Genetic effects of herbicides: Induction of mitotic gene conversion in Saccharomyces cerevisiae

Mutation Research, 22 (I974) I I I - 1 2 o © Elsevier Scientific Publishing Company, A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s

III

G E N E T I C E F F E C T S OF H E R B I C I D E S : I N D U C T I O N OF MITOTIC G E N E CONVERSION IN S A C C H A R O M Y C E S C E R E V I S I A E

D. S I E B E R T AND E. L E M P E R L E *

Forstbotanisches Institut der Universitat Freiburg i. Br., * Staatliches Weinbauinstitut, Freiburg i. Br. ( W. Germany) (Received April 6th, 1973) (Revision received October 23rd, 1973)

SUMMARY

32 herbicides have been tested for their induction of mitotic gene conversion in a diploid strain of the ascomycete Saccharomyces cerevisiae heteroallelic at two loci. Two of these herbicides showed weak genetic activity: Reglone (I,i'-ethylene-2,2'dipyridylium dibromide, Diquat) and U 46 D-Fluid (2,4-dichlorophenoxyacetic acid, 2,4-D).

INTRODUCTION

Among the environmental chemicals with possible hazards to man, the pesticides play an important role, since residues of these substances m a y remain in food and be ingested by man 4. The great number of pesticides used today and their different formulations make it difficult to select those that are genetically active, i.e. induce mutations or recombinations 7. In an earlier study the results of a prescreening method for testing fungicides normally used in viticulture had been published: with a diploid strain of the yeast Saccharomyces cerevisiae heteroallelic in two gene loci (ade2, trp5) an induction of mitotic gene conversions had been investigated **. Here, with the same method, several widely used herbicides were tested. The induction of mitotic gene conversions in diploid yeast is strongly correlated with a mutagenic effect 3~, and this test reacts very sensitively with compounds which induce base-pair substitutions as well as frame-shift mutations la. This system has revealed the genetic activity of a large number of carcinogens 14,~1, pesticidesS,2L drugs*°,2*, ~3 and m a n y other chemical mutagens~l,~7, 32. The induction in mammals of point mutations, which at least partly consist of alterations of the DNA codons, can only be demonstrated with the so-called specific locus method, a very expensive and time-consuming routine test 5. It is not suitable for the investigation of a great number of substances with unknown genetic or nongenetic effects. Therefore the use of a submammalian prescreening method, i.e. with

112

D. S I E B E R T , E. LEMPERLF~

microorganisms or Drosophila, selective for intragenic alterations, has a highly inf o r m a t i v e value. MATERIAL AND METHODS

Strains The i n d u c t i o n of m i t o t i c gene conversion (intragenic recombination) was s t u d i e d with the diploid strain D4 of Saccharomvces cerevisiae heteroallelic at the two loci ade2 a n d trp5. The h a p l o i d c o m p o n e n t strains have the m a r k e r s : D4

t MA 2 o : ~ , gal2, ade2-2, trp5 12 / MD 2o:a, + , ade2-I, trp5 27

h'uI +

This strain requires, for growth, adenine a n d t r y p t o p h a n , b u t gene conversion creates cells no longer requiring either adenine or t r y p t o p h a n . The same strains had been used in earlier experiments 8,%14,2°-al,2~,2~-3a.

Media and culturing Cells were c u l t u r e d in 5 nil Y E P m e d i u m ( I % Difco y e a s t e x t r a c t , 2 % Difco b a c t o p e p t o n e a n d 2 °/o glucose) in test t u b e s at room t e m p e r a t u r e under shaking until t h e y reached late logarithmic phase. Samples of o . i ml from each t u b e were spread onto solid m e d i u m selective for c o n v e r t a n t s to find cultures with a high frequency of s p o n t a n e o u s c o n v e r t a n t s . These cultures were eliminated. Media selective for conv e r t a n t s a n d for scoring s u r v i v a l were s y n t h e t i c m e d i a 2~ s u p p l e m e n t e d with amino acids a n d nucleobases 17 a n d were solidified with Difco b a c t o a g a r (1.5%). Medium selective for c o n v e r t a n t s lacked either adenine or t r y p t o p h a n .

Treatment of cells All herbicides were t e s t e d as commercial p r e p a r a t i o n s because, in most cases only this form gave stable suspensions in water. The pesticide suspensions were prep a r e d with o.I M p o t a s s i u m p h o s p h a t e buffer (pH 7.0) or with citrate-HC1 buffer (pH 4.5). Cells from late logarithmic phase cultures were washed twice in distilled w a t e r ; s e d i m e n t s of a b o u t 5 . i o 7 cells were c o m b i n e d with 2 ml of herbicide suspensions. The m i x t u r e s were i n c u b a t e d in a w a t e r - b a t h at 25 _q= o.I ° with shaking. T r e a t m e n t s were t e r m i n a t e d b y diluting a n d three washings with distilled water. Then suspensions were p r e p a r e d with lO 7 cells/ml to plate on selective m e d i u m for conv e r t a n t s a n d with 2500 cells/ml for complete m e d i u m to d e t e r m i n e survival. Onto 5 plates of each m e d i u m o.I ml per plate were s p r e a d out. All plastic petri dishes were i n c u b a t e d at 25 °. The colonies were c o u n t e d 6 d a y s later. All e x p e r i m e n t s were perf o r m e d three times, a n d r e p r e s e n t a t i v e results are p r e s e n t e d here. RESULTS

32 herbicides f r e q u e n t l y used in practice were tested for an i n d u c t i o n of m i t o t i c gene conversion in two loci of a diploid Saccharomyces cerevisiae. The t r a d e names, c o m m o n a n d s y s t e m a t i c n a m e s as well as the formulae are given in Table I. According to their genetic effects, the herbicides can be d i v i d e d into two m a i n groups: those

GENETIC EFFECTS OF HERBICIDES

113

compounds that increase the conversion frequency per lO 6 survivors significantly over the control level (statistics from the tables of KASTENBAUMAND BOWMAN 12) and those TABLE I HERBICIDES INVESTIGATED (ALPHABETICALORDER) Trade name

Common name

Alipur

Chlorbufam (lO%) Butyn-i-yl-3-N-3chlorophenylcarbamate

Systematic name

C h e m i c a l structure

0It / cH3 '~NH-C-O--C~ /--

C--CH

Ct

Cycluron (15°i,)

Aretit

Dinoseb acetate

N-cyclo-octyl-N',N'dimethylurea

o

NH-C--N(CH3)2 NO2

2-sec. Butyl-4,6dinitrophenylacetate 02N

O-CO-CH 3

" ~ ,,,CzHs CH

NCH~

Basagran

Baygon

Bentazon

"Propoxur

3-Isopropyl-2,1,3-benzothiadiazinone-(4)-2,2dioxide

2- I sopropoxypneny>" " Nmethylcarbamate

~

/CH3

0

[ - / ~ "~"CH ~'~,'~"N "~u2CHI I H

O ~ O _ C _ NHH _ C H 3 O--CH-CH3 I CH3

Betanal

Phenmedipham

3-Methoxycarbonylaminophenyl-N-(3'-

~ - N H - ~ -~ O

carbamate

CH3

N-Butyn-(I )-y1-(3)-¢¢chloroacetanilide

~N-C-CH2CI

methyl-phenyl)Butisan

Caragard

Prynochlor

Terbuthylazin (24%o)

2-Chloro-4-ethylamino6-tert. butylamino-i,3, 5 -

triazine

Casoron

Diehlobenil

0

OCH3 /N~J,

2-Methoxy-4-ethyl"

2,6-Dichlorobenzonitrile

0 II

NH--C-OCH3

anlino-6-tert, butyl-

amino-i,3,5-triazine

Terbumeton (24°o)

0

~

(CH3)3C-N ~N~'N

/C2Hs N

H

Ct N~ N 1- I| /C2H5 (CH3)3C--N-'~N~ N \H CI C~

II 4

D. SIEBERT, E. LEMPERLE

TABLE I (continued) HERBICIDES INVESTIGATE[)(ALPHABETICALORDER) Trade name

Cotoran

Domatol

Common name

Fluometuron

.Systematic n a m e

N-(3-Trifluoromethylphenyl)-N',N'-dimethylurea

Amitrol (19%)

3-Amino-i,2,4-triazole

Simazin (39%)

2-Chloro-4,6-diethylamino- 1,3,5-triazine

C h e m i c a l structure

0

~ --N-C-N-(CH3)z CF3 HN-N I II

H-X',NI-NH2 Ct

N/I%N HsCz--NH--

Dowpon

Dalapon

Sodium-2,2-dichloropropionate

II

I C \N.~--NH-

Ct

I H3C--C--COONo I

Ct

Eptapur

Buturon

N-(4-chlorophenyl )-N'methyl-N'-butyn- I-yl3-urea

CH-CH$ I C=CH

Faneron

Bromophenoxim

3,5_Dibromo_4_hydroxy. benzald°xime-O-(2',4'dinitrophenyl)-ether

Br

O2N-~O-N=CH.~O H NO 2

Flurenol

Flurenol

Br

9-Hydroxyfluorenecarbonic acid-(9)

HO COOH Gatnon

Benzthiazuron

0

N-(2-Benzothiazolyl)N'-methylurea N//

Gesapax

Gesaprim

Ametryn

Atrazin

2-Methylmercapto- 4ethylamino-6-isopropylamino-i,3,5-triazine

2-Chloro-4-ethylamino6-isopropylamino- 1,3,5triazine

.,c /H ~ C - - H N , , ~ N H - C 2 H 5 H3C

C~ HC--NH / ~N~ ~,NH--C2H5 H3C

GENETIC EFFECTS OF HERBICIDES TABLE I

zz5

(continued)

HERBICIDES INVESTIGATED (ALPHABETICALORDER) Trade name

Gesatop

Common name

Simazin

Systematic name

Chemical structure

2-Chloro-4,6-diethylamino- 1,3,5-triazine

¢t

N&N HsC2--NH.~I"N~"-NH--C2H5

Grammoxone Paraquat

Hyvar-X

Bromacil

I, I'-Dimethyl- 4,4'-dipyridyliu mdi- (methylsulphate) 3-sec.

Butyl-5-bromo-6-

methyluracil

÷+ CH3 2(CH]- SO,0-

[H3C--N~-

Br~o'~C~,

HtC

0

CH3

C2Hs

Karmex

Diuron

N-(3'4-Dichl°r°-phenyl)N',N'-dimethylurea

Cl.--~NH-~--N(CH3)2 CN

Merpelan

Benzthiazuron (60 % )

Lenacil (12,5%)

N-(2-Benzothiazolyl)-

b'v'S'.

0I

N'-methylurea

I~/C-NH-~ C--NH--CH3

3-Cyclohexyl-5,6-trimethylene uracil

H2 q-'C2-(N'3~0__

H2 H2 0

Potablan

Monalide

a, ~-Dimethyl-valerianic acid p-chloro-anilide

Prefix

Chlorthiamid

2,6-Dichloro-thiobenzamide

Preforan

Fluorodifen

2'4'-Dinitr°-4-triflu°r°methyldiphenylether

Pyramin

Pyrazon

I-Phenyl-4-amino- 5chloropyridazone- (6)

C[.--~

0 H3 NH-C--C--C,H, CH3

C|

CS-NH2 Ct ~~

t

D. SIEBERT, E. LEMPERLE

116 T A B L E I (continued) ItERBICIDES INVESTIGATED (ALPHABETICAL ORDER)

Trade name

Common name

Reglone

Diquat

1, i '-Ethylene-2,2'-dipyridylium-dibromide

Ronstar

Oxadiazon

2-tert-Butyl-4-(2,4-dichloro-5-isopropyloxyphenyl)- 1,3,4 -oxadiazolin-(5)-one

Sencor

Tenoran

Metribuzin

Chemical structure

Systematic name

ct

(CH3lzHC_O /

"0

C(CH3)3

o

4-Amino-6-tert-butyl-3(nlethylthio)- i, 2, 4triazin-5-one

Chloroxuron

0"/

(CH3)3C ~ J . NH2 N'N SCH3

N-4-(4'-chlorophenoxy)phenyt)-N',N'-dimethyl-

O NH--C-N(CH3)2

C|~'--O--~

Llroa

T o r m o n a 80

2'4'5-T-amylester

2'4'5-Trichl°r°phen°xy-,~l acetate am,.

c~._

oft

O--CH2--C--OCsHn

ct CL

U 46 D-Fluid

2,4-D

2,4-Dichlorophenoxyacetic acid

Ct~C[ O-CH2-COOH

with no effect in the conversion test. Only two compounds, 2,4-D (U 46 D-Fluid) and Diquat (Reglone) increased the conversion frequency: 2,4-D to a 5-fold value in the ade2 locus and 6-fold in the trp5 locus, and Diquat induced a 7- and 4- to 5-fold increase over the respective control (Table II). When these results were compared with those from other known mutagens, including the fungicides Ortho-Phaltan, Dithane-Ultra and Polyram-Combi~L only a weak convertogenic effect was observed with the two herbicides mentioned. Tile other 3o herbicides tested in our experiments gave negative results. In Table I I I we divide them into two groups, the first without any influence upon the conversion frequency, the second with a slight but not significant increase of convertants in one or in both loci. TABLE li Saecharomyces cerevisiae, DIPLOID STRAIN D 4 Induction of mitotic gene conversion in the two unlinked loci ade2 and trp5 by herbicides. The corresponding control values are given in parentheses. Trade

Common

name

name

Reglone Diquat L1 46 D-Fluid 2,4-D

Convertants/zo ~ survivors, loci ade2 trp5

Survival Treatment conditions (%) pH Cone. Time (ppm) (h)

28.2 (3,8)

30.6 (6.8)

78

4"5

IOOO

I6

14.6 (2.8)

11. 4 11.9)

22

4.5

IOOO

16

G E N E T I C E F F E C T S OF H E R B I C I D E S TABLE

III

Saccharomyces cerevisiae, AND

117

DIPLOID STRAIN D 4 , HETEROALLELIC IN THE TYVO UNLINKED LOCI

ade2

trp5

N o n - i n d u c t i o n of m i t o t i c g e n e c o n v e r s i o n b y h e r b i c i d e s , T h e c o r r e s p o n d i n g c o n t r o l v a l u e s a r e g i v e n i n p a r e n t h e s e s . All e x p e r i m e n t s w e r e c a r r i e d o u t a t 25 °. G r o u p 1: c o n v e r s i o n f r e q u e n c y n e i t h e r in t h e acle2 n o r i n t h e trp5 l o c u s d i f f e r e n t f r o m t h e c o n t r o l v a l u e , G r o u p I I : c o n v e r s i o n f r e q u e n c y in o n l y o n e or in b o t h loci s l i g h t l y b u t n o t s i g n i f i c a n t l y i n c r e a s e d .

Trade name

Convertants/Io 6 survivors, loci ade2 trp5

Survival (%)

Treatment conditions C0nc. 7"ime pH (plm) (h)

Group I Caragard Domatol Dowpon Gesaprim Gesatop Karamex Pyramin Ronstar

4.I 4.0 2.9 3.2 2.1 3.4 2.8 3 .6

(7.0) (7.0) (3.5) (4.2) (4.2) (4 .1) (3.3) (4.5)

8.9 ii,I 4-5 8.3 4.9 5.0 5.5 4 .1

(I2.4) (12.4) (6.o) (8.4) (8.4) (8.1) (6.4) (5.4)

69 82 95 99 94 79 90 87

4.5 4.5 4.5 4.5 4.5 7.0 4.5 7.0

iooo iooo IOOO IOOO iooo iooo iooo IOOO

16 16 16 16 16 I6 16 2

5 . 8 (3.8)

8.1 5.8 13.2 lO.9 12.5 7 .8 12.8 9.7 8.6 15.o 30.2 7.6 9-4 lO. 4 11.7 2. 4 5.9 14.2 8.1 2.9 7.4 14. 7

(6.8) (6.8) (8. i) (8.6) (19.2) (3.3) (14.1) (8.4) (12.4) (8.4) (11.2) (8.0) (8.4) (8.4) (12.4) (4.0) (8.0) (18.2) (8.4) (4.0) (8.3) (12.4)

80 68 66 80 24 25 iio 7° 71 5° lO2 76 84 25 46 89 58 lO 4 72 91 94 42

-t'5 4.5 7.0 7.0 4.5 7.0 7.0 4.5 4.5 4.5 7.0 4.5 4.5 7.0 4.5 7.0 7.0 7.0 4.5 7.0 7.0 7.0

1OOO IOO iooo iooo iooo iooo iooo iooo IOOO iooo ioo iooo lOOO iooo iooo iooo iooo iooo iooo iooo IOOO iooo

I0 16 16 i6 16 16 16 16 16 16 4 iO 16 2 16 16 16 16 I6 16 16 16

Group 1I Alipur Aretit Basagran Baygon Betanal Butisan Casoron Cotoran Eptapur Faneron Flurenol Gatnon Gesapax Grammoxone Hyvar X Merpelan Potablan Prefix Preforan Sencor Tenoran T o r m o n a 80

TABLE

5.8 3.6 9.2 14. 3 6.5 5 .0 6.3 7.2 7 .0 +6 5-3 7 .0 6.8 8.1 3.3 5.4 6.9 4.6 6.2 6.1 6. 4

(3.8) (4.1) (7.2) (II.I) (7.5) (4.4) (4.2) (7.o) (4 .2) (4.0) (4 .6) (4 .2) (6.1) (7.0) (3.2) (4 .6) (4.4) (4.2) (3.2) (4.2) (7.o)

IV

Saccharomyces cerevisiae, DIPLOID STRAIN D 4 I n a c t i v a t i o n of y e a s t b y h e r b i c i d e s . All e x p e r i m e n t s w e r e c a r r i e d o u t a t 2 5 .

Trade name

Betanal Butisan Aretit Flurenol Gramnloxone Ronstar

Survival (%)

Treatment conditions pH Conc. (ppm)

Time (h)

24 25 68 II 25 87

4-5 7-o 4-5 7.o 7.° 7.0

i6 16 16 4 2 2

i ooo i ooo i oo iooo IOOO iooo

II8

D. SIEBERT, E. LEMPERLE

An additional non-genetic effect was found with our method. In Table IV those herbicides are collected that show a high toxicity to yeast. Betanal and Butisan were used in a concentration of IOOOppm and with an exposure of 16 tl. A survival of only 24 and 25 % was observed. Under these conditions Aretit, Flurenol, Grammoxone and Ronstar gave no survival at all. Therefore the concentration of Aretit was lowered to ioo instead of IOOOppm and the exposure time of the three other compounds reduced to 2 or 4 instead of 16 h. The toxic effect of these herbicides may have been indirect, and due--through contamination of the soil--to the reduction of the activity of its microorganisms. DISCUSSION

Only a few of the herbicides used in our experiments or at least compounds of the same chemical group--have been previously investigated with cytogenetic or genetic methods: pyridylium salts tested by GUSJKOV et al. ~° and NARKEVITSCHet al. 1~ did not induce point mutations in bacteria, barley or Drosophila. Quaternary pyridylium salts gave positive results on chromosomes when they acted on the S-phase in the mitotic cycle ~. In our experiments Diquat (Reglone) was a weak convertogen. Of the chlorophenoxyacetic acids only the trichloro derivatives had been previously investigated: they produce chromosome aberrations in different plant species (refs. 3, 6, II). In the present paper Tormona was non-convertogenic. In contrast with this derivative, U 46 D-Fluid acted as a weak convertogen. Of the herbicides not included in our list, only 3,4-dichloropropione-anilide proved to be a true mutagen in Aspergillus 16. It is surprising that no cytogenetic or TABLE V HERBICIDES

WITH

CYTOGENETIC

OR GENETIC

EFFECTS

IN SUBMAMMALIAN

TEST SYSTEMS

Trade name

Common name

Nature qf effect

Subject

Ref.

Domatol Domatol

Simazine Simazine Atrazine Atrazine

Gesaprim Hyvar X Propanil Reglone Tormona Tormona

Atrazine isocil 3,4 -dichloropropanilide Diquat 2,4,5-TCA 2,4,5-TCA

barley barley seed barley barley seed barley barley Aspergillus

29 25

Gesaprim Gesaprim

chromosome aberrations n o n - i n d u c t i o n of chlorophyll mutations chromosome aberrations n o n - i n d u c t i o n of ehlorophyll m u t a t i o n s chromosome aberrations a b n o r m a l meiosis back- m u t a t i o n

yeast Allium Drosophila

present paper 2 3

Tormona Tormona U 46 D - F l u i d U 46 D - F l u i d

2,4,5-TCA 2,4,5-TCA 2,4-D 2,4-D

m i t o t i c gene conversion chromosome aberrations d i s t u r b a n c e of early oogenesis, sterility chromosome aberrations a b n o r m a l spindle a p p a r a t u s chromosome aberrations a b n o r m a l mitosis

O il 18 t9

U 46 D - F l u i d

2,4-D m i t o t i c gene conversion q u a t e r n a r y pyridi- c h r o m o s o m e m u t a t i o n s in n i u m salts S-phase p y r i d i n i u m salts n o n - i n d u c t i o n of p o i n t mutations

Vicia Haemanthus Allium Tradescantia Vicia yeast barley

present paper i

Drosophila

Io

3o 25 26 3° 16

GENETIC EFFECTS OF HERBICIDES

119

genetic tests of herbicides have been performed yet with mammalian subjects and methods relevant to man; only bacteria, fungi--including our own results--higher plants and Drosophila have been used. In Table V the data with these methods are cellected from publications known to us. The high number of herbicides which gave negative results in our prescreening test with submammalian organisms cannot yet be regarded as genetically inactive: they also should be tested with relevant methods. Before these experiments are performed no statement should be made about the genetic hazard to man of these herbicides. ACKNOWLEDGEMENT This work was supported

by the Deutsche

Forschungsgemeinschaft.

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