pKM101: detection of 31 oxidative mutagens

Mutation Research 467 Ž2000. 41–53 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres

Mutagenicity of 80 chemicals in Escherichia coli tester strains IC203, deficient in OxyR, and its oxyRq parent WP2 uÕrArpKM101: detection of 31 oxidative mutagens Alicia Martınez, Amparo Urios, Manuel Blanco ) ´ Instituto de InÕestigaciones Citologicas, Fundacion Amadeo de Saboya 4, ´ ´ Valenciana de InÕestigaciones Biomedicas, ´ 46010 Valencia, Spain Received 14 December 1999; received in revised form 8 February 2000; accepted 10 February 2000

Abstract Strain IC203, deficient in OxyR, and its oxyRq parent WP2 uÕrArpKM101 Ždenoted IC188. are the basis of a new bacterial reversion assay, the WP2 Mutoxitest, which has been used in the evaluation of 80 chemicals for oxidative mutagenicity. The following 31 oxidative mutagens were recognized by their greater mutagenic response in IC203 than in IC188: Ž1. peroxides: hydrogen peroxide ŽHP., t-butyl hydroperoxide ŽBOOH. and cumene hydroperoxide ŽCOOH.; Ž2. benzoquinones ŽBQ.: 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3,5,6-tetramethyl-1,4-BQ; Ž3. naphthoquinones ŽNQ.: 1,4-NQ, 2-methyl-1,4-NQ and 2-hydroxy-1,4-NQ; Ž4. phenol derivatives: catechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, t-butylhydroquinone, gallic acid and 4-aminophenol; Ž5. catecholamines: DL- and L-dopa, DL- and L-epinephrine, dopamine and L-norepinephrine; Ž6. thiols: L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol; Ž7. diverse: 3,4-dihydroxyphenylacetic acid, hypoxanthine and xanthine, both in the presence of xanthine oxidase, L-ascorbic acid plus copper ŽII. and phenazine methosulfate. Among these oxidative mutagens, 25 were found to be uniquely positive in IC203. With the exception of BOOH and COOH, mutagenesis by all oxidative mutagens was inhibited by catalase present in rat liver S9, indicating that it is mediated by HP generation, probably in autoxidation reactions. These catalase-sensitive oxidative mutagens were poor inducers of mutations derived from 8-oxoguanine lesions, whereas such mutations were efficiently induced by organic hydroperoxides. The results support the usefulness of incorporating IC203 in the bacterial battery for testing of chemicals. The well-characterized oxidative mutagens available with the use of the WP2 Mutoxitest may serve as a reference in studies on the genotoxicity of oxidative stress. q 2000 Elsevier Science B.V. All rights reserved. Keywords: WP2 Mutoxitest; Oxidative mutagens; OxyR; Peroxide; Quinone; Phenols; Catecholamine; Thiol

1. Introduction

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Corresponding author. Tel.: q34-96-3391252; fax: q34-963601453. E-mail address: [email protected] ŽM. Blanco..

Escherichia coli strains WP2rpKM101 and WP2 uÕrArpKM101 are the basis of the WP2 bacterial reverse mutation assay analogous to the Ames test w1x. These strains carry the trpE65 ochre mutation

1383-5718r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 0 0 . 0 0 0 2 0 - 6

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A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

Table 1 Compounds tested, code and CAS numbers, source and solvents Compound ŽCode No..

CAS No.

Source

Solvent

4-Acetamidophenol Ž48. N-Acetyl-L-cysteine Ž55. Acrolein Ž78. Alloxan Ž2,4,5,6-tetraoxypyrimidine. Ž63. 2-Aminophenol Ž45. 3-Aminophenol Ž46. 4-AminophenolP HCl Ž47. AnilineP HCl Ž50. L-Ascorbic acid Ž60. Bathocuproinedisulfonic acid, disodium salt Ž65. 1,2,4-Benzenetriol Ž24. Benzophenone Ž69. 1,4-Benzoquinone Ž5. Benzoyl peroxide Ž4. t-Butyl hydroperoxide Ž2. t-Butyl hydroquinone Ž25. Caffeic acid Ž34. Ž".-Catechin Ž40. Catechol Ž1,2-benzenediol. Ž20. Chromium trioxide Ž75. Ciprofloxacin Ž64. Cumene hydroperoxide Ž3. Cupric sulfate Ž73. L-Cysteine ethyl ester P HCl Ž53. L-Cysteine methyl ester P HCl Ž52. Deferoxamine mesylate Ž72. Diethyldithiocarbamate, sodium salt Ž57. Diethylstilbestrol Ž41. 1,4-Dihydroxyanthraquinone ŽQuinizarin. Ž17. 2,3-Dihydroxybenzoic acid Ž35. 3,4-Dihydroxyphenylacetic acid ŽDOPAC. Ž33. 2,3-Dimethoxy-1,4-naphthoquinone Ž14. 2,6-Dimethyl-1,4-benzoquinone Ž7. Dimethyl sulfoxide ŽDMSO. Ž66. Dithiothreitol ŽDTT. Ž56. DL-Dopa Ž27. L-Dopa Ž28. DopamineP HCl Ž29. DoxorubicinP HCl Ž16. EDTA–iron ŽIII. –sodium salt Ž74. Emodin Ž6-methyl-1,3, 8-trihydroxyanthraquinone. Ž18. DL-Epinephrine Ž31. L-Epinephrine Ž32. b-Estradiol Ž42. Gallic acid Ž3,4,5-trihydroxybenzoic acid. Ž37. Glyoxal Ž67. L-Histidine Ž70. DL-Homocysteine Ž51. Hydrogen peroxide Ž1. Hydroquinone Ž1,4-benzenediol. Ž22. 5-Hydroxy-2-methyl-1,4-naphthoquinone ŽPlumbagin. Ž13.

103-90-2 616-91-1 107-02-8 2244-11-3 95-55-6 591-27-5 51-78-5 142-04-1 50-81-7 52698-84-7 533-73-3 119-61-9 106-51-4 94-36-0 75-91-2 1948-33-0 331-39-5 7295-85-4 120-80-9 1333-82-0 85721-33-1 80-15-9 7758-98-7 868-59-7 18598-63-5 138-14-7 20624-25-3 56-53-1 81-64-1 303-38-8 102-32-9 527-61-7 67-68-5 27565-41-9 63-84-3 59-92-7 62-31-7 25316-40-9 15708-41-5 518-82-1

Sigma Sigma Sigma Sigma Aldrich Aldrich Sigma Sigma Sigma Sigma Aldrich Sigma Sigma Sigma Sigma Aldrich Sigma Sigma Sigma Sigma Bayer Sigma Merck Sigma Sigma Sigma Sigma Sigma Sigma Sigma Sigma Calbiochem Aldrich Sigma Sigma Sigma Sigma Sigma Pharmacia Sigma Sigma

W W D Ž100 mgrml., then W W D Ž100 mgrml., then W D Ž100 mgrml., then W W D Ž100 mgrml., then W W W W D Ž2 mgrml., then W D Ž10 mgrml., then W D Ž1 mgrml., then W W W D Ž100 mgrml., then W D Ž100 mgrml., then W W W W D Ž10 mgrml., then W W W W W W D Ž0.5 mgrml., then W D Ž0.25 mgrml., then W D Ž100 mgrml., then W W D Ž1 mgrml., then W D Ž10 mgrml., then W W W NaOH Ž0.5 N. Ž50 mgrml., then W NaOH Ž0.5 N. Ž50 mgrml., then W W W W D

329-65-7 51-43-4 50-28-2 149-91-7 107-22-2 71-00-1 52-90-4 7722-84-1 123-31-9 481-42-5

Sigma Sigma Sigma Sigma Sigma Merck Sigma Merck Sigma Sigma

NaOH Ž0.5 N. Ž50 mgrml., then W NaOH Ž0.5 N. Ž50 mgrml., then W D Ž1 mgrml., then W D Ž200 mgrml., then W W W W W W D Ž1 mgrml., then W

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

43

Table 1 Ž continued . Compound ŽCode No..

CAS No.

Source

Solvent

2-Hydroxy-1,4-naphthoquinone Ž11. 5-Hydroxy-1,4-naphthoquinone Ž12. Hypoxanthine Ž58. Melatonin Ž71. 2-Methyl-1,4-benzoquinone Ž6. Methyl gallate Ž39. 2-Methyl-1,4-naphthoquinone ŽMenadione. Ž10. Methyl viologen ŽParaquat. Ž80. 1,4-Naphthoquinone Ž9. o-Nitrophenol Ž43. 2-Nitropropane Ž68. L-Norepinephrine Ž30. L-Penicillamine Ž54. Phenacetin Ž49. Phenazine ethosulfate Ž61. Phenazine methosulfate Ž62. Phenol Žhydroxybenzene. Ž19. Potassium bromate Ž77. n-Propyl gallate Ž38. Protocatechuic acid Ž3,4-dihydroxybenzoic acid. Ž36. Pyrogallol Ž1,2,3-benzenetriol. Ž23. Resorcinol Ž1,3-benzenediol. Ž21. Salicylic acid Ž79. Stannous chloride Ž76. Streptonigrin Ž15. 2,3,5,6-Tetramethyl-1,4-benzoquinone ŽDuroquinone. Ž8. 2,4,6-Trinitrophenol ŽPicric acid. Ž44. DL-Tyrosine Ž26. Xanthine Ž59.

83-72-7 481-39-0 68-94-0 73-31-4 553-97-9 99-24-1 58-27-5 1910-42-5 130-15-4 88-75-5 79-46-9 51-41-2 1113-41-3 62-44-2 10510-77-7 299-11-6 108-95-2 7758-01-2 121-79-9 99-50-3 87-66-1 108-46-3 69-72-7 10025-69-1 3930-19-6 527-17-3 88-89-1 60-18-4 69-89-6

Sigma Sigma Sigma Sigma Aldrich Aldrich Sigma Sigma Aldrich Sigma Aldrich Sigma Sigma Sigma Sigma Sigma Sigma Aldrich Sigma Sigma Sigma Sigma Sigma Merck Sigma Sigma Sigma Sigma Sigma

D Ž5 mgrml., then W D Ž1 mgrml., then W NaOH Ž0.1 N. Ž10 mgrml., then W D Ž100 mgrml., then W D Ž20 mgrml., then W D Ž200 mgrml., then W D Ž1 mgrml., then W W D Ž2 mgrml., then W D Ž100 mgrml., then W D Ž100 mgrml., then W NaOH Ž0.5 N. Ž50 mgrml., then W W D Ž20 mgrml., then W W W W W D Ž100 mgrml., then W D Ž100 mgrml., then W W W D Ž100 mgrml., then W HCl Ž0.1 N. Ž100 mgrml., then W D Ž1 mgrml., then W D Ž1 mgrml., then W W NaOH Ž1 N. Ž100 mgrml., then W NaOH Ž0.1 N. Ž10 mgrml., then W

Solvent: W, water; D, DMSO.

and detect a spectrum of mutagens similar to Salmonella typhimurium strain TA102, which carries the hisG428 ochre mutation w2,3x. In the bacterial test battery for the testing of chemicals, WP2 strains as well as TA102 are recommended for the screening of oxidising mutagens w4,5x. All these strains, however, would be endowed with antioxidant defenses that could interfere with the detection of mutagenesis promoted by reactive oxygen species ŽROS.. In order to increase the sensitivity for the detection of mutations induced by oxidative damage, we have recently developed the new tester strain IC203, a derivative of WP2 uÕrArpKM101 deficient in the OxyR function w6x. The OxyR deficiency prevents the oxidative stress-induced synthesis of antioxidant enzymes such as catalase-peroxidase, alkyl hydroperoxidase and glutathione reductase w7,8x. This defect is assumed to determine an enhancement in the mutagenesis resulting from DNA lesions caused by ROS w6x.

The WP2 mutagenicity test performed with strains WP2 uÕrArpKM101 Žhereafter denoted IC188. and IC203 is called the WP2 Mutoxitest. It has proved to be useful in preliminary validation assays designed to compare the sensitivity of strain IC203 with that of IC188 for the detection of mutagenesis by oxidants like t-butyl hydroperoxide ŽBOOH. and 2methyl-1,4-naphthoquinone Žmenadione. w6x, hydrazine derivatives w9x and thiols w10x. In a further validation study of strain IC203, we have evaluated the mutagenicity of 80 chemicals of diverse classes and structures, preferentially including well-known oxidative agents Ži.e. producing ROS. such as peroxides, quinones, phenol derivatives, catecholamines and thiols. The results confirm the usefulness of the WP2 Mutoxitest for both the detection of oxidative mutagens and the characterization of mechanistic aspects of the activity of these mutagens.

44

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

2. Materials and methods

2.1. Bacterial strains Strains were WP2 uÕrArpKM101, denoted IC188, and its derivative IC203 w6x, carrying the D oxyR30 mutation w11x. Both tester strains carry the trpE65 ochre mutation and can be reverted to Trpq by base substitutions at the A:T base pair in the trpE65 site or at extragenic ochre suppressor loci containing A:T or G:C base pairs at the reversion site Žw10,12x, unpublished results.. The two strains contain the SOS mutagenesis proteins UmuDC, encoded in the chromosome, and MucAB, encoded by the pKM101 plasmid. The following strains devoid of SOS mutagenesis proteins w6x were also used: IC204, a derivative of WP2 uÕrA carrying a D umuDC::cat mutation; IC206, a mutY::kan derivative of IC204; IC208, a D oxyR30 derivative of IC206.

2.2. Media and chemicals Nutrient broth was Oxoid Nutrient Broth No. 2. LA medium contained 5 g NaCl, 10 g Difco Bacto tryptone, 5 g Difco yeast extract and 20 g Difco agar per litre of distilled water. Solid minimal E4 medium contained 15 g Difco agar and 4 g glucose per litre of Vogel-Bonner E medium w13x. ET4 medium was solid minimal E4 medium supplemented with 0.5 mg tryptophan per litre. Top agar contained either 6 g Difco agar and 5 g NaCl per litre of distilled water for mutagenicity assays or 7.5 g Difco agar per litre of distilled water for cytotoxicity tests. S9 liver homogenate was prepared from uninduced rats as described w13x. Compounds tested, code and CAS numbers, and solvents used are indicated in Table 1.

strain, 100 ml of a suitable dilution of the test compound, 50 ml of S9 Žwhen indicated. and 2.5 ml of molten top agar. The mixture was poured on minimal ET4 plates. These were incubated 2 days at 378C. In assays with hypoxanthine and xanthine, 0.1 U per plate of xanthine oxidase ŽSigma. were added. In assays with ascorbic acid, an ascorbic acid q cupric sulfate solution Žmolar ratio 100:1. was used. To evaluate the number of preplating mutants originated during the overnight growth, the zero dose in the mutagenesis assay was screened on unsupplemented E4 plates. Nutrient broth was supplemented with 20 mgrml ampicillin for the overnight cultures of strains containing pKM101. Each compound was tested at least twice with five to six dose levels, including a toxic dose, and the means of Trpq revertants per plate are presented. A positive result is defined as a reproducible, dose-related increase in the number of revertants. The increase should reach at least a doubling of the number of spontaneous revertants. 2.4. Cytotoxicity tests For growth inhibition experiments, 100 ml of overnight broth cultures and 50 ml of S9 Žwhen indicated. were added to 3 ml of molten top agar and poured on LA plates. Paper discs Ž6 mm in diameter. were impregnated with 10 ml of solutions containing test compounds, placed on the solidified top agar plates and allowed to incubate overnight. The diameter of the zone of inhibition Žin mm. was obtained by measuring the diameter Žincluding the disc. of the zone and subtracting the diameter of the disc.

3. Results

2.3. Plate reÕerse mutation assays

3.1. Mutagenicity of 80 chemicals in the WP2 Mutoxitest

Plate incorporation assays were performed by mixing 100 ml of broth cultures Ž100 ml from frozen permanents inoculated into 10 ml of nutrient broth and incubated overnight at 378C. of each tester

Table 2 shows the individual data of mutagenic activity of the 80 compounds evaluated with the WP2 Mutoxitest. For compounds giving either negative or positive non-oxidative responses, only data at

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

the highest dose applied with no observable toxicity are indicated. According to the results of the test, the chemicals were segregated into three classes: Ž1. 31 compounds causing oxidative mutagenesis: the mutagenicity was higher in IC203 than in IC188; Ž2. six compounds causing non-oxidative mutagenesis: the mutagenicity was similar in both tester strains; Ž3. 43 compounds non-mutagenic under the assay conditions. BOOH and menadione, two reference oxidative mutagens w6x, were included as positive controls, BOOH representing oxidants that induce a higher number of revertants in IC203 than in IC188, and menadione representing oxidative mutagens whose response is positive in IC203 but negative in IC188. Also shown in Table 2 is the effect of liver S9 on the mutagenicity occurring in IC203. This effect is assumed to result from the activity of catalase present in the S9 w9,10x. Besides the control BOOH, two peroxides, hydrogen peroxide ŽHP. and cumene hydroperoxide ŽCOOH. reverted IC203 more efficiently than IC188 and were classed as oxidative mutagens. As expected, S9 inhibited mutagenesis by HP but not by BOOH or COOH. Benzoyl peroxide, the fourth peroxide analysed, was non-mutagenic. Of the four benzoquinones ŽBQ. evaluated, 2methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and 2,3,5,6-tetramethyl-1,4-BQ Žduroquinone. were classed as oxidative mutagens, their responses being positive only in IC203 and sensitive to inhibition by S9, whereas 1,4-BQ was non-mutagenic. For the naphthoquinone ŽNQ. derivatives, in addition to the control menadione, 1,4-NQ and 2-hydroxy-1,4-NQ were oxidative mutagens, giving positive responses, sensitive to inhibition by S9, only in IC203. The other three NQ tested, 5-hydroxy-1,4-NQ, 5-hydroxy-2-methyl-1,4-NQ Žplumbagin. and 2,3-dimethoxy-1,4-NQ were non-mutagenic. Two anti-tumor agents, streptonigrin, which has an aminoquinone fragment, and doxorubicin, having both a quinone and a hydroquinone group, induced similar mutagenic responses in IC188 and IC203 and were classed as non-oxidative mutagens. Two anthraquinones, quinizarin, which is structurally related to doxorubicin, and emodin were non-mutagenic. Phenol Žhydroxybenzene. was non-mutagenic, whereas several of its derivatives like catechol Ž1,2benzenediol., hydroquinone Ž1,4-benzenediol., pyro-

45

gallol Ž1,2,3-benzenetriol., 1,2,4-benzenetriol and tbutylhydroquinone were oxidative mutagens, all these compounds except pyrogallol being negative toward IC188. Mutagenesis by these phenols was inhibited by S9, although this inhibition was not complete for pyrogallol. Resorcinol Ž1,3-benzenediol., in contrast to the other phenols with two hydroxyl groups, was non-mutagenic. The six catecholamines analysed, DL- and L-dopa, dopamine, L-norepinephrine and DL- and L-epinephrine were oxidative mutagens. Their mutagenicities were sensitive to inhibition by S9 and none were mutagenic in IC188. 3,4-Dihydroxyphenylacetic acid ŽDOPAC., the main dopamine metabolite, was also an oxidative mutagen exhibiting absence of mutagenicity toward IC188 and inhibition by S9. DLTyrosine, an amino acid precursor of DL-dopa, was non-mutagenic. Of the other catechol-related compounds, gallic acid caused oxidative mutagenesis, being positive only in IC203 and sensitive to inhibition by S9, whereas gallic acid esters like n-propyl gallate and methyl gallate were non-mutagenic. Negative results were also found for caffeic acid, 2,3-dihydroxybenzoic acid and protocatechuic acid, as well as for Ž".-catechin, and two catecholestrogens, diethylstilbestrol and b-estradiol. Among other phenols, 4-aminophenol was an oxidative mutagen giving a positive response, that was inhibited by S9, in IC203, and a weak response in IC188. Other phenol derivatives like o-nitrophenol, 2,4,6-trinitrophenol, 2-aminophenol, 3-aminophenol, 4-acetamidophenol, phenacetin and aniline, were non-mutagenic. The evaluation of seven thiol compounds indicated that four of them, L-cysteine methyl ester, L-cysteine ethyl ester, L-penicillamine and dithiothreitol ŽDTT. were oxidative mutagens. The two cysteine esters and DTT were non-mutagens in IC188 whereas in this strain, L-penicillamine was positive. Mutagenesis by these thiols, except at high L-penicillamine doses, was inhibited by S9. N-acetyl-L-cysteine and the metal chelator diethyldithiocarbamate ŽDETC. were classed as non-mutagenic. DL-Homocysteine was also non-mutagenic although it gave a positive response in the presence of low concentrations of DETC, this response being similar to that reported for cysteine w10x.

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

46

Table 2 Mutagenicity results of 80 compounds in the WP2 Mutoxitest Compound

1

– Hydrogen peroxide

Test result qOX

2

t-Butyl hydroperoxide

qOX

3

Cumene hydroperoxide

qOX

4 5 6

Benzoyl peroxide 1,4-Benzoquinone 2-Methyl-1,4-benzoquinone

y y qOX

7

2,6-Dimethyl-1,4-benzoquinone

qOX

8

2,3,5,6-Tetramethyl-1,4-benzoquinone ŽDuroquinone.

qOX

9

10

11

1,4-Naphthoquinone

qOX

2-Methyl-1,4-naphthoquinone ŽMenadione.

qOX

2-Hydroxy-1,4-naphthoquinone

qOX

12 13 14 15 16 17 18 19 20

5-Hydroxy-1,4-naphthoquinone 5-Hydroxy-2-methyl-1,4-naphthoquinone ŽPlumbagin. 2,3-Dimethoxy-1,4-naphthoquinone Streptonigrin Doxorubicin 1,4-Dihydroxyanthraquinone ŽQuinizarin . Emodin Ž6-methyl-1,3,8-trihydroxyanthraquinone. Phenol Žhydroxybenzene. Catechol Ž1,2-benzenediol.

y y y q q y y y qOX

21 22

Resorcinol Ž1,3-benzenediol. Hydroquinone Ž1,4-benzenediol.

y qOX

23

Pyrogallol Ž1,2,3-benzenetriol.

qOX

24

1,2,4-Benzenetriol

qOX

Dose Žmgrplate.

Number of revertantsrplate IC188 IC203 IC203q S9

– 25 50 100 25 50 100 12.5 25 50 50 10 20 30 40 20 30 40 40 50 60 6 8 16 10 20 30 50 100 150 5 50 60 0.2 40 100 100 1000 1000 2000 3000 3000 50 100 200 25 50 100 25 50 100

145 179 248 289 248 496 864 256 396 717 186 141 151 133 150 145 142 142 133 121 122 126 131 125 150 140 130 110 112 110 124 109 145 1292 826 93 150 90 124 124 94 111 160 162 184 186 230 290 139 128 141

144 378 787 968 699 1375 2178 541 1076 1476 162 150 240 300 316 214 281 489 200 272 376 178 198 413 219 693 957 237 489 677 116 230 265 1210 680 90 149 143 241 414 793 149 219 402 821 332 569 981 214 466 1121

151 183 190 714 1880

1324 1724

234 206 210 146 188 179 187 149 193 163 166 130 138 170 138

158 150 171 198 176 160 293 312 198 214

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

47

Table 2 Ž continued .

25

Compound

Test result

Dose Žmgrplate.

Number of revertantsrplate IC188 IC203 IC203q S9

t-Butylhydroquinone

qOX

50 100 150 1000 500 750 1000 500 750 1000 2500 5000 6000 500 750 1000 250 500 750 250 500 600 500 750 1000 1000 1000 1000 500 750 1000 100 750 5000 25 1000 1000 1000 750 750 250 500 1000 1500 2000 1000 5000 125 250 500

91 129 128 172 194 225 215 211 223 218 157 144 180 180 152 135 173 150 157 145 170 185 153 141 129 112 141 148 175 160 165 280 275 132 134 135 99 126 205 130 155 177 260 268 120 160 109 106 100 105

26 27

DL-Tyrosine DL-Dopa

y qOX

28

L-Dopa

qOX

29

Dopamine

qOX

30

L-Norepinephrine

qOX

31

DL-Epinephrine

qOX

32

L-Epinephrine

qOX

33

3,4-Dihydroxyphenylacetic acid ŽDOPAC.

qOX

34 35 36 37

Caffeic acid 2,3-Dihydroxybenzoic acid Protocatechuic acid Ž3,4-dihydroxybenzoic acid. Gallic acid Ž3,4,5-trihydroxybenzoic acid.

y y y qOX

38 39 40 41 42 43 44 45 46 47

n-Propyl gallate Methyl gallate Ž".-Catechin Diethylstilbestrol ŽDES. b-Estradiol o-Nitrophenol 2,4,6-Trinitrophenol ŽPicric acid. 2-Aminophenol 3-Aminophenol 4-Aminophenol

y y y y y y y y y qOX

48 49 50 51 52

4-Acetamidophenol Phenacetin Aniline DL-Homocysteine L-Cysteine methyl ester

y y y y qOX

147 234 511 179 283 440 502 323 445 529 321 508 917 200 217 370 226 364 964 177 385 780 274 350 696 172 135 188 200 302 616 265 273 152 142 104 129 113 250 170 185 304 1009 245 160 156 159 173 342 1054

155 120 187 215 289 209 249 290 170 130 132 198 193 220 172 195 268 162 168 136 170 173 173

175 192 160

165 180 234

110 122 (continued on next page)

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

48 Table 2 Ž continued . Compound ethyl ester

Test result

Dose Žmgrplate.

Number of revertantsrplate IC188 IC203 IC203q S9

qOX

250 500 750 250 500 1000 1000 100 200 400 100 25 50 100 50 100 200 200 300 400 100 25 50 75 2000 0.025 1000 1000 500 4000 200 5000 5000 500 3 5000 40 1000 100 25 100 1

117 130 110 131 178 345 117 141 145 143 188 193 214 217 203 222 226 140 175 160 133 172 160 170 107 386 109 110 817 477 112 225 122 106 105 200 1244 102 164 122 133 168

53

L-Cysteine

54

L-Penicillamine

qOX

55 56

N-Acetyl-L-cysteine Dithiothreitol ŽDTT.

y qOX

57 58

Diethyldithiocarbamate ŽDETC. Hypoxanthineq XAO Ž0.1 U.

y qOX

59

Xanthineq XAO Ž0.1 U.

qOX

60

L-Ascorbic

61 62

Phenazine ethosulfate Phenazine methosulfate

y qOX

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80

Alloxan Ž2,4,5,6-tetraoxypyrimidine. Ciprofloxacin Bathocuproinedisulfonic acid Dimethyl sulfoxide ŽDMSO. Glyoxal 2-Nitropropane Benzophenone L-Histidine Melatonin Deferoxamine mesylate Cupric sulfate EDTA–iron ŽIII. –sodium salt Chromium trioxide Stannous chloride Potassium bromate Acrolein Salicylic acid Methyl viologen ŽParaquat.

y q y y q q y y y y y y q y y y y y

acid q copper ŽII.

qOX

226 814 1367 290 557 672 119 430 757 1230 195 257 544 1378 306 943 1388 259 851 1280 150 210 297 467 128 358 136 195 880 420 125 185 85 110 118 166 1319 110 193 183 182 187

175 137 138 157 315 519 156 175 131 165 226 240 261 170 136 100 165 225 239 239

qOX, oxidative mutagen; q, non-oxidative mutagen; y, non-mutagen.

In the presence of 0.1 Urplate of xanthine oxidase ŽXAO., both hypoxanthine ŽHXA. and xanthine ŽXA. caused oxidative mutagenesis that was sensitive to inhibition by S9. For HXA, but not for XA, the response was slightly positive toward IC188 at

high doses Žnot shown.. In the absence of XAO, neither HXA nor XA were mutagenic in IC203 Žnot shown.. Mutagenesis by L-ascorbic acid in the presence of copper ŽII. was observed only in IC203 and was

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

abolished by S9. Not shown in Table 2 is a positive response in IC203 for L-ascorbic acid alone that was attained at high doses Ž533 revertants at 2000 mgrplate.. Phenazine methosulfate was classed as an oxidative mutagen since it gives a positive mutagenic

49

response, sensitive to inhibition by S9, only in IC203, whereas phenazine ethosulfate was non-mutagenic. Among other 18 diverse compounds evaluated, none caused oxidative mutagenesis. Four of them, ciprofloxacin, glyoxal, 2-nitropropane and chromium trioxide were judged to be non-oxidative mutagens,

Table 3 Cytotoxic effect of oxidants: influence of rat liver S9 Compound

Dose Žmgrdisc.

Inhibition Žmm. IC188 IC203

IC203q S9

OxidatiÕe mutagens 1 Hydrogen peroxide 2 t-Butyl hydroperoxide 3 Cumene hydroperoxide 6 2-Methyl-1,4-benzoquinone 7 2,6-Dimethyl-1,4-benzoquinone 8 2,3,5,6-Tetramethyl-1,4-benzoquinone ŽDuroquinone. 9 1,4-Naphthoquinone 10 2-Methyl-1,4-naphthoquinone ŽMenadione. 11 2-Hydroxy-1,4-naphthoquinone 20 Catechol Ž1,2-benzenediol. a 22 Hydroquinone Ž1,4-benzenediol. 23 Pyrogallol Ž1,2,3-benzenetriol. 24 1,2,4-benzenetriol 25 t-Butylhydroquinone 27 DL-Dopa 28 L-Dopa 29 Dopaminea 30 L-Norepinephrine 31 DL-Epinephrine 32 L-Epinephrine 33 3,4-Dihydroxyphenylacetic acid ŽDOPAC. a 37 Gallic acid Ž3,4,5-trihydroxybenzoic acid. a 47 4-Aminophenola 52 L-Cysteine methyl ester 53 L-Cysteine ethyl ester 54 L-Penicillamine 56 Dithiothreitol ŽDTT. 58 Hypoxanthineq XAO Ž0.1 U. 59 Xanthineq XAO Ž0.1 U. 60 L-Ascorbic acid q copper ŽII. 62 Phenazine methosulfatea

300 300 300 50 50 500 50 50 50 2000 1000 1000 1000 1000 500 500 2000 1000 1000 1000 500 1000 500 500 1000 1000 500 100 100 1000 50

11 19 16 8 11 0 7 3 0 6 0 13 11 7 4 4 0 6 5 4 0 3 6 0 0 0 0 0 0 2 13

25 37 24 16 20 25 20 22 18 12 8 18 19 13 14 14 15 11 14 14 11 18 13 12 14 14 15 7 7 10 15

0 37 23 12 14 0 13 12 14 7 4 14 13 7 4 4 0 9 4 4 0 3 6 5 10 0 2 2 2 6 13

Non-mutagenic oxidants 5 1,4-Benzoquinone 12 5-Hydroxy-1,4-naphthoquinone 13 5-Hydroxy-2-methyl-1,4-naphthoquinone ŽPlumbagin. 14 2,3-Dimethoxy-1,4-naphthoquinone 51 DL-Homocysteine 55 N-Acetyl-L-cysteinea 80 Methyl viologen ŽParaquat. a

50 100 100 100 500 500 1000

9 7 6 0 0 0 10

17 11 15 9 10 8 14

11 8 14 6 0 0 10

a

Overnight cultures 5-fold diluted in nutrient broth were used.

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

50

whereas the remaining 14 compounds Žsee Table 2., were non-mutagens. 3.2. Cytotoxic effects of oxidants Compounds classed as oxidative mutagens according to the results of Table 2 were tested for cytotoxicity toward strains IC188 and IC203 ŽTable 3.. IC203 was more sensitive to the cytotoxic effects than was IC188 for all the compounds tested, thus confirming the good correlation between mutability and lethality of oxidants. The presence of S9 in

assays with IC203 had a protective effect in all cases except with the two organic hydroperoxides. Seven non-mutagenic compounds, showing enhanced toxicity in IC203, were also included in Table 3. Three NQ, 5-hydroxy-1,4-NQ, 5-hydroxy2-methyl-1,4-NQ Žplumbagin. and 2,3-dimethoxy1,4-NQ were less toxic than the highly mutagenic menadione, and their toxicity was weakly inhibited by S9, suggesting that it did not involve HP. A similar weak inhibition by S9 was found for 1,4-BQ and paraquat. In contrast, cytotoxicity by two nonmutagenic thiols, DL-homocysteine and N-acetyl-Lcysteine, was prevented by S9.

Table 4 Induction of 8-oxoguanine-promoted mutations by the 31 oxidative mutagens in MutY-deficient strains Compound

1 2 3 6 7 8 9 10 11 20 22 23 24 25 27 28 29 30 31 32 33 37 47 52 53 54 56 58 59 60 62

– Hydrogen peroxide t-Butyl hydroperoxide Cumene hydroperoxide 2-Methyl-1,4-benzoquinone 2,6-Dimethyl-1,4-benzoquinone 2,3,5,6-Tetramethyl-1,4-benzoquinone ŽDuroquinone. 1,4-Naphthoquinone 2-Methyl-1,4-naphthoquinone ŽMenadione. 2-Hydroxy-1,4-naphthoquinone Catechol Ž1,2-benzenediol. Hydroquinone Ž1,4-benzenediol. Pyrogallol Ž1,2,3-benzenetriol. 1,2,4-Trihydroxybenzene t-Butylhydroquinone DL-Dopa L-Dopa Dopamine L-Norepinephrine DL-Epinephrine L-Epinephrine 3,4-Dihydroxyphenylacetic acid ŽDOPAC. Gallic acid Ž3,4,5-trihydroxybenzoic acid. 4-Aminophenol L-Cysteine methyl ester L-Cysteine ethyl ester L-Penicillamine Dithiothreitol ŽDTT. Hypoxanthineq XAO Ž0.1 U. Xanthineq XAO Ž0.1 U. Ascorbic acid q copper ŽII. Phenazine methosulfate

Dose Žmgrplate.

Number of revertantsrplate IC204 Ž mutq . IC206 Ž mutY .

IC208 Ž mutY oxyR .

– 100 100 50 40 40 60 16 30 200 2000 300 100 100 150 1000 1000 6000 1000 500 500 1000 1000 1000 500 750 2500 400 100 200 300 100

10 11 21 33 10 9 7 7 9 4 7 10 12 12 10 10 12 8 10 9 12 12 8 7 10 11 15 8 16 18 9 11

32 48 318 254 23 21 26 21 42 46 25 46 80 38 26 36 35 58 45 32 28 35 34 29 44 54 67 60 77 56 48 28

28 35 140 128 33 15 22 16 28 23 14 36 42 38 16 32 30 18 27 14 30 40 21 14 30 32 73 28 41 43 22 27

A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

3.3. Induction of 8-oxoguanine-promoted mutations by the 31 oxidatiÕe mutagens in MutY-deficient strains With the aim of detecting the induction of mutations generated from 8-oxoguanine lesions, we used the MutY-deficient tester strains IC206 Ž mutY . and IC208 Ž mutY oxyR ., as well as the mutq strain IC204 w6x. In these three strains, mutagenesis dependent on the SOS system is abolished by the absence of umuDC and mucAB genes, so that only SOS-independent mutations such as those promoted by 8oxoguanine lesions can be induced. As shown in Table 4, of the 31 oxidative mutagens tested, only the two organic hydroperoxides, BOOH and COOH, were clearly mutagenic in IC206 and IC208, whereas weak mutagenicities were observed in IC208 for pyrogallol, L-penicillamine, and HXA in the presence of XAO.

4. Discussion The new tester strain IC203, a derivative of WP2 uÕrArpKM101 deficient in the OxyR function, was developed for the testing of chemicals causing oxidative mutagenesis w6x. IC203 has been incorporated in the E. coli WP2 mutagenicity assay, and this is then named the WP2 Mutoxitest. In the WP2 Mutoxitest, a mutagenic response in IC203 greater than in WP2 uÕrArpKM101 Žhere denoted IC188. is interpreted as an indicator of oxidative mutagenesis. The validation study presented here shows that from a total of 80 chemicals examined with the WP2 Mutoxitest, 31 were judged to induce oxidative mutagenesis, and of these, 25 were uniquely positive in strain IC203. It is clear therefore, that the incorporation of strain IC203 in the WP2 test, has a large impact on its capacity not only for the detection of oxidative mutagens, but also to further evaluate negative results obtained with the standard strain IC188. The inability of IC188 to detect as mutagens 25 oxidative agents scored with IC203 should be due to the action of antioxidant defenses. These defenses are also present in the S. typhimurium strain TA102 which, although proposed as sensitive to oxidative mutagenesis w2x, failed to detect some of the oxida-

51

tive mutagens such as 4-aminophenol w14x, menadione, DL-epinephrine and phenazine methosulfate w15x, 2-methyl-1,4-BQ, 2,6-dimethyl-1,4-BQ and duroquinone w16x, 2-hydroxy-1,4-NQ w17x, catechol, hydroquinone and DTT Žunpublished results.. In spite of the reduced sensitivity to oxidative mutagenesis exhibited by strains IC188 ŽWP2 uÕrArpKM101. and TA102, these strains are recommended in the OECD guidelines for the screening of oxidising mutagens w4,5x. Our results show that strain IC203 is more appropriate for such screening and support the proposition of incorporating this strain in the bacterial test battery. In this battery, strain IC188 ŽWP2 uÕrArpKM101. is normally included and would serve, in the comparison with IC203, for the assessment of oxidative mutagenesis. This assessment, in tests using only one strain, has been based on the analysis of inhibition of mutagenesis by antioxidant enzymes w17–19x. However, the use of two strains, IC188 and IC203, in the WP2 Mutoxitest allows the screening for mutagenicity and the determination of the oxidative nature of this mutagenicity to be performed in a single step. The fact that the two antitumor agents, doxorubicin and streptonigrin, although mutagenic in the WP2 Mutoxitest, have not been classified as oxidative mutagens, points out the necessity to be cautious with this class of compounds known to generate oxygen radicals. This is especially relevant for streptonigrin, which has been included in several studies as a typical mutagenic oxidant w3,20,21x. Note, however, that the conclusion regarding the mutagenicity of doxorubicin and streptonigrin does not exclude the possibility that they can induce oxidative mutagenesis under certain conditions. Some of the oxidative mutagens Že.g. hydroquinone, L-epinephrine, L-ascorbic acid. were considered as non-alerting chemicals in a study on structure–mutagenicity relationship w22x. This might be explained by the absence of direct reactivity with the DNA of most of the oxidants, which are mutagenic via the formation of ROS able to cause DNA lesions. These lesions, besides a mutagenic effect, could cause cell death and so the mutagenic and cytotoxic effects of oxidative origin are well-correlated end points in testing substances with regard to their oxidative potential. In favor of this correlation is the toxicity observed in strain IC203 at doses just above

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A. Martınez ´ et al.r Mutation Research 467 (2000) 41–53

the maximal value of induced revertants, and the enhanced sensitivity of IC203 over IC188 to the lethal effects produced by all the oxidative mutagens ŽTable 3.. The inhibition by catalase, present in S9, of mutagenesis by all the scored oxidative mutagens, except by the two organic hydroperoxides ŽBOOH and COOH., indicates that it is mediated by HP presumably produced in autoxidation reactions. The occurrence of these reactions appears to require the absence of OxyR since most of the catalase-sensitive oxidative mutagens are positive only in strain IC203. A deficiency in catalase alone would not be enough to promote autoxidation reactions, as is suggested by the fact that, with the exception of the HP mutagenicity and of that resulting from the XAO-catalysed oxidation of HXA and XA, the mutagenicity of the oxidative mutagens is not significatively enhanced in a tester strain carrying a mutation in the catalase-encoding gene katG Žunpublished results.. In strain IC203, autoxidations could be triggered by the cellular pro-oxidant state Že.g. an increased hydroperoxide tone. resulting from the non-inducibility of the synthesis of both alkylhydroperoxidase and catalase. The absence of catalase induction might, in addition, be required to decrease the elimination of the HP generated in the autoxidation process. The results with the IC203 strain demonstrate that the adequate reduction in antioxidant defenses in order to sensitize tester strains to ROS-promoted mutagenesis is achieved by a deficiency in OxyR. The detection of mutations derived from 8oxoguanine lesions requires the absence of the MutY glycosylase w6,12x, so that these lesions would not be involved in the mutagenesis observed in either IC188 or IC203, where MutY is present. The results with the MutY-deficient strains IC206 and IC208 show that oxidants of the hydroperoxide type are good inducers of 8-oxoguanine-promoted mutations ŽTable 4., suggesting a link between lipid peroxidation and production of 8-oxoguanine lesions. In contrast, these lesions do not seem to be efficiently caused by either HP or oxidants acting through the production of HP ŽTable 4., which is in agreement with the lack of effect of both S9 Ži.e. catalase. on the spontaneous mutagenesis in a MutY MutM double mutant w23x, and pure catalase on stationary phase mutations arising in a MutY deficient strain w24x.

In conclusion, the results support the validation of the newly established strain IC203, included in the WP2 Mutoxitest, for the detection of oxidative mutagens. They also show that the WP2 Mutoxitest renders available a set of well-characterized mutagenic oxidants, belonging to different chemical classes, for their use in studies on the genotoxicity induced by oxidative stress, as well as on the prevention of such genotoxicity.

Acknowledgements We are grateful to Dr. Carmen Barrueco for helpful discussions and to Carmen Navarro for technical assistance. This work was supported by CICYT grant SAF97-0076. A.M. is the recipient of a F.P.I.F.V.I.B. Fellowship, and A.U. has a Postdoctoral Bancaixa-F.V.I.B. Fellowship.

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