoxon was slightly mutagenic at concentrations of 500 ␮M and above. 3.3. Mutagenic synergy of OP insecticides with 2AAAF and dietary heterocyclic amines A single concentration of 2AAAF or of each promutagen was selected that induced approximately 200–400 YG1024 revertants/5 × 108 cells plated. This constant concentration of challenge mutagen was titrated against a wide concentration range of each OP insecticide or OP metabolite. The concentrations of the aromatic amine mutagens selected were 500 nM PhIP, 1 nM IQ, 500 nM 2AAAF −S9 and 1 ␮M 2AAAF +S9. Nontoxic concentrations of

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Fig. 1. (A) A log-linear plot of the S. typhimurium cytotoxicity of ethyl parathion or methyl parathion ± mammalian S9 activation. (B) A log-linear plot of the S. typhimurium cytotoxicity of ethyl paraoxon or methyl paraoxon ± mammalian S9 activation. Table 2 Mutagenicity of OP insecticides in S. typhimurium YG1024 Agent

±S9

Treatment range (mM)

ANOVA test

Range of significant response (mM)

Maximum fold increase

Methyl parathion Methyl parathion Methyl paraoxon Methyl paraoxon Ethyl parathion Ethyl parathion

− + − + − +

0.01–1.0 0.01–1.0 0.05–1.0 0.01–1.0 0.01–1.0 0.01–1.0

NS F8,133 = 84.0; P ≤ 0.001 NS F7,15 = 15.4; P ≤ 0.001 NS NS

NA 0.5–1.0 NA 0.5–1.0 NA NA

NA 8.5 NA 1.5 NA NA

NS: not significantly different from the concurrent negative control. NA: not applicable.

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Fig. 2. (A) Top panel: cytotoxicity of methyl parathion and 2AAAF −S9 (䊊) and methyl parathion and 2AAAF +S9 ( ) in S. typhimurium using the microplate cytotoxicity assay. Bottom panel: mutagenic synergy of methyl parathion and 2AAAF −S9 (䊊), methyl parathion and 2AAAF +S9 ( ), and methyl parathion alone plus S9 activation mixture ( ) assayed in S. typhimurium strain YG1024. (B) Top panel: cytotoxicity of methyl parathion and IQ +S9 ( ) and methyl parathion and PhIP +S9 ( ) in S. typhimurium using the microplate cytotoxicity assay. Bottom panel: mutagenic synergy of methyl parathion and IQ +S9 ( ), methyl parathion and PhIP +S9 ( ), methyl parathion and IQ −S9 (䊐), and methyl parathion and PhIP −S9 (䉫), assayed in S. typhimurium strain YG1024. The average spontaneous revertant frequency of the PPB negative control or the S9 negative control was 26.6 ± 2.7 and 29.5 ± 3.2 revertants/5 × 108 cells plated, respectively.

the insecticides or metabolites were chosen from the Salmonella cytotoxicity experiments. 3.3.1. Methyl parathion 2AAAF (500 nM) without S9 activation induced 303.2 YG1024 revertants/5 × 108 cells plated. At concentrations of 50 ␮M and above, methyl parathion significantly increased the mutagenic potency of 2AAAF −S9 (Fig. 2A, Table 3). With S9 activation 1 ␮M 2AAAF induced 119.4 YG1024 revertants/5 × 108 cells plated. A significant increase in revertants was observed with methyl parathion concentrations of 50 ␮M and above in the presence of S9-activated 2AAAF (Fig. 2A, Table 3). PhIP with S9 activation induced 255.6 revertants/5 × 108 cells plated. With concentrations of methyl parathion from 50

to 375 ␮M, a significant increase in revertants was observed (Fig. 2B, Table 4). S9-activated IQ (1 nM) induced 199.6 YG1024 revertants/5×108 cells plated. The mutagenic potency of IQ with S9 activation was significantly increased by methyl parathion concentrations from 10 to 375 ␮M (Fig. 2B, Table 5). There was a requirement of S9 activation for mutagenic synergy to occur with PhIP or with IQ (Fig. 2B, Tables 4 and 5). Methyl parathion alone +S9 was mutagenic at concentrations of 500 ␮M and above. The mutagenicity of methyl parathion alone +S9 is presented in Fig. 2A for comparison. 3.3.2. Methyl paraoxon In the experiments with methyl paraoxon, 2AAAF without activation induced 289.8 revertants/5 × 108

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Table 3 Mutagenic synergy of 2AAAF and OP insecticides or OP metabolites 2AAAF (␮M)

±S9

OP, concentration range (mM)

ANOVA test

Range of significant response (mM)

Maximum fold increase

0.5 1.0 0.5 1.0 0.5 1.0

− + − + − +

Methyl parathion, 0.01–1.0 Methyl parathion, 0.01–1.0 Methyl paraoxon, 0.01–1.0 Methyl paraoxon, 0.01–1.0 Ethyl parathion, 0.005–1.0 Ethyl parathion, 0.01–1.0

F10,81 = 10.1; P ≤ 0.001 F11,68 = 209.0; P ≤ 0.001 F8,69 = 6.7; P ≤ 0.001 F9,80 = 29.4; P ≤ 0.001 F10,55 = 7.5; P ≤ 0.001 F10,87 = 21.9; P ≤ 0.001

0.05–1.0 0.05–1.0 0.25–1.0 0.50–1.0 0.01–0.50 0.10–1.0

3.7 7.4a 2.2 2.5a 1.9 8.1

The contribution of the mutagenicity of methyl parathion +S9 or methyl paraoxon +S9 has been subtracted from the total response in calculations for statistical analyses and maximum fold increases. a

Table 4 Mutagenic synergy of PhIP and OP insecticides or OP metabolites PhIP (nM)

±S9

OP, concentration range (mM)

ANOVA test

Range of significant response (mM)

Maximum fold increase

500 500 500 500 500 500

− + − + − +

Methyl parathion, 0.05–1.0 Methyl parathion, 0.005–1.0 Methyl paraoxon, 0.05–1.0 Methyl paraoxon, 0.005–1.0 Ethyl parathion, 0.05–1.0 Ethyl parathion, 0.005–1.0

NS F10,76 = 14.0; P ≤ 0.001 NS F11,78 = 136.1; P ≤ 0.001 NS F18,92 = 3.2; P ≤ 0.001

NA 0.05–0.375 NA 0.05–1.0 NA 0.01–0.50

NA 1.9a NA 6.2a NA 2.7

The contribution of the mutagenicity of methyl parathion +S9 or methyl paraoxon +S9 has been subtracted from the total response in calculations for statistical analyses and maximum fold increases. a

cells plated. Mutagenic synergy was observed at methyl paraoxon concentrations of 250 ␮M and above (Fig. 3A, Table 3). 2AAAF with activation also exhibited mutagenic synergy with methyl paraoxon with concentrations of 500 ␮M and above (Fig. 3A, Table 3). PhIP with S9 activation induced 248.7 revertants/5 × 108 cells plated. With increasing concentrations of methyl paraoxon (50 ␮M and above)

a significant increase in revertants was observed (Fig. 3B, Table 4). S9-activated IQ induced 204.9 YG1024 revertants/5 × 108 cells plated. There was synergy with IQ and methyl paraoxon at concentrations of 100 ␮M and greater (Fig. 3B, Table 5). Without activation IQ or PhIP were not mutagenic. IQ (−S9) expressed very weak synergy with methyl paraoxon (Fig. 3B).

Table 5 Mutagenic synergy of IQ and OP insecticides or OP metabolites IQ (nM)

±S9

OP, concentration range (mM)

ANOVA test

Range of significant response (mM)

Maximum fold increase

1.0 1.0 1.0 1.0 1.0 1.0

− + − + − +

Methyl parathion, 0.01–0.15 Methyl parathion, 0.005–1.0 Methyl paraoxon, 0.05–1.0 Methyl paraoxon, 0.01–1.0 Ethyl parathion, 0.05–1.0 Ethyl parathion, 0.01–1.0

NS F12,91 = 64.6; P ≤ 0.001 F5,18 = 6.1; P ≤ 0.002 F8,69 = 35.2; P ≤ 0.001 NS NS

NA 0.01–0.375 0.5–1.0 0.1–1.0 NA NA

NA 2.7a 1.7 1.9a NA NA

The contribution of the mutagenicity of methyl parathion +S9 or methyl paraoxon +S9 has been subtracted from the total response in calculations for statistical analyses and maximum fold increases. a

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Fig. 3. (A) Top panel: cytotoxicity of methyl paraoxon and 2AAAF −S9 (䊊) and methyl paraoxon and 2AAAF +S9 ( ) in S. typhimurium using the microplate cytotoxicity assay. Bottom panel: mutagenic synergy of methyl paraoxon and 2AAAF −S9 (䊊), methyl paraoxon and 2AAAF +S9 ( ), and methyl paraoxon alone plus S9 activation mixture ( ) assayed in S. typhimurium strain YG1024. (B) Top panel: cytotoxicity of methyl paraoxon and IQ +S9 ( ) and methyl paraoxon and PhIP +S9 ( ) in S. typhimurium using the microplate cytotoxicity assay. Bottom panel: mutagenic synergy of methyl paraoxon and IQ +S9 ( ), methyl paraoxon and PhIP +S9 ( ), methyl paraoxon and IQ −S9 (䊐), and methyl paraoxon and PhIP −S9 (䉫), assayed in S. typhimurium strain YG1024. The average spontaneous revertant frequency of the PPB negative control or the S9 negative control was 25.2 ± 2.3 and 27.5 ± 2.6 revertants/5 × 108 cells plated, respectively.

3.3.3. Ethyl parathion Ethyl parathion at concentrations from 10 to 500 ␮M significantly enhanced the mutagenic potency of 2AAAF −S9 (Fig. 4A, Table 3). 2AAAF with S9 activation induced 134.3 revertants/5 × 108 cells plated. Ethyl parathion at concentrations of 100 ␮M and above significantly enhanced the mutagenic potency of 2AAAF +S9 (Fig. 4A, Table 3). PhIP with S9 activation induced 222.0 revertants/5 × 108 cells plated. Ethyl parathion at concentrations of 10–500 ␮M caused a significant increase in revertants (Fig. 4B, Table 4). S9-activated IQ induced 160.4 revertants/5 × 108 cells plated; however there was no synergy with ethyl parathion (Fig. 4B, Table 5). A requirement for S9 was observed for synergy to occur with PhIP.

4. Discussion 4.1. Cytotoxicity Ethyl parathion ±S9 was the least cytotoxic agent, much less than its primary metabolite ethyl paraoxon or the closely related methyl parathion. Methyl parathion was slightly more toxic than its metabolite, methyl paraoxon. With the small number of agents examined, it is difficult to determine a trend in toxicity related to structure–function relationships. Overall, our data found that the agents with the larger alkyl group (ethyl) were less toxic than the agents possessing methyl groups. This general rule applies to many insecticides due to an increase in steric hindrance by the larger alkyl group [18]. The notable

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Fig. 4. (A) Top panel: cytotoxicity of ethyl parathion and 2AAAF −S9 (䊊) and ethyl parathion and 2AAAF +S9 ( ) in S. typhimurium using the microplate cytotoxicity assay. Bottom panel: mutagenic synergy of ethyl parathion and 2AAAF −S9 (䊊) and ethyl parathion and 2AAAF +S9 ( ) assayed in S. typhimurium strain YG1024. (B) Top panel: cytotoxicity of ethyl parathion and IQ +S9 ( ), ethyl parathion and PhIP +S9 ( ), ethyl parathion and IQ −S9 (䊐), and ethyl parathion and PhIP −S9 (䉫) in S. typhimurium using the microplate cytotoxicity assay. Bottom panel: mutagenic synergy of ethyl parathion and IQ +S9 ( ), ethyl parathion and PhIP +S9 ( ), ethyl parathion and IQ −S9 (䊐), and ethyl parathion and PhIP −S9 (䉫), assayed in S. typhimurium strain YG1024. The average spontaneous revertant frequency of the PPB negative control or the S9 negative control was 25.5 ± 1.8 and 28.7 ± 2.0 revertants/5 × 108 cells plated, respectively.

exception was ethyl paraoxon +S9 that was the most toxic. 4.2. Mutagenicity of insecticides or metabolites We previously demonstrated that ethyl paraoxon was not directly mutagenic in S. typhimurium strain YG1024 [9] nor was it activated into a mutagen by S9 [10,11]. In this work methyl parathion, methyl paraoxon or ethyl parathion without S9 activation were not mutagenic (Table 2). All of the mutagenicity experiments were conducted under non-cytotoxic conditions. The highest concentration of insecticides in the mutagenicity experiments was 1 mM, considerably lower than the %C1/2 values. Ethyl parathion was not

mutagenic with mammalian S9 activation. With S9 activation methyl paraoxon was slightly mutagenic and methyl parathion was mutagenic at concentrations of 500 ␮M and above. In most microbial systems methyl parathion and ethyl parathion were not mutagenic or were weakly mutagenic [19–23]. 4.3. Mutagenic synergy Methyl parathion increased the mutagenic potency of 2AAAF +S9, 2AAAF −S9, IQ +S9 and PhIP +S9 with maximum fold increases over the heterocyclic amine alone of 7.4, 3.7, 2.7 and 1.9, respectively (Tables 3–5). The direct mutagenicity of methyl parathion +S9 at concentrations of 500 ␮M

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and greater is part of the total synergistic response observed with the S9-activated agents. The direct contribution of methyl parathion +S9 was subtracted from the total response in calculations for statistical analyses and maximum fold increases (Tables 3–5). In general, concentrations of 10–50 ␮M were required for a significant increase over the positive control. Methyl paraoxon increased the mutagenicity of PhIP +S9, 2AAAF +S9, 2AAAF −S9 and IQ +S9 with maximum fold increases of 6.2, 2.5, 2.2 and 1.9, respectively (Tables 3–5). Information in the tables has been adjusted to include the weak mutagenicity of methyl paraoxon +S9. Higher concentrations of methyl paraoxon were required for a significant synergistic response (50–500 ␮M). PhIP −S9 was not mutagenic and did not display synergy with methyl paraoxon. There was a very small, but statistically significant increase with methyl paraoxon and IQ −S9 (Table 5, Fig. 3B). With ethyl parathion, synergy was observed with 2AAAF +S9, PhIP +S9 and 2AAAF −S9 with maximum fold increases of 8.1, 2.7 and 1.9, respectively (Tables 3–5). S9 activation was required for synergy with PhIP. Concentrations from 10 to 100 ␮M were required for synergy. There was no synergy with ethyl parathion and IQ +S9. We previously published an evaluation of the mutagenic synergy between ethyl paraoxon and the identical mutagens analyzed here. Ethyl paraoxon in a concentration range from 20 to 320 ␮M enhanced the mutagenicity of 2AAAF (1 ␮M) −S9 with a maximum fold increase in revertants of approximately five-fold [9]. Mutagenic synergy was observed with ethyl paraoxon (100–300 ␮M) and S9-activated PhIP with a 4.6-fold increase in revertants as compared to PhIP alone [10]. With S9-activated IQ, ethyl paraoxon from 10 to 300 ␮M induced a synergistic response with an approximate doubling of revertants over the positive control [10]. If one considers the maximum fold increase in revertants as an indirect measurement of the potency of the synergistic response, few structural patterns emerge. The highest fold increases were observed in the binary combination of 2AAAF +S9 with ethyl parathion followed by 2AAAF +S9 with methyl parathion. The highest fold increase with 2AAAF −S9 was obtained in the presence of ethyl paraoxon. Methyl paraoxon increased the PhIP-induced revertants to the highest

degree and methyl parathion increased the IQ-induced revertants. In general the level of synergy was greatest between 2AAAF and the OPs followed by PhIP plus the OPs. The lowest synergistic response was with S9-activated IQ and there was no synergy with IQ +S9 and ethyl parathion. This may be due to the lower concentration of IQ (1 nM) used in this study, since IQ is a more potent mutagen in Salmonella than either PhIP or 2AAAF, or it may be due to some characteristic of IQ. From the overall results of the present study we cannot deduce any structural features of the insecticides or their metabolites which contribute to mutagenic synergy. No distinction could be resolved between ethyl versus methyl or between the parent compound (parathion) versus the metabolite (paraoxon). In summary mutagenic synergy was expressed by nearly all heterocyclic amine mutagens in the presence of the OP insecticides or their metabolites. All test agents were analyzed in a concentration range that was nontoxic to S. typhimurium. Mammalian microsomal activation was required for the dietary heterocyclic amines to express mutagenic synergy. For the dietary heterocyclic amines, PhIP was more synergistic than IQ with OPs or their oxon metabolites. For IQ the range of mutagenic synergy was narrow (1.7–2.7×). The mutagenicity of the direct-acting mutagen, 2AAAF was enhanced by all of the OP agents. The primary purpose of this study was to examine the breadth of the phenomenon of mutagenic synergy as exhibited by our earlier studies with ethyl paraoxon. The mechanism for the mutagenic synergy of the heterocyclic amines and the OPs is unknown. There are many possible mechanisms that include, but are not limited to, a reaction between two agents to generate a more potent mutagen, a modification of the stability of an intermediate, an increase in the uptake of the mutagen, an increase in the metabolism of the mutagen, or an alteration of the DNA repair system. Synergy may be the result of primarily one mechanism or of a combination of mechanisms. One mechanism of the OP-mediated mutagenic synergy may be a reaction that generates products with higher mutagenic activity. If there was a simple one to one molecular relationship between the OP and the amine, then a maximum synergistic effect would be reached with equimolar concentrations of both agents. A plateau in mutagenicity would result with higher concentrations of OP. There was no sim-

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ple molecule to molecule relationship between the OPs and the heterocyclic amines in the present study or in previous studies [9–11]. In previous work spectrophotometric analysis was conducted to determine if ethyl paraoxon and 2AAAF reacted to generate novel products that were more mutagenic than the parent compound [11]. The presence of novel products was not detected when ethyl paraoxon was incubated with 2AAAF in phosphate buffer or in a buffer that contained S. typhimurium cytosol. Another widely used pesticide, pentachlorophenol (PCP), enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as 2AAAF when assayed with specific S. typhimurium strains [24]. In spectrophotometric studies PCP significantly reduced the rate of 2AAAF degradation both in phosphate buffer or in S. typhimurium cytosol. This alteration in the rate of degradation may provide a mechanism for the PCP-mediated mutagenic synergy by increasing the stability of 2AAAF and thus allowing more 2AAAF to enter the cell and interact with the bacterial DNA. In a similar manner the hypothesis that ethyl paraoxon may alter the rate of 2AAAF degradation in PPB or in S. typhimurium cytosol was tested. The rate constants for the degradation of 2AAAF with or without ethyl paraoxon were not statistically different, thus this mechanism was not implicated [11]. In previous work we determined that the toxicity of crystal violet was not influenced by the presence of ethyl paraoxon. This suggests that the OP did not alter the porosity of the S. typhimurium cells (unpublished data). The initial step in the metabolic activation of aromatic amine mutagens by cytochrome P450 involves N-oxidation, which results in the generation of N-hydroxyarylamines. AcetylCoA-dependent N-hydroxyarylamine O-acetyltransferases (OATs) in S. typhimurium and N-acetyltransferases (NATs) in mammals are enzymes involved in further activation of N-hydroxyarylamines with the formation of an acetoxy group that can spontaneously degrade into a highly reactive nitrenium ion [25]. The resulting DNA adducts form the basis of DNA misrepair that can lead to mutation. It was reported that the mutagenic activity of plant-activated arylamine products was dependent upon the expression of OAT activity in specific Salmonella tester strains [26–28]. Strain YG1024 over-expresses OAT, TA98 expresses the

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wild-type level of OAT and TA98/1,8-DNP6 carries a frameshift mutation in the OAT gene resulting in a lack of expression of this enzyme [29,30]. The target gene of these strains is reversion at hisD3052. These strains were employed to evaluate the effect of OAT expression on the mutagenic synergy of plant-activated m-phenylenediamine plus ethyl paraoxon. Without OAT expression, no mutagenic activity was observed. At the highest ethyl paraoxon concentration, YG1024 had a 2.2× increase in the mutant yield over that of strain TA98. This suggests that there was an interaction between the acetyltransferase and the plant-activated arylamine substrate [9]. Recently ethyl parathion was shown to significantly augment NAT activity in rats [31]. One mechanism for the OP-mediated mutagenic synergy may be the induction of the enzymes involved in the activation of the amine. The significance of OP-mediated mutagenic synergy in mammalian systems is currently undefined. Using the single cell gel electrophoresis (SCGE) assay with human lymphocytes, the genotoxic potency of mPDA, IQ or PhIP was assayed for ethyl paraoxon-mediated modulation [10]. Ethyl paraoxon potentiated the genotoxicity of mPDA, 15-fold at the highest ethyl paraoxon concentration. IQ or PhIP when incubated with ethyl paraoxon exhibited an initial increase in DNA damage demonstrating genotoxic synergy. A comparative study examined the effect of methyl parathion on human lymphocytes from healthy subjects as well as from alcoholics and smokers. Methyl parathion induced chromosome aberrations in human lymphocytes of alcoholics and smokers but not from healthy subjects. Sunil et al. [32] suggested that the damage induced by methyl parathion was potentiated by smoking and alcohol intake. Another widely used OP insecticide malathion enhanced fish S9 metabolic activation of 2-aminoanthracene and 2-acetylaminofluorene [33]. This mutagenic enhancement was not related to the induction of CYP1A1. This may be another example of an OP-mediated mutagenic synergy. Mutagenic synergy and in the broader sense, genotoxic synergy, is a major concern. Synergistic responses are rarely incorporated in risk assessment models; however, such responses are extremely important in establishing the true toxicological charac-

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teristics of agents that impact upon the environment and the public health [34–38]. Pesticides represent a major source of global contamination; annually approximately 2.9 × 109 kg of active ingredients are consumed world-wide [39]. Organophosphorus ester insecticides are globally employed and are environmental contaminants [40]. Aromatic amines are ubiquitous global environmental and dietary contaminants and are potent animal-activated and plant-activated promutagens. The enhanced genotoxic phenomenon associated with agents that are global environmental contaminants may have an insidious impact not only upon the public health but also on biological systems in the environment.

[8]

[9]

[10]

[11]

Acknowledgements This research was funded with support from C-FAR grant 00I-017-5 and the Department of Crop Sciences. Support for undergraduate research was from the UIUC Campus Honors Program, UIUC Environmental Council, Radian Corporation, Howard Hughes Undergraduate Research Program and Colgate-Palmolive Co.

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