Mutagenicity Assay with Salmonella typhimurium Revealing Biotransformation of Antiphytoviral Substances by Cell-free Plant Extract

Zentral bl. Mikrobiol. 144 (1989), 197-202 VES Gustav Fischer Verlag Jena

[Karl-Marx-Universitat Leipzig, Sektion Biowissenschaften, Wissenschaftsbereich Pflanzenphysiologie und Mikrobiologie, Leipzig, OOR; Zentralinstitut fur Genetik und Kulturpflanzenforschung der Akademie der Wissenschaften der OOR, Gatersleben]

Mutagenicity Assay with Salmonella typhimurium Revealing Biotransformation of Antiphytoviral Substances by Cell-free Plant Extract ADa EL-TARRAS, ROLF BRAUN, ECKART STENZ and GOTTFRIED SCHUSTER

Summary The mutagenic activity of four antiphytoviral substances was tested in reversion mutagenicity assays with a set of histidine auxotrophic strains of Salmonella typhimur ium by means of the preincubation method . A possible metabolic activation of the substances by cell free fractions from maize seedlings (5-l4-fraction) and for comparison from mouse liver (5-9 mix) was examined . None of the guanidine , phenyl urea and thiadiazole compound s exerted mutagenic activity in the bacterial strains in experiments without metabolic activation. Cyanoguanidine -and N-phenyl-Ncarboxyphen ylurea became mutagenic for Salmonella strain TA98 after metabolic activation by the 5-14 plant fraction . Both substances were not mutagenic in the presence of 5-9 mix made from mouse liver. The promutagen cyclophosphamide proved highly mutagenic in experiments with S-14 mediated plant metabolic activation.'This kind of bacterial mutagenicity assay is valuable in invest igations of potential agrochemicals, as the examples have shown.

Zusammenfassung Die mutagene Wirkung von 4 antiphytoviralen Substanzen wurde in Reversion stests an verschiedenen histidinauxotrophen Stammen von Salmonella typhimurium mit der Vorinkubationsmethode des Ames-Te stes untersucht. Es wurde die mogliche Aktivierung der Substanzen durch zellfrei e Fraktionen aus Maiskeimlingen (S- 14Fraktion) und zum Verglei ch aus Mauseleber (S-9-Mix) gepriift. Keine der Guanid in-, Phenylhamstoff- und Thiadiazol -Verbindungen hatte in den Bakterien stammen ohne metabolische Aktivierung mutagene Wirkung . Cyanoguanidin und N-Phenyl-N-carboxyphenyl -hams toff wurden fur den Salmonella-Stamm TA 98 mutagen nach metabolischer Aktivierung durch die S-14-Pfl anzenfraktion. Seide Substanzen waren unter Einwirkung von S-9-M ix aus Mauseleber fur keinen Bakterienstamm mutagen . Oas Promutagen Cyclophosphamid erwies sich als hoch mutagen bei metabolischer Aktivierung durch die pflanzliche S-14-Fraktion . Oiese Methode der bakteriellen Mutagenitatsprufung mit zellfreiem Pflanzenextrakt lst, wie die Ergebn isse zeigen, fur die Untersuchung potentieller Agrochemikalien bedeutsam.

Mutagenicity tests based on microbial indicators.are widely used in genetic toxicology, as they are simple to perform and results can be obtained rapidly. Incorporation of S-9 mix made from mammalian liver homogenate into bacterial systems is an established procedure to simulate mammalian metabolism (AMES et al. 1973; GARNER et al. 1972; Mq'CANN et al. 1975). Substitution of S-9 fraction by a fraction of cell-free plant extract is a further development (GENTILE et al. 1977, 1985) and can provide metabolic activation capacity of plans cells to a certain extend. Inthe present study, an attempt is made in Salmon ella typhimurium to assess the mutagenicity of a few antiphytoviral agents employing S-14 fraction from maize extract and for comparison S-9 mix from mouse liver homogenate .

198

A. EL-TARRAS et al.

Materials and Methods The bacterial strains used were Salmonella typhimurium his mutants TA 100 and TA 1535 which carry a basepair substitution as genetic marker and TA 98, TA 1537 and TA 1538 which carry frameshiftmutations. These strains derived from the culture collection of the Zentralinstitut fiir Genetik und Kulturpflanzenforschung Gatersleben (G .D.R.) and were kindly provided by Prof. B. N. AMES, Berkeley (U.S.A.). The following compounds with antiphytoviral activity (SCHUSTER 1988) were tested for mutagenicity in the bacterial strains: cyanoguanidine (CG, Chemiekombinat Bitterfeld, G.D.R.), guanidine-2,4-dichlorphenoxyacetate (G2,4-D, ZIMET lena, G.D.R.), N-phenyl-N-carboxyphenyl-p-urea (NPU, Fahlberg-List, Magdeburg, G.D.R.) and anilin-adamantyl-thiadiazol (AAT, Berlin-Chemie, G.D.R.). The direct acting mutagens dinitro-o-cresol (DNOC) and bis-(2-chloroethyl)-ammoniumchloride (NorHN2), as well as the promutagens N-nitrosomorpholin (NM), Nacetylaminofluorene (AAF) and cyclophosphamide (CP) were employed as positive controls. All compounds were dissolved in dimethylsulfoxide (DMSO) and this solvent was used as negative control too. Concentrations ranging from 0.01 to 10 mg per plate of each chemical were tested. S-9 mix was obtained according to the procedure of GARNER et al. (1972) and MARON and AMES (1983) using male mice. S-14 fraction from maize seedlings was prepared as detailed by WILDEMAN and NAZAR (1982) and WEAVER et al. (1983). The bacteria (0.2 ml of an overnight culture, about 3 X 109 cells/ml) were preincubated with 0.2 ml solution of test substance and 1.0 ml buffer, S-14 fraction or S-9 mix, respectively. After shaking (30 min, 37°C) 0.7 ml of this culture were mixed with 2 ml biotin-histidine supplemented soft agar and poured onto a minimal agar layer. The plates were incubated at 37°C for 48 h and then the revertant colonies were counted. Twofold increase of revertant colony counts compared to the negative control and a reproducible result were taken as indicative for mutagenic activity. Reduced numbers of bacterial colonies and the presence of microcolonies were interpreted as indicators of toxicity (AMES et al. 1975; MARON and AMES 1983). All tests were done with at least two parallel petri dishes and repeated once or more. For media and other details see EL-TARRAS et al. (1989).

Results The data on toxicity and mutagenicity of the antiviral substances CG, G2,4-D, NPU and AAT tested on five strains of S. typhimurium are given in Tables I to 4. CG slightly reduced the numbers of bacterial colonies in plates with 10, 5 or 2.5 mg/plate, respectively (Table 1). It did not exert mutagenic effects on tester strains incubated either in the presence or absence of activation system S-9 mix, although a small increase of colonies was observed in strain TA 1535 when tested with O. I, 0.5 and 1.0 mg ofCG per plate. Activation with S-14 maize fraction yielded positive results in the strain TA 98 when exposed to concentrations of 0.1, 1.a and 5.0 mg/p1ate CG. These effects were quite below those caused by the standard promutagen CP but reproducible and with colony counts more than threefold as compared to control counts. G2,4-D in higher concentration (2.5 to 5 mg/plate) decreased the number of revertant colonies in 4 of the 5 Salmonella strains when tested without metabolic activation indicative for toxicity (Table 2). The toxic effect was reduced when S-9 mix from mouse liver was present. In the presence of S-14 fraction, this compound exhibited concentration dependent toxicity down to I mg per plate. G2,4-D never increased the revertant count and is classified to be neither a mutagen nor a promutagen. Observations on the effect of NPU on the bacterial strains are summarized in Table 3. At concentrations of 5 and 10 mg per plate a decrease of the number of revertant colonies in strains TA 1535, TA 1538, TA98 and TA 100 was seen. The toxic effect was enhanced after activation with 5-14 or S-9 mix as indicated by the occurrence of microcolonies. At lower concentrations NPU was found to be without any effect. The substance exhibited slight mutagenic effects only in S. typhimurium TA 98 and only after activation with S-14 fraction from maize in one test concentration. AAT was found to be toxic at a dose of 5 or 10 mg per plate in strain TA 98 (Table 4). After metabolic activation of AAT with 5-14 maize fraction in the bacterial strain TA 100 a decrease in revertant counts occurred. It did not exhibit mutagenicity in any strain neither without metabolic activation nor in presence of 5-9 mix from mouse liver or 5-14 fraction from maize.

199

Mutagenicity Assay with Salmonella typhimurium

Table I. Effect of cyanoguanidine (CG) on his strains of Salmonella typhimurium after preincubation with and without cell free fractions for metabolic activation. Revertant colonies per plate after no activation

Strain of S. typhimurium

TA 1535

o (rug/plate)

32 40 38 79 49 53 9 IS

0.01 0.05 0.1 0.5 1.0 25 5.0 10.0 Positive control

958 a

TA 1537 12.0 12.5 13.0 16.5 16.0 11.5 5.0 6.5 220 b

TA 1538

TA 98

28.5 33.0 23.0 27.0 31.5 23.0 17.0 16.0 558 b

27.0 34.0 29.5 30.5 24.5 34.5 25.0 22.0 398 b

TA 100 133.5 118.0 118.5 137.0 102.0 123.0 119.0 71.5 985"

activation with mouse liver S-9 mix

activation with maize fraction S-14

TA 98

TA 98

TA 100

TA 100

10.0

72.5

13.0

78.0

10.0 9.0 9.5 7.0 9.5

70.0 70.0 63.5 81.5 61.5

47.0

85.5

42.0

70.5

53.0 19+M

80.5 60.5

946"

197d

231 e

404 e

M: microcolonies, a: NorHN2 0.1, b: DNOC 0.1, c: AAF 0.1, d: NM 0.58, e: CP 2.0 mg/plate. Table 2. Effect of guanidine-2,4-dichlorphenoxyacetate (G2,4-D) on his strains of Salmonella typhimurium after preincubation with and without cell free fractions for metabolic activation. Revertant colonies per plate after no activation

Strain of S. typhimurium

o (mg/plate) 0.01 0.05 0.1 0.5 1.0 2.5 5.0

TA 1535 34.0 27.0 34.5 55.0 37.5 44.0 19.0 11.0

TA 1537 10.5 15.5 7.5 7.5 10.5 11.5 4.0 10.5

TA 1538 18.0 17.5 18.0 21.5 18.0 15.0 15.0

6.5

TA 98 23.5 28.5 21.5 30.0 25.5 23.5 13.0 9.0

TA 100 101.5 93.0 94.0 106.0 93.5 86.0 66.5 26.5

activation with mouse liver S-9 mix

activation with maize fraction S-14

TA 98

TA 98

TA 100

7.0

88

23.0

142

10.0 9.5 5.0 11.5 4.5

76 110 77 88 64

14.5

118

10.5 4.0

30 8

10.0 Positive control

950 a

203 b

585 b

393 b

895 a

TA 100

177c

176d

152e

485"

a-e: see Table 1.

Discussion The data achieved with Salmonella typhimurium strains and presented in Tables I to 4 demonstrate that the promutagens AAF, NM and CP were transformed into mutagens not only with the standard mouse liver S-9 mix, but also with the S-14 fraction from maize seedlings. The activation of promutagens by plants has been discussed since 1966 (Vuu and GRANT 1966, 1967;

200

A.

EL-TARRAS

et ai.

Table 3. Effect of N-phenyl-N-carboxyphenyl-p-urea (NPU) on his strains of Salmonella typhimurium after preincubation with and without cell free fractions for metabolic activation. Revertant colonies per plate after no activation

Strain of S. typhimurium

o (mg/plate) 0.01 0.05 0.1 0.5 1.0 5.0 10.0 Positive control

TA 1535

TA 1537

TA 1538

9.0 6.5 3.5 6.0 8.0 9.5 8.0 9.5

21.5 23.0 23.0 23.5 18.0 16.0 16.0 8.0

16.0 16.5 15.5 12.0 21.0 31.5 19.0 7.0 1039"

212b

726b

TA 98 21.5 21.5 24.5 17.0 12.0 23.5 10.5 6.5 900 b

TA 100 112.0 107.0 87.5 103.5 93.5 92.5 70.0 149.0 502"

activation with mouse liver S-9 mix

activation with maize fraction S-14

TA 98

TA 98

TA 100

10.5

88.0

13.5 69.0 10.5 113.0 6.0 110.5 2+M 32+M O+M O+M 196c

214d

TA 100

13.0

78.0

16.5

95.5

32.5 21.5 8+M

90.5 92.0 O+M

213e

404 e

a-e: see Table 1. M: microcolonies. Table 4. Effect of anilin-adamantyl-thiadiazol (AAT) on his strains of Salmonella typhimurium after preincubation with and without cell free fractions for metabolic activation. Revertant colonies per plate after no activation

Strain of S. typhimurium

TA 1535

o (mg/plate)

18 11 13 16 14 13

0.1 0.5 1.0 2.5 5.0 10.0 Positive control

380"

TA 1537

TA 1538

8.5 4.5 7.5 11.0 7.5 6.5

21.0 16.0 23.0 21.0 18.0 22.0

250b

840b

TA 98 23.0 15.0 13.5 23.5 17.5 13.5 980 b

TA 100 87.0 102.0 71.0 88.5 98.5 78.5 680"

activation with mouse liver S-9 mix

activation with maize fraction S-14

TA 98

TA 98

9.0 6.5 7.5 6.5 10.0 6.5 15.0 196c

TA 100 108.0 101.0 99.0 93.5 116.0 133.0 19.0 214d

TA 100

22.5 19.5

171.0 62.5

18.0

78.0

152e

485 e

a: NorHN2 0.01, b: DNOC 0.1, c: AAF O.I, d: NM 0.58, e: CP 2.0 mg/plate.

LIANG et al. 1967), but the first systematic study by use of a microbial assay was done in 1976 by PLEWA and GENTILE. The in vivo biotranformation of promutagens into mutagens was also found to be possible by plants (PLEWA et al. 1984). The conversion of promutagen CP into mutagens by the cell free plant extract as demonstrated in our studies is a new contribution to the phenomenon of plant mediated mutagenicity of promutagens. Preliminary investigations done by short term heat treatment (10 min at 100°C) of S-14 fraction resulted in loss of its biotransformation capacity, suggesting evidence for an enzyme mediated activation. The enzymatic attack to CP may be an oxidative hydroxylation leading to a bifunctional alkylating metabolite (SLADEK 1973). For final conclusion it is, however, necessary to

Mutagenicity Assay with Salmonella typhimurium

201

investigate the role of various co-factors in the activation reaction of5-14 fraction from plant extracts. WILDEMAN and NAZAR (1982) reported that the metabolic effects provided by 5-14 fraction and by 59 mix from mouse liver could be to a certain extend similar. Our results (this paper and EL-TARRAS et al. 1989) suggest that the metabolic activation of promutagens by the plant 5-14 fraction is somewhat different compared to that of 5-9 mix. In the activity of5-9 mix cytochrome P-450 had been shown to be an essential component for activation (MARON and AMES 1983). We, like PLEWA et al. (1983), did not succeed in detecting cytochrome P-450 in metabolic active plant fraction 5-14. The data given for antiphytoviral agents demonstrate that CG, G2,4-D, NPU and AAT are not mutagenic in their original structure. Antiviral substances often are thought to interfere with nucleic acid biosynthesis and, therefore, could be mutagens. As shown, however, only CG and NPU exerted mutagenic acitivity in Salmonella typhimurium provided the substances are tested in the presence of maize fraction 5-14. Also some antiviral chemicals of the nucleobase analogue type were per se not mutagenic but might during metabolic activation with 5-14 fraction of maize seedlings or liver 5-9 mix, become mutagenic for Salmonella strains (EL-TARRAS et al. 1989). Ourresults with standard mutagens thus support the use of metabolic activation systems as derived from maize for bacterial mutagenicity assays. There are, however, some differences in the specific capacity of plant cell fraction 5-14 as compared to mouse Ii ver 5-9 mix as shown by the given data for CG and NPU. In conclusion, the 5-14 fraction from plant in microbial mutagenicity test systems should especially been used when testing agrochemicals.

References AMES, B. B., DURSTON, W. E., YAMASAKI, E., LEE, F. D.: Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc. Nat. Acad. Sci. 70 (1973), 2281-2285. - MCCANN, J., YAMASAKI, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalianmicrosome mutagenicity test. Mut. Res. 31 (1975), 347-364. EL-TARRAS, A., BRAUN, R., STENZ, E., SCHUSTER, G.: Mutagenicity of antiviral substances of nucleobase analogue type in Salmonella typhimurium employing metabolic activation by mouse liver homogenate or cell-free plant extracts. Zbl. Mikrobiol. 144 (1989), 191-196. GARNER, R. C., MILLER, E. c., MILLER, J. A.: Liver microsomal metabolism of aflatoxin B I to a reactive derivative toxic to Salmonella typhimurium TA 1530. Cancer Res. 32 (1972),2058-2066. GENTILE, J. M., GENTILE, G. J., TOWNSEND, S., PLEWA, J. M.: In vitro enhancement of the mutagenicity of-l-nitro-ophenylenediamine by plant 5-9. Environm. Mutagenesis 7 (1985), 73-85. - WAGNER, E., PLEWA, M. J.: The detection of weak recombinogenic activities in the herbicides alachlor and propachlor using a plant activation bioassay. Mut. Res. 48 (1977), ]]3-]]6. LJA NG,G. H. , FELTNER, K. C.; LIANG, T. S., MORILL, J. L. :Cytogenetic effects and responses of agronomic characters in grain sorghum (Sorghum vulgare PERS.) following atrazine application. Crop Sci. 7 (1967),245-248. MARON, D. M., AMES, B. N.: Revised methods for the Salmonella typhimurium test. Mut. Res. 113 (1983),173-215. MCCANN, J., SPINGARN, N. E., KOBORI, J., AMES, B. N.: Detection of carcinogens as mutagens: bacterial tester strains with R-factor plasmids. Proc. Nat. Acad. Sci. 72 (1975), 979-983. PLEWA, M. J., GENTILE, J. M.: The mutagenicity of atrazine: a maize-microbe bioassay. Mut. Res. 38 (1976), 287-292. WAGNER, E. D., GENTILE, G. J., GENTILE, J. M.: An evaluation of the genotoxic properties of herbicides following plant and animal activation. Mut. Res. 136 (1984),233-245. WEAVER, D. L., BLAIR, L. c., EGAN, B., LHOTKA, M., GENTILE, J. M.: The plant cel\lmicrobecoincubation assay. Environm. Mutagenesis 5 (1983),374. SCHUSTER, G.: Synthetic antiphytoviral substances. In: KURSTAK, E., MARUSYK, R. G., MURPHY, F. A., VAN REGENMORTEL, M. H. V. (eds.), Applied Virology Research, Vol. I, New York, London 1988, 265-283. SLADEK, N. E.: Evidence for an aldehyde possessing alkylating activitiy as the primary metabolite of cyclophosphamide. Cancer Res. 33 (1973), 651-658. Vuu, K. D., GRANT, W. F.: Morphological and somatic chromosomal aberration induced by pesticides in barley. Canad. J. Genet. Cytol. 8 (1966),31-34. - - Chromosomal aberration induced by pesticides in meiotic cell of barley. Cytologia 32 (1967),31-34. WEAVER, D. L., PLEWA, M. J., BLAIR, L. C., GENTILE, J. M.: A comparison of the activation of 2-aminofluorene among four species of cultured plant cells. Environm. Mutagenesis 5 (1983),374. 14 Zentralbl. Mikrobiol., Bd. 144. 3

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WILDEMAN, A. G., NAZAR, R. N.: Significance of plant metabolism in the mutagenicity and toxicity of pesticides. Canad. J. Genet. Cytol. 24 (1982), 437-449. Authors' addresses: Doz. Dr. sc. ECKART STENZ, Prof. Dr. sc. GOTTFRIED SCHUSTER, Sektion Biowissenschaften der Karl-MarxUniversitat Leipzig, WB Pflanzenphysiologie und Mikrobiologie, TalstraBe 33, Leipzig, DDR - 7010; Dr. ADEL ELTARRAS, Cairo University, Faculty of Agriculture, Department of Genetics, Cairo, AR Egypt; Prof. Dr. sc. ROLF BRAUN, Zentralinstitut fur Genetik und Kulturpflanzenforschung der AdW der DDR, CorrensstraBe 3, Gatersleben, DDR-4325.

Zentralbl. Mikrobiol. 144 (1989),202 VEB Gustav Fischer Verlag Jena

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