An evaluation of the genotoxic properties of herbicides following plant and animal activation

Mutanon Research, 136 (1984) 233-245 Elsevier

233

MTR 00884

An evaluation of the genotoxic properties of herbicides following plant and animal activation Michael J. Plewa a Elizabeth D. Wagner a, Glenda J. Gentile b and James M. Gentile b a Instttutefor Envtronmental Studws, Unwerstty ofllhnots, Urbana, I L 61801 (U S.A ) and b Department of Bzology, Hope College, Holland, M I 49423 (U S A ) (Recewed 15 September 1983) (Revision recewed 16 January 1984) (Accepted 6 February 1984)

Summary Commercial and technical grades of 11 herbicides and 13 combinations of commercial grade herbicides were evaluated for their genotoxic properties with Salmonella typhimurtum, Saccharomyces cerevislae directly and following plant and animal activation, or with Zea mays. The herbicides were related by their use in commercial corn (maize) production. Commercial grade formulations of each herbicide and combination of herbicides were also evaluated in situ with the pollen waxy locus assay of Z. mays. Eradicane and bifenox were negative in all assays. Alachlor, propachlor, procyazine and SD50093 (a formulation of cyanazine plus atrazine) were positive in one assay. Cyanazine, dicamba and metolachlor were positive in 2 assays. Atrazine, simazine and butylate were tested only in situ. Atrazine and simazlne were positive and butylate was negative. Of the combinations of herbicides evaluated with the 3 genetic assays, alachlor plus bifenox and procyazine plus metolachlor were positive in I assay and metolachlor plus atrazine was positive in 2 assays. Of the combinations of herbicides evaluated only in situ, butylate plus atrazine, eradicane plus atrazine, eradicane plus cyanazine and metolachlor plus cyanazine were positive while butylate plus cyanazine was negative.

The extensive use of pesticides in modern agriculture reqmres that the compatibility of these agents with the environment and the public health be determined. In this paper data are presented on the genotoxic properties of herbicides used in commercial corn (maize) production. A previous paper (Gentile et al., 1982) provided data on 10 insecticides used in the same cropping system. We conducted this investigation because little information exists concerning the genotoxic properties of All correspondence: Dr. Mxchael Plewa, Associate Professor of GeneUcs, InsUtute for Enwronmental Studies, Enwronmental Research Laboratory, 1005 W. Western Avenue, Urbana, IL 61801 (U.S.A). 0165-1218/84/$03.00 © 1984 Elsewer Science Pubhshers B.V.

pesticides after animal and/or plant activation. We devised and tested an in situ (in field) assay to measure the possible mutagenic effects of a pesticide on a crop plant under field conditions. The incorporation of a comprehensive scheme of metabolic activation is important when investigating the mutagenic properties of pesticides. The activation of a promutagen was first described by Mailing (1966). It is now a standard practice to use in vitro activation in microbial mutagen assays (Mailing, 1971; Ames et al., 1973, 1975). With the dispersal of hundreds of millions of kilograms of pesticides on hundreds of millions of hectares of land in the United States, prudence requires that a variety of activation systems be used in the de-

234 termination of the mutagenic properties of these agents. Pesticides must be analyzed with plant activation protocols. The term 'plant activation' connotes the process by which a non-mutagenic agent (promutagen) is transformed by the biological action of a plant into a mutagen (Plewa and Gentile, 1982; Plewa et al., 1983). Since virtually all crop plants are exposed to pesticides we believe it is important to complement the standard mammalian microsome activation systems with a plant activation system to insure a comprehensive analysis of the genotoxic properties of such agents. Materials and methods

Chemwals The commercial grade formulations of the herbicides were provided by the manufacturers. Technical grade herbicides were obtained from the manufacturers and from the U.S. Environmental Protection Agency. The herbicides evaluated in this study are listed in Table 1. Chemical supphes were purchased from Sigma Chemical Co., St. Louis and microbiological supplies were purchased from Difco Biological Co., Detroit.

Preparatton of rat-liver $9 actwatlon mixture Sprague-Dawley male rats of approximately 200 g each were induced with Aroclor 1254 according to the methods of Ames et al. (1975). The procedures of Garner et al. (1972) were used in the preparation of the rat fiver homogenates. The procedures for the preparation of the rat-liver $9 activation mixture were presented in Gentile et al. (1982).

Preparatwn of maize 1S fractwns Kernels of Z. mays inbred B37 were placed in waxed paper containers filled with vermiculite. Equal volumes of water or water plus chemical were added to each container. The containers were placed in an environmental chamber with a 14-h photoperiod at an average illumination of 300/~E m -2 sec-1 P R R and day and night temperatures of 25 and 20°C, respectively. At the 3-leaf stage, plants within each concentration group were harvested and handled separately. Kernel remnants were removed, and the roots, stems and leaves were weighed and homogenized in distilled

TABLE 1 HERBICIDES EVALUATED FOR GENOTOXICITY Name

Chemicalname

Alachlor

2-chloro-2',6'-dlethyl-N(methoxymethyl)acetamlide

Atrazane

2-chloro-4(ethylamano)-60sopropylamlno)-s-tnazlne

Blfenox

Methyl5(2,4dlchlorophenoxy)2-mtrobenzoate

Butylate

S-ethyl-N,N-dnsobutylthaocarbamate

Cyanazane 2-((4-chloro-6-(ethylamlno)-s-tnazan-2-yl)ammo)-2-methylpropamtrlle Dlcamba

CAS Regastry Number 15972-60-8 1912-24-9 42576-02-3 2008-41-5 21725-46-2

1918-00-9

2-methoxy-3,6-dlchlorobenzolc acld-3,5-dlchloro-o-anaslc acid

Eradlcane S-ethyldlpropylthlocarbamate

759-94-4

Metolachlor 2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxyethyl)acetarmde

51218-45-2

Procyazme 2-((4-chloro-6-(cyclopropylanuno)-s-tnazane-2-yl)anuno)2-methylproplomtnle

32889-48-8

1918-16-7

Propachlor 2-chloro-N-lsopropylacetamhde

122-34-9

Slmazane 2-chloro-4,6-bls(ethylanuno)s-tnazane SD50093

Formulationof cyanazme+ atrazane

water (5 w / v ) . The homogenate was filtered to remove debris and the filtrate centrifuged at 1000 × g for 10 min. The supernatant fluid (1S fraction) was removed, lyophilized, and stored at - 18°C.

Mlcroorgamsm genettc assays Bactertal assay. Salmonella

typhtmurium

strains TA1535, TA1537, TA1538, TA98 and TA100 were provided by Dr. B.N. Ames, Berkeley. The mutability of the strains was routinely tested with 2-nltrofluorene (TA1538 and TA98), sodium azide (TA1535 and TA100), and 9-aminoacridme (TA1537). The range of spontaneous mutant colonies per plate for each strain in our laboratory

235 was as follows: TA1535 (5-20), TA1537 (3-15), TA1538 (10-20), TA98 (30-115) and TA100 (75-150). Positive control mutagens were run concurrently for each strain in each test. When $9 was used, aflatoxin B 1 (AFB1) was used as a positive control for $9 activity with strain TA100. In those cases where positive mutagen or $9 controls d~d not give the expected results, the data from that particular experiment were disregarded. In general the range of concentrations assayed per test agent included three orders of magnitude. The procedures used for the plate incorporation assay were presented in Gentile et al. (1982). A slight modification of the procedures for the plate-incorporation assay was used when analyzing 1S fractions for mutagemcity. Lyophihzed 1S fractions were resuspended in ethanol, and then diluted in sterile water to a final concentration of approximately 20 mg material per ml. Varying ahquots of this resuspentted 1S material were put into soft-agar tubes containing specific bacterial strains. Plates were incubated, scored and revertants verified as previously described (Gentile et al., 1982). Mutagenlcity assays were conducted in triplicate for each chemical tested, and a minim u m of 3 plates was used for each strain in each experiment. Yeast assay. Saccharomyces cerevistae strain D4 (ade 2-1/ade 2-2; trp 5-12/trp 5-27) was provided by Dr. F.K. Zimmermann, Darmstadt. Mitotic gene conversion at both loci was routinely monitored using N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) as a positive control (Zimmermann, 1975). Spontaneous gene conversion frequencies in our laboratory were 0.5-5 × 105 survivors for the ade locus and 0.3-5 × l0 s surwvors for the trp locus. M N N G was run concurrently as a positive control for each locus in each test. When $9 was used in a test, AFB 1 was used as a positive control for $9 activity. In those cases where the M N N G or the AFB 1 $9 controls did not g~ve the expected results, the data from that particular experiment were disregarded. An appropriate amount of test agent was dissolved in D M S O and diluted in water in steps of 1 : 3, 1 : 10, 1 : 30 and 1 : 100. The protocols for inducing gene conversion in strata D4 were described by Gentile et al. (1982).

Maize genettc assay The plants used for the maize waxy locus assay were inbred W22 homozygous for the wx-C allele. This inbred line was originally obtained from the Maize Genetics Cooperation Stock Center at the Universxty of Illinois and was propagated in the genetic nursery of M. Plewa. The wx-C heteroallele was reconfirmed by conducting a cts-trans test with a homozygous wx-C line, inbred M14 supplied by Dr. O.E. Nelson, Madison. The m situ field plots were constructed at the South Farms of the University of Illinois. Each plot was approximately 10 m × 3 m and consisted of 3 parallel 10 m long rows. The outer 2 rows were planted with a commercial corn variety while the center row of each plot was planted with inbred W22 w x - C / w x - C kernels. A commercial grade formulation of each herbicide was applied prior to the emergence of the maize seedlings. Since all of the herbicides were not evaluated in a single year and since some of the weaker inbred maize plants did not survive during a season, three separate plantings (A, B and C) in 3 seasons during 1976, 1977 and 1978 were conducted. The herbicides and the combination of herbicides and their rates of application are presented in Table 5. Control plots were distributed within each field. Plants were allowed to grow until early anthesis (12-14 weeks). A tassel was harvested when only a few anthers were dehisced and the majority of florets unopened. Tassels were dehydrated in 70% ethanol for 2 days and then placed in labeled 1-1 jars filled with 70% ethanol. The wx locus controls the synthesis of the carbohydrate amylose. Pollen grains containing the recessive allele wx stain tan with an ~odine stain whereas pollen grains containing the dominant allele starchy (Wx) stain black. Therefore a reversion (back-mutation of wx to Wx) can be detected by scoring for pollen grains obtained from sporophytes that are homozygous wx that stain a black color after treatment with an iodine solution. For a more detailed description, see Plewa and Wagner (1981). Slides of pollen grains were prepared by a procedure developed by Nelson (1959). Anthers were dissected from unopened florets, homogenized with a gelatin-iodine stain, and strained through cheese-cloth onto a microscope slide. The black-

236 staining pollen grains were counted. The total number of pollen grains on a slide was estimated by counting the number of pollen grains in 20 randomly chosen 1-mm 2 areas and multiplying this value by an appropriate area factor. The frequency of revertant pollen grains was calculated by dividing the total number of W x pollen grains by the total estimated number of viable pollen grains. Statistical analysis Microbial data. The data for each herbicide were evaluated using three statistical parameters: the doubling frequency over spontaneous controls of either revertants per plate in the Salmonella test or gene convertants per 105 survivors in the Saccharomyces test (Ames et al., 1975), a 0 test for significance in the Salmonella test or a • test for significance in the Saccharomyces test (Katz, 1978, 1979), and a concentration-dependent response in one or more of the Salmonella strains or at one of the two loci tested in Saccharomyces (de Serres and Shelby, 1979). For the Salmonella assay, a test agent was appraised as positive if it induced an increase in the number of revertants per plate that was twice the control value or indicated a significant 0 value. For the Saccharomyces assay, a test agent was considered positive if it induced an increase in the number of convertants per 105 survivors that was twice the control value or indicated a significant 4, value. In addition, for both assays, the agent had to reduce a reproducible dose-dependent response. Maize data. The data for each herbicide were evaluated using three statistical parameters: the rate of the wx mutation among gametophytes, a t-test between an appropriate control group for each independent treatment group, and a ~ test for significance. The doubling of the mutation rate among gametophytes was calculated by using the best estimate for the average mutation rate among gametophytes for the control plants and multiplying the value by 2. If the average mutation rate among gametophytes for a treatment group was greater than twice that in the controls, then the herbicide was considered to be mutagenic. Engels (1979) developed a series of equations that may be used to estimate the mutation rate among gametes when premeiotlc mutational events are involved. Such events may result in a distribution of excep-

tional gametophytes that is 'clustered' as opposed to binomial. The true mutation rate among gametophytes, u, (in contrast to a mutation rate per locus per generation of somatic cells can be estimated as a weighted average ( p w ) or as an unweighted average (pu). The best estimator is the average with the smallest variance. When the effect of clustering was pronounced, the unweighted average mutation rate provided the best approximation of the true mutation rate among gametophytes (Engels, 1979). The comparison of each treatment group for an average mutation rate that was double or more than the average mutation rate of control pollen grains was conducted using the estimator with the least variance. A test agent was appraised as positive if it induced an increase in the mutation rate among gametophytes that was twice the best estimate of the spontaneous control value or indicated a significant ff value. A ~ of 4.42 was used to define a significant difference between the mutation rates of the control and treatment groups. This high value was chosen because the test does not include clustering of mutant germ cell lines induced by premeiotic mutation events and the variance estimate is limited by the expansion of the binomial. Note that the only average mutation rate among gametophytes used in the ~ test is the weighted average. In addition, a significant t-test was required.

Results and discussion Commercial and technical grades of herbicides used in commercial corn production were tested for genotoxicity in S. typhimurlum and S. cerevislae with liver microsome activation, plant activation and no activation system. The commercial grades of the herbicides were also assayed for inducing mutations at the wx locus of Z. mays. A summary of the statistical analysis of the data is presented in Table 2. When assayed with S. typhimurtum, S. cerevlsiae and Z. mays no genotoxic properties were detected for the commercial and technical grades of bifenox, eradicane, the commercial grade of dicamba and procyazine and the technical grade of metolachlor. The positive responses of herbicides or combi-

237

nations or herbicides tested with S. typhtmurtum are presented in Table 3. Both commercial and technical grades of cyanazine were positive only after plant activation. The technical grade of procyazine was positive after $9 activation in strain TA1535, however, a stronger positive response was

TABLE

observed in strains TA98 and TA100 after plant activation. The technical grade dicamba was mutagenic without activation in strain TA1535 and following plant activation in strains TA1538 and TA100. The commercial grade of metolachlor was positive in TA1538 and positive after $9 activation

2

SUMMARY

OF STATISTICAL

Herbicide

ANALYSIS

OF HERBICIDE

DATA

Genetic assay SalmoneUa $9 .

Z mays

Saccharomyces

D a .

1S

Alachlor(C) b

.

Alachlor(T)

-

-

.

Blfenox(C)

-

-

Blfenox(T)

-

-

-

Cyanazane(C)

-

-

+

Cyanazane(T)

-

-

+

Dlcamba(C)

-

-

-

Dlcamba(T)

+

-

+

Eradlcane(C)

--

--

--

Eradlcane(T)

-

-

-

Metolachlor(C)

+

+

-

Metolachlor(T)

-

-

-

Procyazine(C)

-

-

Procyazane(T)

-

+

Propachlor(C)

-

-

-

Propachlor(T)

-

-

-

-

-

D _1_

-

--

$9 c

1S

m

m

+

Atrazane(C)

Butylate(C) d

--

+

--

+

+

Slmazine(C) d Atrazane(C)/Cyanazme(C) Alachlor(C)/Blfenox(C)

+

Alachlor(C)/Dlcamba(C) Butylate(C)/Atrazane(C)

d

Butylate(C)/Cyanazane(C)

d

Cyanazane(C)/Alachlor(C)

+

Eradlcane(C)/Atrazane(C)

d

Eradlcane(C)/Cyanazane(C)

d

Metolachlor(C)/Atrazane(C) Metolachlor(C)/Cyanazme(C)

+ d +

Metolachlor(C)/Dlcamba(C) Procyazane(C)/Metolachlor(C)

+

Propachlor(C)/Cyanazane(C)

+

a NO act~vatmn system. b Commercial ¢ -,

(C) or technical (T) grade.

negauve response,

d Tested m Z

+, positive response

mays o n l y .

238 in TA100. The combinations herbicides activation plus

that

were

were alachlor

alachlor,

of commercial grade

positive

propachlor

only

after

plus bifenox, plus

lachlor plus

plant

cyanazine

cyanazine,

atrazine

lachlor. Note

and

procyazine

plus meto-

t h a t all o f t h e h e r b i c i d e

combina-

tions that are positive in Salmonella required plant

meto-

activation.

TABLE 3 DATA OF POSITIVE RESULTS IN T H E SALMONELLA ASSAY Herbicide

Highest positive dose

Metabohc activation $9

1S

Cyanazane(C) c Cyanazane(C) Cyanazlne(C) Cyanazlne(C)

o d d d

---

+ + + +

Cyanazane(T) c Cyanazane(T) Cyanazane(T) Dlcamba(T)

c ¢ e 10/~g

-

Dlcamba(T) Dlcamba(T) Metolachlor(T) Metolachlor(T)

f f 10 #g 10 #g

Procyazane(T) Procyazine(T) Procyazme(T) Procyazane(T) Alachlor(C)/ Blfenox(C) Cyanazane(C)/ Alachlor(C) Propachlor(C)/ Cyanazane(C) Propachlor(C)/ Cyanazane(C) Metolachlor(C)/ Atrazane(C) Procyazme(C)/ Metolachlor(C)

Strain

Colonies

Statistical evaluation

per plate (reduced/control)

0a

DR b

TA1535 TA1537 TA1538 TA100

42/23 41/19 52/21 955/335

2.2 27 24 12.9

+ + + +

+ + + -

TA1537 TA98 TA100 TA1535

55/10 340/158 375/140 176/38

54 10 3 49 60

+ + + +

+

+ + -

TA1538 TA100 TA1538 TA100

14/4 314/110 11/4 322/133

17 98 2.1 6.1

+ + + +

10 pg g s g

+ -

+ + +

TA1535 TA1535 TA98 TA100

49/18 61/12 212/61 252/110

3.6 56 90 55

+ + + +

h

--

+

TA100

283/129

2.8

+

l

-

+

TA1535

37/4

7.1

+

J

-

+

TA1538

369/11

18 3

+

J

-

+

TA98

843/119

67

+

k

--

+

TA98

262/131

2.3

+

+

TA98

337/184

1.9

+

1

-

-

-

-

a The values for O were calculated according to the methods of Katz (1978, 1979). Slgmficance was set at the 0.05 level with a 0 value greater than 1 65 reqmred for a test result to be considered significant b A + sign m&cates that a dose-dependent response was observed. ¢ Commercial (C) or techmcal (T) grade. d Z m a y s plants were treated with 5.17 × 10 -3 M cyanazane and 3 mg 1S were added per plate. c Z . m a y s plants were treated with 2 51 x 10 -2 M cyanazlne and 3 mg 1S were added per plate. f Z m a y s plants were treated with 5.35 × 10 -5 M &camba and 3 mg 1S were added per plate s Z m a y s plants were treated with 1.92 × 10 -3 M procyazine and 3 mg 1S were added per plate h Z . m a y s plants were treated with 1.4 × 10 -5 M/6.6 × 10 -4 M alachlor/blfenox and 3 mg 1S were added per plate 1 Z m a y s plants were treated with 1 2 x 10-3 M / 1 4 × 10-4 M cyanazane/alachlor and 3 mg 1S were added per plate. J Z m a y s plants were treated with 1 16 × 10 -3 M/1.25 x 10 -3 M propachlor/cyanazane and 3 mg lS were added per plate. k Z m a y s plants were treated with 1.66 × 10 -5 M/1.37 × 10 -4 M metolachlor/atrazme and 3 mg 1S were added per plate. I Z m a y s were treated with 1 50 X 10-s M / 1 85 × 10-5 M procyazme/metolachlor and 3 mg 1S were added per plate.

239

Table 5. Among the single commercial formulations assayed, only the s-triazine compounds atrazine, simazine, cyanazine and SD50093 were positive under field conditions. The combinations of herbicides that elicited a positive response in maize were metolachlor plus atrazine, metolachlor plus cyanazine, eradicane plus atrazine, eradicane plus cyanazine, and butylate plus atrazine. All of the combinations of herbicides that induced mutant pollen grams in maize had a s-triazine compound as one of the components m the combination. The data presented in this paper generally agree with the literature especially if both mammalian $9 activation and plant activation are involved m the evaluation. The positive responses of the acetamide and the s-triazlne herbicides compare favorably with information in the literature. Alachlor and propachlor did not induce mutations in S. typhtmurtum with or without rat hepatic

The positive responses of herbicides or combinations of herbicides assayed with S. cerewstae are presented in Table 4. A commercial formulation of alachlor induced a significant increase in gene conversion at both the ade and trp loci. A technical grade of alachlor was positive in yeast after plant activation or mammalian $9 activation. Another acetamide herbicide, propachlor was positive only after plant activation in both the commercial and technical grades. The technical grade of &camba was positive after mammalian $9 activation and the commercial grade of metolachlor reduced a positive response after $9 activation. The combination of commercial grades of metolachlor and dicamba induced a positive response when directly administered to D4 and after plant activation. The results of herbicides or combinations of herbicides assayed with Z. mays are presented in TABLE 4

D A T A OF POSITIVE RESULTS IN T H E S A C C H A R O M Y C E S ASSAY Herbicide

Highest a posmve

Metabohc activation

dose

$9

1S

Alachlor(C) e Alachlor(C) Alachlor(T)

33/tg 33/~g f

-

+

ade

Alachlor(T) Dlcarnba(T) Metolachlor(C)

33/~ g 33/~g 1/~g

+ + +

-

trp ade

Propachlor(C) Propachlor(T) Propachlor(T)

~

-

+

ade

h

--

+

ade

'

-

+

J

-

J

-

Metolachlor(C)/ Dmamba (C) Metolachlor(C)/ Dlcamba (C)

Locus b

Convertants (reduced/control) × l 0 s survwors

Statistical Evaluation 0c

DR d

3.22/1.24 3 33/0 7 1 71/0.6

2.1 25 31

+ + +

0.81/0.3 1 5 8 / 0 56 13 71/7.7

1.7 1.8 18

+ + +

trp

31 0 / 5 . 0 38.0/5.3 4.7/1.2

6.9 17.2 6.1

+ + +

+

ade

2.3/1.1

25

+

+

trp

0 8/0 1

6.3

+

trp ade

trp

a # g chemical × &luUon factor. b Adenine (ade) or tryptophan (trp). c The values for ~ were calculated according to the methods of Katz (1978, 1979) Stgmficance was set at the 0.05 level with a ~ value greater than 1 65 reqmred for a test result to be considered slgmflcant. d A + sign m&cates a dose-dependent response e Commercial (C) or techmcal (T) grade. f Z . m a y s plants were treated wtth 1.4 × 10-3 M alachlor and 3 mg 1S were added per plate. s Z m a y s plants were treated with 1.306 × 10-3 M propachlor and 3 mg 1S were added per plate. h Z m a y s plants were treated with 1 306 × 10 -3 M propachlor and 3 mg 1S were added per plate ' Z . m a y s plants were treated with 1.306 × 10 - 4 M propachlor and 3 mg 1S were added per plate. J Z m a y s plants were treated with 1.67 x 1 0 - s M / 4 . 3 0 × 10-6 M m e t o l a c h l o r / & c a m b a and 3 mg 1S were added per plate

240

$9 activation (Andersen et al., 1972; Shirasu et al., 1976; Eisenbeis et al., 1981). However, the data presented in this paper on alachlor and propachlor are consistent with our earlier findings that these agents require plant activation to be converted into a genotoxin (Gentile et al., 1977). Also ala-

chlor was demonstrated to induce mutations in the blue-green alga N o s t o c m u s c o r u m (Singh et al., 1979). These data suggest that certain acetamide herbicides may have some mutagenic potential after plant activation. The s-triazine herbicides are the most heavily

TABLE 5 MAIZE wx LOCUS ASSAY FOR COMMERCIAL G R A D E F O R M U L A T I O N S OF HERBICIDES OR COMBINATIONS OF HERBICIDES Herbicide or combmatlon of herbicides

Apphcatlon rate (kg/ha)

Estimated number of pollen grams analyzed

Estimated mutation rate among

( × 10 6)

gametophytes ( × 1 0 -5)

Stanstlcal evaluation 2X

t

NA

NA

Experlmental plots A Control Blfenox Cyanazane

0 2.24 3 58

2.59 1 39 0 47

5 28 9 05 28 25

Procyazme SD50093 Metolachlor + Dlcamba

3.58 4 48 2 24 + 0.56

0.24 0.37 0.91

4.64 39 37 8.22

Procyazme + Metolachlor Alachlor + Dicamba Blfenox + Alachlor Bffenox + Alachlor

2 24 _+2 24 2.34 + 0.56 1.12 + 2.34 1 68 + 2.24

0 61 1 44 1 38 1 53

9 83 3.82 7.01 4.29

Control Atrazane Cyanazane

0 3.84 4 80

2.39 0.95 0 94

4 24 8.53 14.76

Metolachlor Eradlcane Alachlor

8.40 7.20 6 00

1.09 0 81 1.35

2 84 4.31 4 21

SD50093 Slmazane Butylate

4 80 3.80 7.20

1.14 0.99 0 91

16.25 10.86 5 72

Metolachlor + Atrazane Metolachlor + Dlcamba Metolachlor + Cyanazane

3.00 + 2.40 3.00 + 0.60 4.80 + 4.80

0 79 0 75 0.52

12 27 7.31 10.84

Eradlcane + Atra2ane Eradlcane + Cyanazme Propachlor + Cyanazlne

3.60 + 1.92 3 60 + 2.40 4.80 + 2 24

1 16 0.88 1.16

14.20 8 61 6 29

Butylate + Atrazane Butylate + Cyanazane

4.80 + 1.92 4.80 + 2.40

1 24 1 02

0 0.56 3 36

1.17 1.44 1.13

--

+

NA --

+

P < 0001

+(1613)

+

P < 0.05

+(19.34)

--

+

--

--

+

--

Expertmental plots B NA + +

NA P < 0.005 P < 0.001

NA + (4.90) +(10.47)

+ +

P < 0001 P < 001

+(716) +(7 16)

+

P < 0.001

+(8.15)

--

+

--

+

P < 0.001

+ (5 92)

+ +

P < 0.001 P < 0 001

+ (11.53) + (4.87)

8 25 5.90

-

P < 0.001

+ (4

2 91 2.43 3 20

NA

NA

NA

Expertmental plots C Control Dlcamba Propaehlor

96)

241 and widely used pesticides in the United States (Hileman, 1982). These agents induce geneuc damage in a wide variety of organisms. The results of cytogenetic studies on s-triazine herbicides indicate that these agents induce both mitotic and meiotic damage. In plants, atrazine and simazine induced mitotic chromosome aberrations in roottip cells of Hordeum vulgare (Wuu and Grant, 1966; Stroev, 1968; 1970) and Vtcta faba (Wuu and Grant, 1967b). Cyanazine caused chromosome aberrations in root-tip cells of Tradescantta reflexa and V. faba (Ahmed and Grant, 1972). The herbicide aventox SC, a s-triazine herbicide contaming simazine and trietazine (2-chloro-4-diethylamino6-ethylamino-s-triazlne), induced chromosome aberrations in root-tip cells of Alhum cepa and V. faba (Badr, 1983). Atrazine and simazine caused chromosome aberrations in meiotic cells of H. vulgare (Wuu and Grant, 1967a) and atrazine induced chromosome damage in microsporocytes of Sorghum vulgare (Liang et al., 1967; Liang and Liang, 1972; Lee et al., 1974). Grant (1972) stated that atrazine induced semisterility in maize plants in field experiments due to the induction of chromosome translocations. The herbicides atrazine and 2,4-D (2,4-dichlorophenoxyacetic acid) each induced damage to meiotic chromosomes of grain sorghum. However, after the plants were simultaneously treated with both herbicides the frequency of meiotic chromosome aberrations increased due to a synergisuc interaction of the herbicides (Liang et al., 1969). At variance with the above findings, two studies indicated that atrazine did not induce cytogenetic damage in treated plants (Sawamura, 1965; Muller et al., 1972). The s-triazine herbicides have been shown to induce other types of cytogenetic anomalies in plants besides mitotic and meiotic chromosome aberrations. Griffiths (1979) analyzed 48 chemicals with a system to detect the production of aneuploid products of meiosis in Neurospora crassa. The frequency of 'pseudo-wild type' (PWT) heterokaryotic cultures was the index of aneuploid frequency. The PWT frequency for the control and atrazine, cyanazine or simazine treated Neurospora was, 5.5 x 10 -5, 10.3 × 10 -5, 6.5 x 10 -5 and 5.7 x 10 -5, respectively. Griffiths concluded that atrazine demonstrated major, consistent and statistically significant effects. Thus, atrazine induces

nondisjunction in N. crassa. Finally, Chou and Weber (1981) reported that atrazine induced sister chromatid exchanges in root-tips of treated maize kernels. This result suggests that atrazine has a recombinogenic effect on plant somatic chromosomes. Only a few papers exist in which the reduction of chromosome damage or loss in ammals by s-triazine herbicides have been reported. Murmk and Nash (1977) reported that atrazine and simazine induced an increased frequency in the 'loss of X or Y chromosomes. However, cyanazine did not significantly increase sex chromosome loss, breakage or nondisjunction. Atrazine did not.lnduce chromosome aberrations or sister chromatid exchanges in chinese hamster ovary ceils. However, at high in vivo doses in mice (1500 and 2000 mg/kg), atrazine induced a significant increase in the frequency of chromosome aberrations in bone marrow ceils as compared to control mice (Loprieno and Adler, 1980; Adler, 1980). The genetic impact of herbicides was evaluated using agricultural workers who were employed as custom applicators of herbicides, including atrazine. Yoder et al. (1973) found that these applicators had a maximum of a 4-fold increase in chromosome aberrations detected in lymphocyte cultures from their blood as compared to a control population. The s-triazine herbicides induced point mutations in plants and animals. Atrazine and simazine induced recessive mutations in H. oulgare uncovered in the F2 generation (Wuu and Grant, 1966). Atrazine, simazine and cyanazine, at concentrations employed in modern agricultural practice effected reverse mutations at the wx locus in maize pollen grains (Plewa and Gentile, 1976; Plewa and Wagner, 1981). Recently Schairer and Sautkulis (1982) reported that atrazine at concentrations of 6.90 × 10 -5 M induced a sigmficant increase in the frequency of somatic mutation in stamen hair cells of Tradescantia clone 4430. In animals, atrazine, simazine and cyanazine increased the rate of apparent dominant lethal mutations in Drosophzla melanogaster; however, the reduction in egg hatch may have been due to toxicological aspects rather than mutation (Murnik and Nash, 1977). Atrazine and simazine were reported to induce sex-linked recessive mutations in D.

242

melanogaster (Murnik and Nash, 1977). A longterm study on the genotoxicity of pesticides conducted under the auspices of the U.S. Environmental Protection Agency agreed with the above finding in that simazine was positive in the sexlinked recessive mutation assay in D. melanogaster (Waters et al., 1982). However, one study found atrazine refractory in the same assay (Loprieno and Adler, 1980). In mammahan systems, atrazine and simazane caused genetic damage. Atrazlne induced dominant lethal mutations in mice at high concentrations (1500-2000 mg/kg) (Loprieno and Adler, 1980; Adler, 1980). However, Seiler (1977), using an assay that measured the inhibition of mouse testlcular DNA as an index of genotoxicity, reported no effect by 1000 mg/kg of simazine administered orally to male mice. In mammalian cell culture tests atrazine was refractory in V79 Chinese hamster cells with or without rat hepatic $9 microsomal activation and in affecting unscheduled DNA synthesis in human EUE cells. After plant activation using potato mlcrosomes, 3.0 mM atrazine induced forward mutation in V79 cells at the 6TG R locus and the same concentration of atrazine plus potato rmcrosomal activation caused unscheduled DNA synthesis in EUE cells. Apparently plant activation is a requirement for the metaboic conversion of atrazine to a mutagen in these cell lines (Loprieno and Adler, 1980; Adler, 1980). It is interesting to note that simazine induced forward mutation in cultured mouse lymphoma cell line L5178Y without plant activation (Waters et al., 1982). The analysis of the mutagenicity of s-triazine herbicides and related compounds using microbial assays at first seem contradictory. With the advent of activation protocols using plant systems (Plewa and Gentile, 1982) a pattern emerges from these incongruous data. Microbial assays using the H17 Rec + and M45 Rec- strains of Bactllus subtths, WP2 strains of Eschermhta coh, strains G46, TA1530, TA1532, TA1534, TA1535, TA1536, TA1537 and TA1538 of S. typhlmurtum, all without metabolic activation, demonstrated that atrazine, simazine and other s-triazine herbicides did not induce point mutations (Andersen et al., 1972; Seiler, 1973; Shirasu et al., 1976). Seiler

(1973) reported very weak mutagenic activity in S. typhimunum with 2-hydroxy-4,6-bls(ethylamino)s-triazine and 2-chloro-4,6-diamino-s-triazine. Atrazine was tested with Salmonella strains TA98, TA100, TA1535 or TA1538 using rat hepatic rmcrosomal suspensions ($9) derived from aroclor 1254 induced rats (Lusby et al., 1979; Bartsch et al., 1980; Loprieno and Adler, 1980) or with $9 derived from 3-methylcholanthrene induced rats (Bartsch et al., 1980). Atrazine was not activated into a mutagen by mammalian microsomes. Lusby et al. (1979) assayed 16 s-triazine compounds in addition to atrazine with Salmonella. These compounds included hydroxyatrazine, cyprazine, cyanazine, simazine, 2,4-dichlorohexahydro-l,3,5-triazine, 2,4-diamino-s-triazine, 2,5,6-tripyridyl-s-triazine, 5-azauracil, 5-azacytosine, ammeline, cyanuric chloride, cyanuric acid, tri-thiocyanuric acid, s-triazine and triethylenemelamine. Only triethylenemelamine was mutagenlc to Salmonella. Clearly s-triazine herbicides and a number of their metabolites are not mutagens or promutagens activated by mammalian microsomal suspensions. With microbial genetic indicator organisms the activation of atrazine, simazine and cyanazine requires plant activation. The term 'plant activation' connotes the process by which a nonmutagenic chemical is converted into a mutagen by the biological activity of a plant. These processes are analogous to the familiar mammalian microsomal activation system that is routinely employed in many short-term microbial systems. To establish that a chemical is a plant promutagen, one must be able to separate the activation process from the genetic end point used to assay for genotoxicity (Plewa and Gentile, 1982). The activation of atrazine was first demonstrated by Plewa and Gentile (1976) using gene conversion at the ade-2 and trp-5 locl in Saccharomyces cerevtstae. Atrazine alone was not recombinogenic to yeast, however, extracts from maize seedlings treated with 0-25 ppm atrazine induced a concentration-dependent increase of gene convertants. Extracts of untreated maize seedlings did not induce an increase in gene convertants. The first observations of the plant activation of atrazlne into a mutagen using repair-deficient E. coh and forward mutation in E. coh and atrazine or

243

cyanazine in Salmonella strains TA98 and TA100 was conducted by S. Rogers and G. Warren (personal communication, 1976; 1977). These data were not confirmed in an unrelated study that involved field grown corn (Bakshi et al., 1981). However, the plant activation of atrazine using cultured plant cells (Nicotlana alata) was demonstrated by Benign1 et al. (1979). Atrazine alone, plant tissue culture medium alone, atrazine plus tissue culture medium or homogenized cultured plant cells were not mutagenic when assayed with Aspergdlus mdulans. However, a homogenate of N. alata cells that were exposed to atrazine induced forward mutation of resistance to 8-azaguanine in A. ntdulans. Benigni and his colleagues also assayed for mitotic crossing over or nondisjunction by scoring for a recessive trait, resistance to p-fluorophenylalamine, in a diploid strain heterozygous for fpa r (Bignami et al., 1974). Atrazine was positive for these genetic end points only after plant activation. Singh et al. (1982) isolated a genotoxic fraction from maize seedlings exposed to atrazine using high-pressure liquid chromatography (HPLC). The treatment of the maize and the preparation of the lyophilized water extract of the seedlings was essentially the procedure developed by Plewa and Gentile (1976). Atrazine at concentrations of 0, 30, 60 and 90 ppm were administered to control and treatment groups, respectively. The plant extracts were assayed for their ability to induce gene conversion at the ade-2 and trp-5 loci in S. cerevtstae strain D4. A concentration-dependent increase in the frequency of gene convertants at both loci was observed. A 2.7- and 2.2-fold increase over the control at the ade-2 and trp-5 loci, respectively, were induced by extracts from seedlings treated with 60 ppm atrazine. The plant extracts were analyzed with HPLC and the fractions were collected in water, 50% methanol and 100% methanol. The HPLC fractions of the mmze extracts of each control and treatment group were assayed with S. cerevtstae. Concentration-dependent genotoxic responses were obtained w~th the 50% methanol fraction from the treatment groups as compared to the control. This indicates that a plant-activated mutagen associated with atrazine treatment was recovered in that fraction. Finally m a recently completed study by Means et al. (1983) water-

soluble extracts from maize seedlings exposed to atrazine, simazine or cyanazine were mutagenic in S. typhlmurium strain TA100. These herbicides with or without mammalian $9 were refractory when assayed with strains TA98 or TA100 of Salmonella. Analysis with lyophilized, filtered plant extracts by gel permeation column chromatography indicated the mutagen is a small molecule (MW 1000). Fractionation of plant extracts from plants exposed to 100 ppm atrazine, simazine or cyanazine by reverse phase HPLC yielded a single fraction (50% methanol) that contamed enriched levels of mutagen. This HPLC fraction was not mutagenlc when obtained from control plant extracts. Experiments that involved the in vivo metabolism of 14C-labeled atrazine indicated over 90% of the label incorporated into the maize seedlings was present in the plant extracts and 97% of this activity was conserved in the filtered, lyophilized plant extracts used in the HPLC analysis. Approximately 89% of the HPLC chromatographicable 14C-ring label was detected in the mutagenic fraction. These data present a strong, direct and experimental link between these s-triazine herbicides and their metabolism into plant activated mutagens. The data presented in this paper agree with the general conclusion that atrazme, simazine and cyanazine are plant activated promutagens. It is of special importance that the mutagenlc properties of these herbicides are detectable in the field experiments using reverse mutation at the wx-C locus in maize as the genetic endpoint. Thus, these data clearly demonstrate that the mutagenicity of these agents is not only a laboratory phenomenon but is present in fields under the conditions of modern agricultural practice. In light of the data presented m this paper and the information contained in the literature we believe that the determination of the mutagenic properties of pesticides is necessary to reduce the mutagenic impact upon the environment due to modern agricultural practice. We stress the need for plant systems to be incorporated in the battery of biological assays used m the evaluation of the mutagenic properties of pesticides. The plant kingdom constitutes a significant component of the environment and to ignore plant systems during

244 the e v a l u a t i o n o f a g r i c u l t u r a l c h e m i c a l s is ill advised. W e also suggest t h a t in situ g e n e t i c assays f o r m o n i t o r i n g a g r i c u l t u r a l fields b e e m p l o y e d in t h e a p p r a i s a l o f the g e n o t o x i c p r o p e r t i e s o f pesticides. T h e analysis o f these a g e n t s at c o n c e n t r a t i o n s a n d u n d e r c o n d i t i o n s c o m m o n to a g r i c u l t u r e a r e i m p o r t a n t c o m p o n e n t s in a r e a s o n a b l e assessm e n t of e n w r o n m e n t a l m u t a g e n i c h a z a r d s .

Acknowledgements T h e a u t h o r s g r a t e f u l l y a c k n o w l e d g e the t e c h n i cal a s s i s t a n c e o f Ms. C. W e l l s a n d Ms. M. H o . T h e field facilities w e r e p r o v i d e d b y t h e D e p a r t m e n t of A g r o n o m y , U n i v e r s i t y o f Illinois. W e e s p e c i a l l y appreciate the contributions of Drs. D.E. A l e x a n d e r a n d F . W . Slife of t h e D e p a r t m e n t o f A g r o n o m y a n d Dr. M . D . W a t e r s , D i r e c t o r , G e n e t i c Toxicology Division, U.S.EPA. The authors thank Ms. E. v o n H a l l e , E n v i r o n m e n t a l M u t a g e n I n f o r m a t i o n C e n t e r , for h e r a s s i s t a n c e w i t h the r e v i e w o f t h e literature. T h i s r e s e a r c h was s u p p o r t e d b y U . S . E P A c o n t r a c t 68-02-2704.

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