Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes

Mutation Research 395 Ž1997. 159–171

Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes Anna B. Hill 1, Phillip R. Jefferies, Gary B. Quistad, John E. Casida

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EnÕironmental Chemistry and Toxicology Laboratory, Department of EnÕironmental Science, Policy and Management, UniÕersity of California, 114 Wellman Hall, Berkeley, CA 94720-3112, USA Received 29 May 1997; revised 18 August 1997; accepted 18 August 1997

Abstract Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor Žderived from 2,6-diethylaniline. and metolachlor and acetochlor Žsynthesized from 2-ethyl-6-methylaniline.. This investigation tests the hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as diethylbenzoquinoneimine, a purported alachlor metabolite. Syntheses are reported here for the corresponding 2,6-dialkylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites. The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100 with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations. The frequency of sister chromatid exchange ŽSCE. in Chinese hamster ovary cells was increased by alachlor at 10 m M and diethylaniline but not ethylmethylaniline at 30 and 3 m M. Isolated and cultured peripheral lymphocytes Žmostly T cells. were used from two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells. In tests at 10 m M, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor Ža 2,6-dimethyl analog. and dimethenamid Žan analog based on 2,4-dimethyl-3-thienylamine.. At 0.3 m M in cultured human lymphocytes, alachlor, the corresponding chloroacetanilide Ž N-dealkyl-alachlor. and aniline metabolites Žand their 4-hydroxy derivatives., and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1–0.3 m M the SCE ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and dimethylbenzoquinoneimines. All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index and increased duration of the cell cycle were not directly linked with SCE induction. Previous investigations have suggested

X

Abbreviations: BrdU, 5-bromo-2 -deoxyuridine; CHO, Chinese hamster ovary; CI, chemical ionization; DMSO, dimethyl sulfoxide; EI, electron impact; GSH, glutathione; HBSS, Hank’s buffered saline solution; M1, M2 and M3, first-, second- and third-division metaphases; MI, mitotic index; MS, mass spectrometry; MTT, 3-Ž4,5-dimethylthiazol-2-yl.-2,5-diphenyltetrazolium bromide; PRI, proliferation index; SCE, sister chromatid exchange; SCE index, number of SCEs in treated cells % number of SCEs in control cells ) Corresponding author. Tel.: q1 Ž510. 642-5424; Fax: q1 Ž510. 642-6497; E-mail: [email protected] 1 Present address: Metabolex, Inc., 3876 Bay Center Place, Hayward, CA 94545, USA. 1383-5718r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 3 - 5 7 1 8 Ž 9 7 . 0 0 1 6 3 - 0

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that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dialkylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in human lymphocytes. q 1997 Elsevier Science B.V. Keywords: Acetochlor; Alachlor; Butachlor; Herbicide; Metolachlor; Sister chromatid exchange

1. Introduction Three chloroacetanilide herbicides Žalachlor, acetochlor and metolachlor. are used in large amounts Ž) 100 000 000 lbsryr. for economical weed control in corn, soybean, sorghum and other major crops and a fourth chloroacetanilide Žbutachlor. is an important rice herbicide ŽFig. 1.. These compounds are oncogenic in rats w1–6x and alachlor is classified as a probable human carcinogen w1–3x. Alachlor manufacturing workers had possible elevated rates of colorectal cancer w7,8x. Residues of the chloroacetanilide herbicides are present in groundwater, including that used in some cases for human consumption w1,9,10x. It is, therefore, important to consider possible mechanisms for the genotoxicity of these four chloroacetanilide herbicides based on metabolic considerations and short-term assays.

Fig. 1. Structures and names for five 2-chloro-N-Ž2,6-dialkylphenyl.-N-Žalkoxyalkyl.acetamide and two other chloroacetamide herbicides studied.

The four oncogenic chloroacetanilide herbicides are derived from 2,6-diethylaniline Žfor alachlor and butachlor. or 2-ethyl-6-methylaniline Žfor acetochlor and metolachlor. and vary in the N-alkoxyalkyl substituents ŽFig. 1.. A related herbicide Ždimethachlor. is derived from 2,6-dimethylaniline ŽFig. 1. and is used for weed control in oilseed rape w6x. Two analogs important as herbicides with limited evidence of carcinogenicity are dimethenamid w11x and propachlor w6x ŽFig. 1.. Although these relationships focus attention on the 2,6-dialkylaniline moiety, there is a notable lack of structure-activity data on the genotoxicity of these herbicides. Metabolic activation may be important in the genotoxicity of alachlor and possibly of some of its analogs. Although several mechanisms have been considered for alachlor Žsee Section 4., the favored proposal w3x is that the activity in inducing nasal turbinate tumors in rats is due to metabolic activation to the electrophilic 3,5-diethyl-p-benzoquinone-4imine Ždiethylquinoneimine. which readily binds to cellular proteins w12–14x. More generally, it appears possible that the oncogenic activity of the 2,6-dialkylaniline-derived herbicides may be due to dialkylquinoneimine metabolites. This hypothesis can be examined directly by comparing the genotoxicity of the relevant herbicides and their major metabolites. Selecting a genotoxicity assay for alachlor and its analogs and metabolites is facilitated by extensive studies on alachlor w1,2x which have used bacteria w15x, mouse bone marrow w16,17x, Chinese hamster ovary ŽCHO. cells w18x, and human lymphocytes w16,19–22x as experimental models. The latter studies show that alachlor induces genotoxic effects wsister chromatid exchanges ŽSCEs., chromosomal aberrations and micronuclei formationx in human lymphocytes at 4–138 m M. Acetochlor w4x, butachlor w23x and metolachlor w24x also induce chromosomal aberrations in human lymphocytes. Studies on SCE induction are particularly convenient for potential structure-activity investigations on analogs and

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161

Fig. 2. Metabolic activation of alachlor to diethylquinoneimine showing possible intermediates from oxidative and hydrolytic pathways. For brevity the compounds are designated by chemical type deleting the diethyl portion. Additional metabolic reactions are also involved Žsee Section 4.. Potencies for induction of sister chromatid exchanges in cultured human lymphocytes are rated from negative Žy. to distinctly positive Žqqq . at 0.3 m M. A compound rated negative Žy. at 0.3 m M may be positive at a higher concentration, e.g. alachlor is rated as Žqqq . at 10 m M.

metabolites because of high sensitivity and ease of scoring w25x. In addition, peripheral lymphocytes circulate for long periods and are a useful indicator of cytogenetic effects of occupational exposure to pesticides w26,27x. The studies reported here have examined whether diethylquinoneimine is mutagenic in bacteria and whether the oncogenic chloroacetanilide herbicides and their analogs and metabolites induce SCEs in CHO cells and human lymphocytes. The relationships between the candidate metabolites, using

alachlor as an example, and the names used are shown in Fig. 2. 2. Materials and methods 2.1. Chemicals Sources for the chemicals Žand their CAS numbers. were: alachlor Ž15972-60-8., butachlor Ž2318466-9., metolachlor Ž51218-45-2. and propachlor Ž1918-16-7. from Chem Service ŽWest Chester, PA.;

Fig. 3. General synthesis scheme for selected metabolites of alachlor and its analogs.

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acetochlor Ž34256-82-1. and dimethachlor Ž5056336-5. from Zeneca Agrochemicals ŽRichmond, CA.; dimethenamid Ž87674-68-8. from Sandoz Agro, Inc. ŽPalo Alto, CA.; 2,6-diethylaniline Ž579-66-8., 3,5dimethylphenol Ž108-68-9. and 3-ethylphenol Ž62017-7. from Aldrich Chemical Co. ŽMilwaukee, WI.. 2-Chloro-N-Ž2,6-diethylphenyl.acetamide Ž6967-299. Žreferred to as the chloroacetanilide metabolite. was prepared as described w28x Žpurity 99%.. NMR spectra were measured with a Bruker AM300 spectrometer ŽCDCl 3 .. Chemical ionization ŽCI. and electron impact ŽEI. mass spectra ŽMS. were obtained in the Department of Chemistry ŽUniversity of California at Berkeley.. Purities of the synthetic compounds were determined by TLC and 1 H-NMR and they were stored anhydrous at y208C. 2.2. Syntheses (Fig. 3) 2.2.1. 3,5-Dimethyl-4-aminophenol (3096-70-61) Following the general method w29x, 3,5-dimethylphenol was treated with diazotized sulphanilic acid and the azo dye reduced with fresh sodium hydrosulfite at 658C. The product was purified on silica with CHCl 3-methanol mixtures, mp 181–1838C Žplates from CHCl 3 . Žliterature w29x: 183–1848C. Žpurity 99%.. 2.2.2. 3-Ethyl-5-methyl-4-aminophenol (112730-648) 3-Ethyl-5-methylphenol w30x was treated as above to give 3-ethyl-5-methyl-4-aminophenol, mp 167– 1708C Žplates from ethyl acetate. as reported w31x Žpurity 99%.. 2.2.3. 3,5-Diethyl-4-aminophenol (108451-25-8) (hydroxyaniline) 3,5-Diethylphenol was obtained from 3-ethylphenol using a published method w32x modified by increasing the aluminum chloride by 30%. The diethylphenol was treated as above to give 3,5-diethyl-4aminophenol, mp 105–1078C Žneedles from toluene–hexane. as reported w12x Žpurity 99%.. 2.2.4. 3,5-D im ethyl-p-benzoquinone-4-im ine (132298-15-8) 3,5-Dimethyl-4-aminophenol Ž30 mg. in ethyl acetate Ž3 ml. was treated dropwise with lead tetraac-

etate Ž140 mg. in ethyl acetate Ž5 ml. at 0–58C. After completion Ž10 min. 8% NaHCO 3 solution Ž2 ml. was added. The organic phase gave dimethylquinoneimine, mp 84–858C Žcream needles from ether. Žliterature w33x mp 81–838C.. 1 H-NMR d 6.33 Žs, H-2 and -6., 2.15 Žs, Me.. 13 C-NMR d 187.2 ŽC-1., 167.6 ŽC-4., 145.8 br ŽC-3 and -5., 130.8 ŽC-2 and -6., 17.5 ŽMe.. MS ŽCI., mrz 136 ŽM q 1. Žpurity 99%.. 2.2.5. 3-Ethyl-5-methyl-p-benzoquinone-4-imine 3-Ethyl-5-methyl-4-aminophenol was oxidized with lead tetraacetate as described above to give ethylmethylquinoneimine, mp 63–648C Žneedles from hexane.. 1 H-NMR d 6.35 and 6.32 Žss, H-2 and -6., 2.17 Žs, Me., 2.57 Žq, J s 7 Hz. and 1.19 Žt, J s 7 Hz. ŽEt.. 13 C-NMR d 187.4 ŽC-1., 166.9 ŽC-4., 150.8 br ŽC-3., 146.2 br ŽC-5., 130.6 and 128.7 ŽC-2 and -6.. MS ŽCI., mrz 150 ŽM q 1. Žpurity 99%.. 2.2.6. 3,5-Diethyl-p-benzoquinone-4-imine (10845126-9) (quinoneimine) Oxidation of 3,5-diethyl-4-aminophenol as above gave diethylquinoneimine, mp 35–368C Žprisms from hexane at y208C.. 1 H-NMR d 6.33 Žs, H-2 and -6., 2.56 Žq, J s 7.2 Hz. and 1.18 Žt, J s 7.2 Hz, Et.. 13 C-NMR d 187.4 ŽC-1., 166.2 ŽC-4., 150 br ŽC-3 and -5., 128.4 ŽC-2 and -6., 23.2 and 12.2 ŽEt.. MS ŽCI., mrz 164 ŽM q 1. Žpurity 99%.. Diethylquinoneimine in pH 7.4 100 mM phosphate at 258C has a half-life of about 2 h but reacts completely within a few minutes on addition of a slight molar excess of glutathione ŽGSH.. 2.2.7. 2,6-Diethyl-p-benzoquinone (50348-20-4) (quinone) Chromatography of diethylquinoneimine on silica with ethyl acetate–hexane Ž20:1. gave diethylbenzoquinone, mp 36–378C Žyellow needles from pentane. Žliterature w34x: mp 358C. Žpurity 99%.. 2.2.8. N-Chloroacetyl-3,5-dimethyl-4-aminophenol 3,5-Dimethyl-4-aminophenol Ž100 mg. in toluene Ž3 ml., tetrahydrofuran Ž0.3 ml., and triethylamine Ž0.75 ml. were cooled to 08C and chloroacetyl chloride Ž0.35 g. in toluene Ž0.7 ml. was added dropwise with stirring. After 10 min, the solution was washed

A.B. Hill et al.r Mutation Research 395 (1997) 159–171

with water, dried and evaporated. TLC of the product on silica in CHCl 3-methanol Ž50:1. gave the corresponding hydroxychloroacetanilide, mp 168– 1708C Žprisms from CHCl 3 .. 1 H-NMR d 6.37 Žs, H-2 and -6., 4.07 Žs, COCH 2 ., 2.02 ŽMe.. 13 C-NMR ŽCDCl 3rCD 3 OD. d 166.7 ŽC5O., 156.4 ŽC-1., 137.1 ŽC-3 and -5., 115.2 ŽC-2 and -6., 42.0 ŽC-2X ., 17.3 ŽMe.. MS ŽEI., mrz 213, 215 ŽMq. Žpurity 99%.. 2.2.9. N-Chloroacetyl-3,5-diethyl-4-aminophenol (hydroxychloroacetanilide) This compound was prepared as for the dimethyl analog, mp 146–1488C Žplates from ethyl acetate– hexane.. 1 H-NMR d 6.42 Žs, H-2 and -6., 4.29 Žs, COCH 2 ., 2.53 Žq, J s 7.5 Hz. and 1.19 Žt, J s 7.5 Hz. ŽEt.. 13 C-NMR ŽCDCl 3rCD 3 OD. d 167.3 ŽC5O., 156.4 ŽC-1., 137.1 ŽC-3 and -5., 115.2 ŽC-2 and -6., 42.7 ŽCOCH 2 ., 25.0 and 17.6 ŽEt.. MS ŽEI., mrz 241, 243 ŽMq. Žpurity 99%.. 2.2.10. N-Chloroacetyl-3,5-dimethyl-p-benzo quinone-4-imine N-Chloroacetyl-3,5-dimethyl-4-aminophenol was treated with lead tetraacetate as above. The noncrystalline product hydrolyzed to the quinone on attempted chromatography Žsilica, florisil. but was purified by precipitation of impurities from ether with hexane and sublimation. 1 H-NMR d 6.46 Žs, H-2 and -6., 4.32 Žs, COCH 2 ., 2.15 Žs, Me.. MS ŽEI., mrz 211, 213 ŽMq. 204 ŽM–Cl.. Minor peaks Ž10%. were observed in the 1 H-NMR at 6.6 and 4.2 Žpurity 90%.. 2.2.11. N-Chloroacetyl-3,5-diethyl-p-benzoquinone4-imine (chloroacetylquinoneimine) Oxidation of N-chloroacetyl-3,5-diethyl-4aminophenol gave chloroacetyldiethylquinoneimine which was purified by sublimation. 1 H-NMR d 6.43 Žs, H-2 and -6., 4.32 Žs, COCH 2 ., 2.41 Žq, J s 7.3 Hz. and 1.15 Žq, J s 7.3 Hz, Et.. MS ŽEI., mrz 239, 241 ŽMq. , 204 ŽM–Cl.. Minor peaks Ž10%. were observed in the 1 H-NMR at 6.6 and 4.2 Žpurity 90%.. 2.3. Bacterial assays The preincubation modification of the standard Ames test was performed at SRI International Toxi-

163

cology Laboratory ŽMenlo Park, CA. with two Salmonella typhimurium tester strains ŽTA98 and TA100. w35x for diethylquinoneimine at 0, 1, 3, 10, 30 and 100 m grplate in the absence of metabolic activation using three plates per dose. TA100 reveals base-pair substitution mutations and TA98 detects both frameshift and base-pair mutations. The sample Žstored at y208C. was dissolved and diluted in dimethyl sulfoxide ŽDMSO. and immediately added to the test system. Solvent and positive controls were within acceptable limits and sterility controls showed no microbial contamination. 2.4. Cells assays 2.4.1. CHO cells and cultures All tissue culture supplies and reagents were from Grand Island Biological Co. ŽGrand Island, NY.. CHO cells from American Tissue Type Culture Collection ŽRockville, MD. were cultured in Hamm’s F12 medium and 10% fetal bovine serum. Alachlor cytotoxicity was measured using a colony formation assay and a 3-Ž4,5-dimethylthiazol-2-yl.-2,5-diphenyltetrazolium bromide ŽMTT. ŽSigma Chemical Co., St. Louis, MO. dye reduction procedure w36x for mitochondrial activity in viable cells. For colony formation studies 100 cells were plated in 100 mm dishes and the next day the medium was removed and fresh medium added with varying concentrations of alachlor dissolved in DMSO Žup to 1% final concentration.. After 6 days incubation the colonies were fixed by 3:1 methanol: acetic acid and stained with 1% Giemsa ŽpH 7.4.; visible colonies were scored and an LD50 was determined. For MTT studies, 2500 cells per well were seeded in 96-well plates. The medium was removed after 24 h and replaced with medium containing varying concentrations of alachlor. After incubation for 72 h viable cells were assayed with MTT. For cytogenetic studies, 5 = 10 5 cells were plated in T-25 flasks. After 24 h the cultures were treated with 10 m M 5-bromo2X-deoxyuridine ŽBrdU. ŽAldrich Chemical Co., Milwaukee, WI. and with test compounds Žadded in DMSO; 1% final concentration. then incubated for 48 h at 378C with 5% CO 2 . The 48-h assay time was chosen to obtain a high percentage of second-division metaphases ŽM2. in both the control and treated cultures, i.e. 64–66% and 72–92%, respectively.

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2.4.2. Human lymphocytes and cultures For human lymphocyte studies, white blood cells were isolated from two different non-smoking males Ž30–40 years old., neither of whom was taking medication. The blood Ž20 ml from each. was drawn into heparinized tubes and diluted 1:1 with Ca2qand Mg 2q-free Hank’s buffered saline solution ŽHBSS.. Isolated lymphocytes were used rather than whole blood since the two donors differed significantly in white blood cell numbers and the same number of cells could then be innoculated for each culture. Diluted blood was centrifuged in Lymphoprep w tubes and the white blood cells in the upper phases were then washed with HBSS, and the cell number determined by staining with Trypan Blue, followed by hemocytometer counting. RPMI 1640 medium wsupplemented with 15% fetal bovine serum, penicillin-streptomycin Ž100 unitsrml and 100 m grml, respectively., 2 mM glutamine, and 1% phytohemagglutinin Mx was used for lymphocyte cultures. Although both B and T cells were present in the isolated lymphocytes, phytohemagglutinin M is primarily a T-cell mitogen Ž25. resulting in a dividing cell population highly enriched in Tlymphoblasts. Isolated lymphocytes Ž1.0–1.5 = 10 6 . were seeded in flasks, 25 m M BrdU was added, and cells were incubated in 5 ml of medium for 24 h at 378C with 5% CO 2 . Then the test herbicides and metabolites were added as fresh solutions in DMSO. Mitomycin C ŽSigma. freshly dissolved in water was used as a positive control for SCE induction. Lymphocyte cultures were incubated for 72 h after addition of test compounds or carrier solvent alone Žcontrol.. This duration allowed comparison of M2 metaphases in controls and all treated cultures Ždiscussed later.. A lymphocyte-based MTT assay of cytotoxicity was developed from the protocol of Schiller et al. w37x. Briefly, lymphocytes were incubated with the test compounds for 72 h as above, then cells from an aliquot Ž1 ml. of each culture were collected by centrifugation at 1000 = g. Fresh medium Ž200 m l. containing MTT Ž0.1 mgrml. was added to the cells in each tube followed by incubation for 2 h at 378C in 5% CO 2 . The cells were recovered by centrifugation and 200 m l DMSO added to dissolve the purple formazan product Žfrom reduction of MTT. for measurement at 570 nm using a UVmax Kinetic Mi-

croplate Reader ŽMolecular Devices, Menlo Park, CA.. 2.4.3. Chromosome and cell cycle analyses In both CHO and lymphocyte experiments, colcemid Ž0.1 m grml. was added during the last 2 h of incubation to arrest mitotic division for metaphase chromosome examination. Chromosome preparations made by standard procedures Žhypotonic treatment, followed by methanol: acetic acid fixation. were stained using the fluorescence plus Giemsa technique w38x. Twenty-five M2 cells were used for each treatment to score SCEs unless noted otherwise. Each lymphocyte experiment was designated by a letter ŽA–H. and donor number Ž1–2.. A two-tailed Student’s t-test determined whether the SCE frequency of treated cultures differed from that in the corresponding controls. SCE induction in lymphocytes was quantitated by calculating an SCE index Žnumber of SCEs in treated cells % number of SCEs in control cells.. The mitotic index ŽMI. Žpercent of mitotic cells. was determined for human lymphocytes using 2 000 cells per culture. Fifty metaphases per treatment were used to determine the percent value of cells in the first division ŽM1., second division ŽM2. and third division ŽM3. in both CHO and lymphocyte cultures. The proliferation index ŽPRI. was calculated using the following formula: PRI s w1Ž%M1. q 2Ž%M2. q 3Ž%M3.xr100 w26x. PRI values can range from 1.00 Žall metaphases

Table 1 Diethylquinoneimine as a mutagen in Salmonella typhimurium strains TA98 and TA100 Compound

Dose, m grplate Revertantsrplate, mean"SE TA98

Diethylquinoneiminea 0 10 30 2-Nitrofluorene 0 Žpositive control. Sodium azide Žpositive control.

5 0 5

a

29"3 22"2 28"2 28"3

TA100 159"12 170"4 335"12

1884"11 167"6 1152"43

Diethylquinoneimine was not significantly different from the control Ž0 m g. when tested at 1 and 3 m g and toxic at 100 m g.

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Table 2 Alachlor and two dialkylanilines as inducers of sister chromatid exchanges and effects on mitotic index and cell cycle progression in Chinese hamster ovary cells Treatment

Control Alachlor Diethylaniline Ethylmethylaniline

mM

– 10 c 30 3 30 3

SCErchromosome" SE a

0.31 " 0.01 0.40 " 0.01) ) 0.40 " 0.02 ) 0.37 " 0.02 ) 0.35 " 0.01 0.36 " 0.02

Cell cycle b M1 Ž%.

M2 Ž%.

M3 Ž%.

PRI

2 4 2 0 2 2

64 66 72 78 92 60

34 30 26 22 6 38

2.32 2.26 2.24 2.22 2.04 2.36

a

Based on 25 second-division metaphases. Exposure for 48 h. Based on 50 metaphases. c No metaphases at 30 mM. ) p F 0.05; ) ) p F 0.01. b

Table 3 Alachlor and analogs as inducers of sister chromatid exchanges in cultured human lymphocytes; structures are given in Fig. 1 Compound

mM

Expt. donor

SCErcell" SE a

SCE index b

Controls

0 0 0 0 0.01 0.01 0.01

F1 G1 G2 H1 F1 G1 G2

4.36 " 0.30 5.56 " 0.36 6.00 " 0.35 4.44 " 0.32 8.00 " 0.44 ) ) ) 17.9 " 1.3 ) ) ) 20.5 " 1.3 ) ) )

1.83 3.22 3.42

F1 G2 H1 F1 G2 F1 G2 F1 G2 F1 G2 F1 G2 F1 G2

6.16 " 0.36 ) ) ) 7.68 " 0.48 ) ) 6.96 " 0.46 ) ) ) 5.20 " 0.33 6.72 " 0.51 4.00 " 0.33 5.89 " 0.44 4.56 " 0.36 6.00 " 0.35 6.40 " 0.57 ) ) 6.68 " 0.50 4.84 " 0.28 5.96 " 0.37 4.72 " 0.39 5.60 " 0.31

1.41 1.28 1.57 1.19 1.12 0.92 0.98 1.05 1.00 1.47 1.11 1.11 0.99 1.08 0.93

F1 G2 G1

3.88 " 0.27 5.56 " 0.29 7.48 " 0.47 ) )

0.89 0.93 1.35

Mitomycin C Žpositive control.

Derivatives of 2,6-dialkylanilines Žand alkyl substituents. Alachlor ŽEt,Et. 10 10 10 3 3 1 1 Butachlor ŽEt,Et. 10 10 Acetochlor ŽMe,Et. 10 10 Metolachlor ŽMe,Et. 10 10 Dimethachlor ŽMe,Me. 10 10 Derivatives of other arylamines Dimethenamid Propachlor c a

10 10 1

n s 25 except G2 for alachlor at 3 m M n s 19. Values by experiment and donor as mean" SE. Mean for treatment % mean for corresponding control. c No metaphases at 10 m M ŽF1 and G1. and 1 m M ŽG2.. )) p F 0.01; ) ) ) p F 0.001 b

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undergoing first division. to 3.00 Žall metaphases undergoing third division..

ylquinoneimine is weakly mutagenic in the TA100 but not the TA98 strain.

3. Results

3.2. Cytotoxicity and genotoxicity of alachlor and metabolites in CHO cells

3.1. Bacterial mutagenicity studies Two Salmonella tester strains were used to determine whether diethylquinoneimine reverts hisy mutations to hisq ŽTable 1.. In both, 100 m grplate was toxic. In TA98, diethylquinoneimine at 10 and 30 m grplate was not significantly different than the negative control w hereas 5 m g of 2nitrofluorenerplate Žpositive control. resulted in a mutation frequency that was 65-fold greater than the negative control. In contrast, TA100 showed a twofold increase in mutation frequency with diethylquinoneimine at 30 m grplate; because this dose induced some toxicity, representative colonies were grown on hisy plates, confirming the heritability of the mutations. Sodium azide Ž5 m grplate. as a positive control for TA100 gave a 7-fold increase in hisq revertants. It is therefore concluded that dieth-

The LD50 of alachlor was ) 30 m M in both the MTT and colony formation assays. To test for possible genotoxicity, alachlor and two dialkylanilines were incubated for 48 h in exponentially-growing CHO cultures with 10 m M BrdU ŽTable 2.. Mitotic index depression was a more sensitive measure of cytotoxicity than the MTT assay since no mitotic figures were observed with alachlor at 30 m M; however, they were present with alachlor at 10 m M and diethylaniline and ethylmethylaniline at 30 and 3 m M. Diethylaniline Ž30 and 3 m M. and ethylmethylaniline Ž30 m M. increased the percent of M2 cells, with a corresponding decrease of M3 cells, and decreased the PRI values which indicate that these treatments extend the cell cycles. The SCE rate for M2 cells was increased by alachlor at 10 m M Ž p 0.01. and by diethylaniline Ž p - 0.05. but not ethylmethylaniline at 30 and 3 m M.

Table 4 Selected alachlor metabolites as inducers of sister chromatid exchanges in cultured human lymphocytes; structures are given in Fig. 2 Compound

mM

Expt. donor

SCErcell" SE a

Controls

0 0 0 0.01 0.3 0.3 0.3 0.3 0.3 0.3 0.1 0.3 0.3 0.3 0.3 0.1 0.3 0.3 0.03

A1 B1 E2 E2 A1 E2 A1 E2 A1 E2 A1 A1 E2 A1 E2 A1 B1 E2 B1

4.80 " 0.40 4.04 " 0.30 5.84 " 0.37 13.2 " 0.8 ) ) ) 4.88 " 0.35 5.20 " 0.30 6.12 " 0.47 ) toxic 5.88 " 0.42 5.76 " 0.21 5.92 " 0.37 ) 6.08 " 0.37 ) 5.08 " 0.37 5.32 " 0.36 5.92 " 0.42 5.28 " 0.42 4.48 " 0.25 5.88 " 0.48 3.56 " 0.21

Mitomycin C Žpositive control. Alachlor Chloroacetanilide Hydroxychloroacetanilide

Aniline Hydroxyaniline

Quinone

a

n s 25. Values by experiment and donor as mean " SE. Mean for treatment % mean for corresponding control. ) p F 0.05; ) ) p F 0.001. b

SCE index b

2.26 1.02 0.89 1.28 1.23 0.99 1.23 1.27 0.87 1.11 1.01 1.10 1.11 1.01 0.88

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3.3. Genotoxicity of alachlor and analogs in human lymphocytes The first experiments Žeach with two different blood donors. determined whether alachlor and six analogs Ž butachlor, acetochlor, metolachlor, dimethachlor, dimethenamid and propachlor. induced SCEs using 10 m M as the test dose ŽTable 3.. Metaphase chromosomes were observed in all treatments except for cultures incubated with propachlor at 10 m M for both donors and 1 m M for donor 2. M2 cells were used to quantitate SCEs. Only alachlor at 10 m M consistently and significantly increased the SCE rate while acetochlor at 10 m M and propachlor at 1 m M Žonly donor 1. were less active. Butachlor, metolachlor, dimethachlor and dimethenamid did not induce SCEs at 10 m M. Positive controls treated with 0.01 m M mitomycin C showed a 2.8-fold increase in SCEs. 3.4. Cytotoxicity and genotoxicity of alachlor and selected metabolites in human lymphocytes Two alachlor metabolites, the chloroacetanilide and the aniline, were highly toxic to primary lymphocytes with LD50 s of approximately 1 m M based on the MTT assay. To directly compare the genotoxicity of alachlor with selected metabolites, the test concentrations used were therefore 0.3, 0.1 and 0.03 m M ŽTable 4.. Alachlor was not genotoxic at 0.3 m M whereas three metabolites Žchloroacetanilide and aniline at 0.3 m M and hydroxychloroacetanilide at 0.1 but not at 0.3 m M. induced a small but significant increase in SCEs in donor 1 cells Žexperiment A only.. The chloroacetanilide was toxic to donor 2’s lymphocytes and the hydroxyaniline and quinone did not increase SCEs with either donor. These results indicated weak genotoxicity for three of the alachlor metabolites. Diethylquinoneimine is proposed to be a metabolite of alachlor and possibly therefore of butachlor. By analogy, ethylmethylquinoneimine is a candidate metabolite of acetochlor and metolachlor and dimethylquinoneimine of dimethachlor. Their genotoxicity and that of the corresponding chloroacetylquinoneimines is therefore of special interest. Dialkylquinoneimines Ždiethyl, ethylmethyl and dimethyl. and chloroacetyldialkylquinoneimines

167

Table 5 Dialkylquinoneimines and chloroacetyldialkylquinoneimines as inducers of sister chromatid exchanges in cultured human lymphocytes. Structural types are given in Fig. 2 Compound m M Expt. donor SCErcell"SE a Controls

0 0 0 0 0 0

A1 B1 C2 D1 D2 E2

SCE index b

4.80"0.40 4.04"0.30 4.36"0.34 5.68"0.37 4.20"0.20 5.84"0.37

Dialkylquinoneimines Ž2,6-Substituents. Et, Et 0.3 A1 7.80"0.70 ) ) ) B1 6.00"0.31) ) ) C2 6.56"0.43 ) ) ) D1 7.45"0.96 ) ) D2 6.32"0.27 ) ) ) 0.1 A1 6.88"0.63 ) ) D1 8.48"0.53 ) ) ) D2 6.56"0.26 ) ) ) 0.03 B1 4.24"0.23 E2 6.08"0.47 Et, Me 0.3 C2 5.60"0.37 ) D1 6.48"0.37 ) D2 6.28"0.34 ) ) ) 0.1 C2 5.32"0.38 D1 6.52"0.61 D2 5.48"0.31) ) ) Me, Me 0.3 C2 6.00"0.33 ) ) ) D1 6.96"0.44 ) D2 5.64"0.39 ) ) 0.1 C2 5.92"0.38 ) ) D1 7.52"0.54 ) ) D2 4.92"0.45

1.63 1.49 1.50 1.31 1.50 1.43 1.49 1.56 1.05 1.04 1.28 1.14 1.50 1.22 1.15 1.30 1.38 1.23 1.34 1.36 1.32 1.17

Chloroacetyldialkylquinoneimines Ž2,6-Substituents. Et, Et 0.3 B1 6.16"0.41) ) ) 1.52 C2 5.28"0.44 1.21 D1 6.12"0.24 1.08 D2 4.72"0.25 1.12 0.03 B1 3.84"0.21 0.95 Me, Me 0.3 C2 5.52"0.27 ) 1.27 D1 5.96"0.46 1.05 D2 4.40"0.18 1.05 a

ns 25 except C2 for the first compound ns18 and D1 for the third compound ns 20. Values by experiment and donor as mean"SE. b Mean for treatment % mean for corresponding control. ) p- 0.05; ) ) pF 0.01; ) ) ) pF 0.001.

Ždiethyl and dimethyl. were compared at 0.03–0.3 m M for activity in induction of SCEs ŽTable 5.. The SCE index was always higher for diethylquinonei-

A.B. Hill et al.r Mutation Research 395 (1997) 159–171

168

Table 6 Diethylquinoneimine and chloroacetyldiethylquinoneimine effects on mitotic index and cell cycle progression in cultured human lymphocytes Treatment

Controls Mitomycin C Žpositive control.

Diethylquinoneimine

Chloroacetyldiethylquinoneimine

Expt. donor

D2 E2 D2 E2 E2 D2 D2 E2 D2

mM

– – 0.2 0.1 0.01 0.3 0.1 0.03 0.3

Cell cycle a MI b

%M1

%M2

%M3

PRI

1.35 1.25 0.20 2.55 2.60 0.65 1.20 1.20 0.80

12 20 N.D.c 12 6 34 34 10 26

24 12 N.D.c 22 10 24 24 20 32

64 68 N.D.c 66 84 42 42 70 42

2.52 2.48 N.D.c 2.54 2.78 2.08 2.08 2.60 2.16

a

Based on 50 metaphases. Mitotic cellsr100; based on 2000 cells. c Too few metaphases to analyze. b

mine than for ethylmethyl- and dimethylquinoneimines at 0.3 and 0.1 m M, while diethylquinoneimine was not genotoxic at 0.03 m M. Chloroacetyldiethyland dimethylquinoneimines were not consistently genotoxic at 0.3 m M, indicating that the chloroacetyl group lowers the genotoxicity of the dialkylquinoneimines. 3.5. E ffects of diethylquinoneim ine and chloroacetyldiethylquinoneimine on mitotic index and cell cycle progression in human lymphocytes Metaphases from two experiments were used to determine MI values and percentages of M1, M2 and M3 cells for cultures treated with diethylquinoneimine, chloroacetyldiethylquinoneimine and mitomycin C Žpositive control. ŽTable 6.. Diethylquinoneimine and chloroacetyldiethylquinoneimine at 0.3 m M, but not the former compound at 0.1 and 0.03 m M, inhibited mitosis by 40–50%. In contrast, mitomycin C increased the MI by two-fold at 0.01 and 0.1 m M but depressed it by 85% at 0.2 m M. Diethylquinoneimine at 0.1 and 0.3 m M and chloroacetylquinoneimine at 0.3 m M reduced the M3 cells to 42% from 64–68% in the controls. Mitomycin C, a potent inducer of SCEs, did not delay cell division at 0.1 m M or lower. The proliferation index ŽPRI. is another measure of cell cycle delay. Control values were not different

from those of cells treated with 0.03 m M diethylquinoneimine and 0.1 m M mitomycin C, which at 0.01 m M gave a substantially higher PRI value than control cells ŽTable 6.. Significant cell cycle delay ŽPRI values of 2.08–2.16. was observed in cultures treated with 0.1 and 0.3 m M diethylquinoneimine and 0.3 m M chloroacetyldiethylquinoneimine. Thus, a long incubation time with test compound Ž72 h. was necessary to have an adequate number of M2 cells ŽPRI was reduced. for careful analysis of SCEs. With mitomycin C a significant number of SCEs can be induced without cell cycle delays. We conclude that diethylquinoneimine and chloroacetyldiethylquinoneimine induce cell cycle delays to the same extent, even though the genotoxicity is much higher for diethylquinoneimine. Thus, depression of the MI and increased duration of the cell cycle by diethylquinoneimine are not directly linked with SCE induction.

4. Discussion Alachlor is genotoxic in CHO cells and human lymphocytes based on our studies determining SCE frequency and other investigations showing increased SCEs w20,22x, chromosomal aberrations w16,18,21x and micronuclei formation w19x with alachlor at a high level Žca. 4–40 m M.. Single

A.B. Hill et al.r Mutation Research 395 (1997) 159–171

stranded DNA breaks are also evident in primary rat hepatocytes w39x and Chinese hamster V79 cells w20x exposed to G 100 m M alachlor. The metabolic activation of alachlor to diethylquinoneimine is illustrated in Fig. 2 based on the candidate metabolites tested in this research for induction of SCEs in cultured human lymphocytes. Alachlor is active at high concentration Ž10 but not 3 m M. possibly due to metabolic activation, which is known to occur with cyclophosphamide and benzow axpyrene in purified human lymphocyte cultures w40x. The findings support the proposal that diethylquinoneimine is the reactive intermediate not only for derivatizing GSH and nasal turbinate proteins w12–14x but also for induction of SCEs. Additional pathways possibly relevant to genotoxicity from alachlor involve GSH conjugation to detoxify diethylquinoneimine w12–14x, oxidation of diethylaniline to diethylnitrosobenzene w41x, and fragmentation of the methoxymethyl moiety w42x. Although the overall metabolic pathway is very complex w43x, that portion leading to diethylquinoneimine has special toxicological relevance. The oxidative bioactivation of alachlor and several of its analogs and metabolites has some similarities to that of the analgesics 4X-hydroxyacetanilide Žacetaminophen. and phenacetin w44,45x, the latter causing nasal carcinoma in rats w46x. 2,6-Dialkylquinoneimines are the purported ultimate genotoxicants in the herbicide series and Nacetylquinoneimine is the proposed hepato- and nephrotoxicant from the analgesics and in each case direct reaction of the quinoneim ine or acetylquinoneimine with GSH is a major detoxification mechanism. Alachlor induces nasal turbinate tumors in rats but possibly not man based on comparative metabolism studies with rats and monkeys w3,14x. This proposal can now be tested more directly comparing nasal mucosa cells of rats and humans w47x for genotoxic effects of alachlor and its analogs and metabolites. The structure-activity relationships observed for inducing SCEs in human lymphocytes are interpretable in part in relation to activation and detoxification of the herbicides and their metabolites within the cells. Relative potencies are consistent with metabolic formation of diethylquinoneimine as the reactive metabolite of alachlor since this constitutes

169

an activation factor of about 100-fold, i.e. there is a similar SCE index of 1.4–1.5 at 10 versus 0.1 m M and inactive levels of 3 versus 0.03 m M, respectively. The potencies of other alachlor metabolites relative to the starting material and diethylquinoneimine do not clearly define an activation sequence. The observed potency for diethylquinoneimine is a balance of activation and detoxification pathways. It has been observed as a metabolite of diethylaniline by rat liver microsomal enzymes w12x but not of alachlor in isolated rat hepatocytes w39x. A portion of the diethylquinoneimine undoubtedly undergoes chemical degradation Žsee Section 2.2., reacts in the medium Že.g. with fetal bovine serum. or undergoes conjugation with GSH in the cytosol. Lymphocytes are very low in GSH content w4x which otherwise would detoxify diethylquinoneimine w12 x. The potency of the quinoneimine for inducing SCEs would presumably be lower in systems with more GSH or other detoxification factors; on an analogous basis, acetochlor induces a greater number of chromosomal aberrations in isolated T-cells compared to T-cells cultured in the presence of whole blood w4x. Alachlor is more active than its chloroacetanilide metabolite and herbicidal chloroacetamide analogs in inducing SCEs in human lymphocytes. This may result from serving as a better metabolic precursor for dialkylquinoneimine formation. The higher activity of alachlor than butachlor, and of acetochlor than metoachlor and dimethachlor, may be related to the ease of metabolically removing the alkoxyalkyl moiety w42x. The greater potency of diethyl- than ethylmethyl- or dimethylquinoneimine may also be a factor. Although acetochlor, butachlor and metolachlor are less active than alachlor in inducing SCEs, they are genotoxic with the more sensitive endpoint of chromosomal aberrations w4,23,24,48x. The low activity or inactivity of dimethenamid is not surprising since it could not generate a metabolite directly analogous to the dialkylquinoneimines. The observed cytotoxicity and genotoxicity of propachlor may result from an unrelated mode of action. The mechanism by which SCEs are induced by alachlor and diethylquinoneimine and some of their analogs Žthis study. involves DNA damage and repair, possibly leading to long-lived genetic change and mutation w48x. The DNA damage may be associ-

170

A.B. Hill et al.r Mutation Research 395 (1997) 159–171

ated with protein derivatization w4x. The weak mutagenicity of diethylquinoneimine Žand of diethylaniline with activation w41,49x. in S. typhimurium strain TA100 indicates induction of base-pair substitution mutations. In summary, these studies show that when cultured human lymphocytes are exposed to alachlor and some of its analogs and metabolites, the resulting DNA damage and repair induce an increased frequency of SCE.

Acknowledgements The study described was supported by Grant P01 ES00049 from the National Institute of Environmental Health Sciences, NIH. Presented in part at the American Association for Cancer Research Annual Meeting in San Diego, CA, April 12–16, 1997. Proc. Am. Assoc. Cancer Res. 38 Ž1997. abstract 819.

w10x w11x w12x

w13x

w14x

w15x

w16x

w17x

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