- Email: [email protected]
[68] Elevated mutagenesis in bacterial mutants lacking superoxide dismutase
646
ORGAN, TISSUE, AND CELL DAMAGE
[68]
[68] E l e v a t e d M u t a g e n e s i s in B a c t e r i a l M u t a n t s L a c k i n g Superoxide Dismutase
By DANIELE TOUATI and SPENCER B. FARR Introduction Reactive oxygen species (superoxide radicals, hydrogen peroxide, hydroxyl radicals) are responsible for damage to DNA, 1-3 causing strand breaks and base alterations that can lead to cell death or mutations/,5 Although oxygen and a wide variety of oxidants have now been shown to be mutagenic, ~ it is often difficult to prove the role of an oxygen derivative in promoting or exacerbating the mutagenic activity of a substance. In particular, a role for superoxide radicals in mutagenesis has been questioned, since intraceUular superoxide radical generators, such as paraquat, have not been detected as mutagens by the usual mutagenicity tests, which otherwise have detected numerous oxidants as mutagens. 9 Different assays, however, although not completely convincing, have suggested a role for superoxide radicals in mutagenesis. 1°,11 Difficulties in evaluating the mutagenic potency of reactive oxygen species arise at least from two factors: (1) Rarely is consideration given to the nature of the oxidative DNA lesion(s) (the premutagenic lesions) that can lead to mutations, nor to the repair mechanism that will specifically act on lesions to convert them to mutations. This might lead to nondetection of the oxidative mutagenic events, when certain specific mutagenesis tests are used. (2) The generation of superoxide radicals or hydrogen peroxide induces in bacterial tester strains the corresponding detoxifying
i H. Sies, Angew. Chem. 25, 1058 (1986). 2 j. A. Imlay and S. Linn, Science 240, 1302 (1988). 3 W. A. Pryor, Free Radical Biol. Med. 4, 219 (1988). 4 B. N. Ames, Science 204, 587 (1979). 5 B. N. Ames, in "Mutagens in Our Environment" (M. Sorsa and H. Vainio, eds.), p. 3. Alan R. Liss, New York, 1982. 6 D. E. Lewin, M. Hollstein, M. F. Christman, E. A. Schwiers, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 79, 7445 (1982). 7 G. Storz, M. F. Christman, H. Sies, and B. Ames, Proc. Natl. Acad. Sci. U.S.A. 84, 8917 (1987). s j. T. Greenberg and B. Demple, EMBO J. 7, 2611 (1988). 9 D. E. Levin, M. Hollstein, M. F. Christman, and B. N. Ames, this series, Vol. 105, p. 249. to H. M. Hassan and C. S. Moody, this series, Vol. 105, p. 254. tl C. I. Wei, K. Allen, and H. P. Misra, J. Appl. Toxicol. 5, 315 (1985).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[68]
ELEVATED MUTAGENESIS IN E. coli LACKING S O D
647
enzymes, superoxide dismutase (SOD) 12or catalase,~3 masking a potential mutagenic effect which could appear in a different environment. The construction of an Escherichia coli K 12 double mutant completely lacking both Fe and Mn superoxide dismutase ~4permitted us to assess the role of superoxide radicals in the absence of their scavenger.~5 Results show that the increase in the intracellular flux of superoxide radicals leads to increased mutagenesis, demonstrating the protective role of superoxide dismutase of lowering the rate of spontaneous mutagenesis. 15 Consequently, the superoxide dismutase-deficient E. coli mutants constitute a unique tool to specifically assay the role of superoxide radicals in the mutagenic activity of some chemical agents or various forms of radiation. Material and Methods Strains. GC4468 is the parental E. coli K12 strain, considered as wild type. QC779 is like GC4468, but carries insertional mutations to the sodA (for Mn-SOD) and sodB (for Fe-SOD) structural genes. Markers encoding resistance factors are carried by the insertions (chloramphenicol resistance for sodA and kanamycin resistance for sodB), allowing the transfer of sodA and sodB into a different genetic background, if needed. The transfer can be achieved by two successive Pl-mediated transductions selecting for chloramphenicol resistance (associated with sodA) and kanamycin resistance (associated with sodB). Cultures are grown in LB medium (I0 g Bacto-tryptone, 10 g NaCI, 5 g Bacto yeast extract per liter, pH 7.2) in a New Brunswick Scientific Co. rotary shaker bath (200 rpm) at 37°. The ratio of Erlenmeyer flask volume to culture volume is between 7 : 1 and 10: 1; 20 p~g/ml chloramphenicol and 40 ~g/ml kanamycin sulfate are added to QC779 precultures. Chemicals. Chloramphenicol, kanamycin, trimethoprim, and paraquat (methyl viologen) may be purchased from Sigma (St. Louis, MO). Mutagenicity Test Mutagenicity tests using the sodA sodB mutant require growth and plating on complete medium, as the strain is unable to grow on minimal medium. ~4 The test described below measures forward mutations from Thy÷ to Thy- (thymine-requiring mutants). Thy- mutants are resistant to t2 H. M. Hassan and I. Fridovich, J. Biol. Chem. 252, 7667 (1977). t3 p. C. Loewen, J. Switala, and B. L. Triggs-Raine, Arch. Biochem. Biophys. 243, 144 (1985). 14 A. Carlioz and D. Touati, EMBO J. 5, 623 (1986). t5 S. B. Farr, R. D'Ari, and D. Touati, Proc. Natl. Acad. Sci. U.S.A. 83, 8268 (1986).
648
ORGAN, TISSUE, AND CELL DAMAGE
[68]
the drug trimethoprim and can be selected from a Thy ÷ population. 16 Other mutagenicity tests performed in rich medium could alternatively be used, following a similar procedure (e.g., the test described by Lucchesi e t al.17). Procedure. Cell precultures of GC4468 and QC779 are grown in LB medium at 37° until they reach an optical density at 600 nm (OD60o) of 1; the cultures are chilled on ice, and kept in the cold (up to 48 hr) to be used for inoculate. Prewarmed LB medium is inoculated at an OD600 of 0.020.03 from the preculture. When cells reach an OD600 of about 0.1, the culture is subdivided (15 ml/portion) into 100-ml flasks and further incubated until an OD600 of 0.2 is reached. Compound to be tested is added at that time, at various concentrations. Cells are further incubated until the corresponding untreated culture reaches an OD600 of 1, and the cells are chilled on ice for plating. Plating. One-tenth milliliter of bacterial dilutions (1 / 10, 1/40, 1/ 100) in cold 10 mM MgSO4 is uniformly spread on dried trimethoprim agar plates (1.5% Difco agar, 18/zg/ml trimethoprim, and 50/~g/ml thymine in LB medium; trimethoprim stock solution is made 5 mg/ml in 50% ethanol and kept at -20°). Three plates are made for each dilution. Plates are incubated at 37° for 18 to 24 hr, and Thy- colonies which appear on a more or less thin lawn of Thy ÷ bacteria are counted. Spreading should be done very carefully to obtain an uniform lawn. A glass spreader can be used. We preferentially use sterile glass beads, 4 mm diameter, usually utilized for separatory columns. Five or six beads are distributed on plates before adding a sample of cells. Plates are gently shaken until the culture sample has been completely adsorbed onto the solid medium. Plates are inverted and the beads removed from the cover before incubation. Up to six plates can be piled up and shaken together. Alternatively, bacteria can be spread using soft agar. In this method, 0.1 ml bacterial dilution is added to 2.5 ml molten soft agar (0.6% Bacto agar in water) maintained at 48° in a heat block, gently vortexed, and
16 J. Miller, "Experiments in Molecular Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1972. 17 The test described by Lucchesi et al. [P. Lucchesi, M. Carraway, and G. Marinus, J. Bacteriol. 166, 34 (1986)] measure forward Tet s to Tet R mutations. Strains to be tested are transformed with a plasmid, pPY98, a derivative of pBR322 in which the tet gene is under the mnt-regulated ant promotor of P22. Cells containing the wild-type plasmid are Amp R, Tet s. Mutations in the rant gene or its operator confer tetracycline resistance to the cell. We have recently developed this test in the laboratory to assay the mutagenic effect of superoxide radicals. The strains in which to use it have been constructed. Protocols and strains are available. This test is easier to handle than the test described here, but its use is somewhat restricted.
[68]
ELEVATED MUTAGENESlS IN E. coli LACKING S O D
649
poured rapidly onto a plate. The plate is rocked gently to ensure uniform distribution. Plates are left on the bench to harden for 15 min, after which they are inverted and incubated at 37°. Colonies embedded into agar are smaller; therefore, plates should be incubated 36 hr before counting, using this procedure. Cell Counting. Titration for viable cell counts is obtained by plating on LBT (1.5% Bacto agar and 50/.~g/ml thymine in LB), in duplicate, 0.1 ml of 10-~ and 2 × 10 -6 bacterial dilutions in cold 10 mM M g S O 4 . Accurate titration is essential, particularly when toxic substances are used. We recommend two independent cell titrations for each experiment. An OD600 reading of I corresponds to about 4 x 108 cells/ml for untreated cells. Remarks and Cautions A major difficulty in using mutants lacking SOD in mutagenesis testing is that such mutants grow more slowly than the corresponding wild-type cells: 45 min generation time for sodA sodB versus 28 min for GC4468 in LB at 37°. The mutants are also much more sensitive to compounds that generate reactive oxygen species, and excessive lethality may possibly mask mutagenesis. For these reasons, the procedure described above does not give quantitative results for the mutation frequency of a compound. However, reliable qualitative results can be obtained, with the following precautions. (1) Thy- cells should not show any growth advantage or disadvantage over Thy ÷ cells. This has to be checked after introducing sodA sodB in a new genetic background. This was verified for the strains used previously in the assay as follows: Thy-/Thy ÷ mixed cultures can be grown for more than 12 generations without measurable change in the initial ratio of Thy- to Thy ÷. The Thy-/Thy ÷ ratio is established by replica plating on a minimal medium with or without 50/.tg/ml thymine. For sodA sodB, minimal medium should be supplemented with 20 amino acids (0.5 mM). (2) Conditions should be chosen to minimize killing by toxic compounds. Concentrations as low as possible should be used for detection. A linear dose-response curve should be obtained. (3) Oxygen increases mutagenesis in sodA sodB mutants. Therefore one should be very careful about keeping uniform conditions during culture aeration in experiments, i.e., the same flask/culture volume ratio, same shaking conditions. The initial volume should be large enough so that withdrawing samples during the experiment will not seriously affect the initial flask/culture volume ratio. (4) For each strain used, at least 20 trimethoprim-resistant colonies should be checked for Thy- phenotype to ensure that Thy- mutants are counted.
650
ORGAN, TISSUE, AND CELL DAMAGE
[68]
(5) Every experiment should be repeated at least twice with similar results to be taken into consideration. If enhancement of mutagenicity in the sodA sodB versus the wild-type strain is observed under these conditions, further tests to verify the results can be made. Further Tests for Determining Role of Superoxide Radicals in Mutagenicity The enhancement of spontaneous mutations in sodA sodB mutants is completely oxygen dependent. It is also largely dependent on a functional exonuclease IIPs (encoded by the xthA gene), suggesting that this enzyme is specifically involved in converting the premutagenic lesions produced (directly or more likely indirectly) by superoxide radicals to mutations. The introduction of a multicopy plasmid carrying an SOD + allele into sodA sodB reduces the mutation rate to that of the wild type.~5 Therefore, enhancement of mutagenicity of any compound in an sodA sodB mutant owing to the intracellular increased flux of superoxide radicals should be suppressed either by performing the assay under anaerobic conditions or in an sodA sodB strain deficient in exonuclease III activity (sodA sodB xthA mutant), or by introduction of an SOD + plasmid (sodA sodB/psod + strain). Anaerobic experiments should be performed in an anaerobic chamber. In the absence of an anaerobic chamber, however, microaerobic conditions can be obtained by purging a filled 5-ml flask with nitrogen for 10 min, capping the flask, and allowing the cells to grow at 37° without shaking. One percent glucose should be added to the medium for anaerobic growth. In any case, successive precultures should be grown anaerobically for 48 hr before beginning the experiment to ensure anaerobic conditions. A set of isogenic strains can be used to test the effect of the xthA mutation. They include BW35, the parental strain (wild type); BW295, as BW35 but carrying the xthA mutation (the two former strains were provided by B. Weiss); QC910, as BW35 but sodA sodB; QC915, as BW295 but sodA sodB. Using these strains in the assay, one would expect to find enhanced mutagenicity in QC910 versus wild type but not (or slightly enhanced) in QC915. Both strains have approximately the same generation time. Strains bearing plasmids expressing Fe-SOD (pHS1-8) or Mn-SOD (pDTI-5) are also available: QC1093 is QC779/pHS1-8 and QC1094 is QC779/pDT1-5. In these strains mutagenicity should be the same as the ts S. G. Rogers and B. Weiss, this series, Vol. 65, p. 201.
[69]
ASSAY O F X A N T H I N E O X l D A S E A C T I V I T Y
651
TABLEI T h y + ~ Thy-FORWARDMOTATtON" Plus
Strain QC4468 QC779 QC1093 QC1094
Relevant genotype Wild sodA sodA sodA
type sodB sodB/sodB ÷ sodB/sodA ÷
Aerobic
Plus paraquat 50 tzM, 200 p.M
plumbagin (500/zM)
Oxygenated
Anaerobi~
20 81 21 19
21,19 105,160 NT NT
32 382 NT 25
31 1321 NT NT
15 13 NT NT
Data are numbers of Thy- mutants per 107 cells (as determined by viable counts) plated on LBq plus trimethoprim. A Forma Scientific Chamber, Model 1024, was used for anaerobic experi ments. Hyperoxygenation was achieved by bubbling oxygen constantly through the culture. Th~ efficiency of plating was 90% for oxygen-treated, wild type cells, 50% for sodA sodB; for para quat-treated cells it was 100% for 50/zM, 70% for 200/zM in wild type, 50% for 50/zM and 35~ for 200/zM in sodA sodB, for plumbagin-treated cells it was 70% for wild type, 30% for soda sodB. NT, Not tested.
wild-type levels. Generation times of sodA s o d B / p s o d ÷ strains are approximately that of the wild type (see Table I). Acknowledgments This work was supported by a grant from the Association pour la Recherche sur le Cancer (No. 6791). We thank A. Eisenstark for careful critical reading of the manuscript.
[69] M e a s u r e m e n t o f X a n t h i n e Oxidase in Biological Tissues
By LANCE S. TEgnDA, JONATHANA. LEFF, and JOHN E. REPINE Xanthine oxidase (EC 1.1.3.22) is found in many tissues and a variety of species ranging from bacteria to humans. ~,2 Despite the ubiquity of xanthine oxidase, its exact role in cellular physiology is unclear; however, one of its functions is thought to involve purine metabolism, and this forms the basis for most standard assays which follow the rate of formation of uric acid from xanthine. Additionally, xanthine oxidase has a i D. A. Parks and D. N. Granger, Acta Physiol. Scand. Suppl. $~18, 87 (1986). 2 T. A. Krenitsky, J. V. Tuttle, E. L. Cattau, and P. Wang, Comp. Biochem. Physiol. 49B, 687 (1974).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ORGAN, TISSUE, AND CELL DAMAGE
[68]
[68] E l e v a t e d M u t a g e n e s i s in B a c t e r i a l M u t a n t s L a c k i n g Superoxide Dismutase
By DANIELE TOUATI and SPENCER B. FARR Introduction Reactive oxygen species (superoxide radicals, hydrogen peroxide, hydroxyl radicals) are responsible for damage to DNA, 1-3 causing strand breaks and base alterations that can lead to cell death or mutations/,5 Although oxygen and a wide variety of oxidants have now been shown to be mutagenic, ~ it is often difficult to prove the role of an oxygen derivative in promoting or exacerbating the mutagenic activity of a substance. In particular, a role for superoxide radicals in mutagenesis has been questioned, since intraceUular superoxide radical generators, such as paraquat, have not been detected as mutagens by the usual mutagenicity tests, which otherwise have detected numerous oxidants as mutagens. 9 Different assays, however, although not completely convincing, have suggested a role for superoxide radicals in mutagenesis. 1°,11 Difficulties in evaluating the mutagenic potency of reactive oxygen species arise at least from two factors: (1) Rarely is consideration given to the nature of the oxidative DNA lesion(s) (the premutagenic lesions) that can lead to mutations, nor to the repair mechanism that will specifically act on lesions to convert them to mutations. This might lead to nondetection of the oxidative mutagenic events, when certain specific mutagenesis tests are used. (2) The generation of superoxide radicals or hydrogen peroxide induces in bacterial tester strains the corresponding detoxifying
i H. Sies, Angew. Chem. 25, 1058 (1986). 2 j. A. Imlay and S. Linn, Science 240, 1302 (1988). 3 W. A. Pryor, Free Radical Biol. Med. 4, 219 (1988). 4 B. N. Ames, Science 204, 587 (1979). 5 B. N. Ames, in "Mutagens in Our Environment" (M. Sorsa and H. Vainio, eds.), p. 3. Alan R. Liss, New York, 1982. 6 D. E. Lewin, M. Hollstein, M. F. Christman, E. A. Schwiers, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 79, 7445 (1982). 7 G. Storz, M. F. Christman, H. Sies, and B. Ames, Proc. Natl. Acad. Sci. U.S.A. 84, 8917 (1987). s j. T. Greenberg and B. Demple, EMBO J. 7, 2611 (1988). 9 D. E. Levin, M. Hollstein, M. F. Christman, and B. N. Ames, this series, Vol. 105, p. 249. to H. M. Hassan and C. S. Moody, this series, Vol. 105, p. 254. tl C. I. Wei, K. Allen, and H. P. Misra, J. Appl. Toxicol. 5, 315 (1985).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[68]
ELEVATED MUTAGENESIS IN E. coli LACKING S O D
647
enzymes, superoxide dismutase (SOD) 12or catalase,~3 masking a potential mutagenic effect which could appear in a different environment. The construction of an Escherichia coli K 12 double mutant completely lacking both Fe and Mn superoxide dismutase ~4permitted us to assess the role of superoxide radicals in the absence of their scavenger.~5 Results show that the increase in the intracellular flux of superoxide radicals leads to increased mutagenesis, demonstrating the protective role of superoxide dismutase of lowering the rate of spontaneous mutagenesis. 15 Consequently, the superoxide dismutase-deficient E. coli mutants constitute a unique tool to specifically assay the role of superoxide radicals in the mutagenic activity of some chemical agents or various forms of radiation. Material and Methods Strains. GC4468 is the parental E. coli K12 strain, considered as wild type. QC779 is like GC4468, but carries insertional mutations to the sodA (for Mn-SOD) and sodB (for Fe-SOD) structural genes. Markers encoding resistance factors are carried by the insertions (chloramphenicol resistance for sodA and kanamycin resistance for sodB), allowing the transfer of sodA and sodB into a different genetic background, if needed. The transfer can be achieved by two successive Pl-mediated transductions selecting for chloramphenicol resistance (associated with sodA) and kanamycin resistance (associated with sodB). Cultures are grown in LB medium (I0 g Bacto-tryptone, 10 g NaCI, 5 g Bacto yeast extract per liter, pH 7.2) in a New Brunswick Scientific Co. rotary shaker bath (200 rpm) at 37°. The ratio of Erlenmeyer flask volume to culture volume is between 7 : 1 and 10: 1; 20 p~g/ml chloramphenicol and 40 ~g/ml kanamycin sulfate are added to QC779 precultures. Chemicals. Chloramphenicol, kanamycin, trimethoprim, and paraquat (methyl viologen) may be purchased from Sigma (St. Louis, MO). Mutagenicity Test Mutagenicity tests using the sodA sodB mutant require growth and plating on complete medium, as the strain is unable to grow on minimal medium. ~4 The test described below measures forward mutations from Thy÷ to Thy- (thymine-requiring mutants). Thy- mutants are resistant to t2 H. M. Hassan and I. Fridovich, J. Biol. Chem. 252, 7667 (1977). t3 p. C. Loewen, J. Switala, and B. L. Triggs-Raine, Arch. Biochem. Biophys. 243, 144 (1985). 14 A. Carlioz and D. Touati, EMBO J. 5, 623 (1986). t5 S. B. Farr, R. D'Ari, and D. Touati, Proc. Natl. Acad. Sci. U.S.A. 83, 8268 (1986).
648
ORGAN, TISSUE, AND CELL DAMAGE
[68]
the drug trimethoprim and can be selected from a Thy ÷ population. 16 Other mutagenicity tests performed in rich medium could alternatively be used, following a similar procedure (e.g., the test described by Lucchesi e t al.17). Procedure. Cell precultures of GC4468 and QC779 are grown in LB medium at 37° until they reach an optical density at 600 nm (OD60o) of 1; the cultures are chilled on ice, and kept in the cold (up to 48 hr) to be used for inoculate. Prewarmed LB medium is inoculated at an OD600 of 0.020.03 from the preculture. When cells reach an OD600 of about 0.1, the culture is subdivided (15 ml/portion) into 100-ml flasks and further incubated until an OD600 of 0.2 is reached. Compound to be tested is added at that time, at various concentrations. Cells are further incubated until the corresponding untreated culture reaches an OD600 of 1, and the cells are chilled on ice for plating. Plating. One-tenth milliliter of bacterial dilutions (1 / 10, 1/40, 1/ 100) in cold 10 mM MgSO4 is uniformly spread on dried trimethoprim agar plates (1.5% Difco agar, 18/zg/ml trimethoprim, and 50/~g/ml thymine in LB medium; trimethoprim stock solution is made 5 mg/ml in 50% ethanol and kept at -20°). Three plates are made for each dilution. Plates are incubated at 37° for 18 to 24 hr, and Thy- colonies which appear on a more or less thin lawn of Thy ÷ bacteria are counted. Spreading should be done very carefully to obtain an uniform lawn. A glass spreader can be used. We preferentially use sterile glass beads, 4 mm diameter, usually utilized for separatory columns. Five or six beads are distributed on plates before adding a sample of cells. Plates are gently shaken until the culture sample has been completely adsorbed onto the solid medium. Plates are inverted and the beads removed from the cover before incubation. Up to six plates can be piled up and shaken together. Alternatively, bacteria can be spread using soft agar. In this method, 0.1 ml bacterial dilution is added to 2.5 ml molten soft agar (0.6% Bacto agar in water) maintained at 48° in a heat block, gently vortexed, and
16 J. Miller, "Experiments in Molecular Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 1972. 17 The test described by Lucchesi et al. [P. Lucchesi, M. Carraway, and G. Marinus, J. Bacteriol. 166, 34 (1986)] measure forward Tet s to Tet R mutations. Strains to be tested are transformed with a plasmid, pPY98, a derivative of pBR322 in which the tet gene is under the mnt-regulated ant promotor of P22. Cells containing the wild-type plasmid are Amp R, Tet s. Mutations in the rant gene or its operator confer tetracycline resistance to the cell. We have recently developed this test in the laboratory to assay the mutagenic effect of superoxide radicals. The strains in which to use it have been constructed. Protocols and strains are available. This test is easier to handle than the test described here, but its use is somewhat restricted.
[68]
ELEVATED MUTAGENESlS IN E. coli LACKING S O D
649
poured rapidly onto a plate. The plate is rocked gently to ensure uniform distribution. Plates are left on the bench to harden for 15 min, after which they are inverted and incubated at 37°. Colonies embedded into agar are smaller; therefore, plates should be incubated 36 hr before counting, using this procedure. Cell Counting. Titration for viable cell counts is obtained by plating on LBT (1.5% Bacto agar and 50/.~g/ml thymine in LB), in duplicate, 0.1 ml of 10-~ and 2 × 10 -6 bacterial dilutions in cold 10 mM M g S O 4 . Accurate titration is essential, particularly when toxic substances are used. We recommend two independent cell titrations for each experiment. An OD600 reading of I corresponds to about 4 x 108 cells/ml for untreated cells. Remarks and Cautions A major difficulty in using mutants lacking SOD in mutagenesis testing is that such mutants grow more slowly than the corresponding wild-type cells: 45 min generation time for sodA sodB versus 28 min for GC4468 in LB at 37°. The mutants are also much more sensitive to compounds that generate reactive oxygen species, and excessive lethality may possibly mask mutagenesis. For these reasons, the procedure described above does not give quantitative results for the mutation frequency of a compound. However, reliable qualitative results can be obtained, with the following precautions. (1) Thy- cells should not show any growth advantage or disadvantage over Thy ÷ cells. This has to be checked after introducing sodA sodB in a new genetic background. This was verified for the strains used previously in the assay as follows: Thy-/Thy ÷ mixed cultures can be grown for more than 12 generations without measurable change in the initial ratio of Thy- to Thy ÷. The Thy-/Thy ÷ ratio is established by replica plating on a minimal medium with or without 50/.tg/ml thymine. For sodA sodB, minimal medium should be supplemented with 20 amino acids (0.5 mM). (2) Conditions should be chosen to minimize killing by toxic compounds. Concentrations as low as possible should be used for detection. A linear dose-response curve should be obtained. (3) Oxygen increases mutagenesis in sodA sodB mutants. Therefore one should be very careful about keeping uniform conditions during culture aeration in experiments, i.e., the same flask/culture volume ratio, same shaking conditions. The initial volume should be large enough so that withdrawing samples during the experiment will not seriously affect the initial flask/culture volume ratio. (4) For each strain used, at least 20 trimethoprim-resistant colonies should be checked for Thy- phenotype to ensure that Thy- mutants are counted.
650
ORGAN, TISSUE, AND CELL DAMAGE
[68]
(5) Every experiment should be repeated at least twice with similar results to be taken into consideration. If enhancement of mutagenicity in the sodA sodB versus the wild-type strain is observed under these conditions, further tests to verify the results can be made. Further Tests for Determining Role of Superoxide Radicals in Mutagenicity The enhancement of spontaneous mutations in sodA sodB mutants is completely oxygen dependent. It is also largely dependent on a functional exonuclease IIPs (encoded by the xthA gene), suggesting that this enzyme is specifically involved in converting the premutagenic lesions produced (directly or more likely indirectly) by superoxide radicals to mutations. The introduction of a multicopy plasmid carrying an SOD + allele into sodA sodB reduces the mutation rate to that of the wild type.~5 Therefore, enhancement of mutagenicity of any compound in an sodA sodB mutant owing to the intracellular increased flux of superoxide radicals should be suppressed either by performing the assay under anaerobic conditions or in an sodA sodB strain deficient in exonuclease III activity (sodA sodB xthA mutant), or by introduction of an SOD + plasmid (sodA sodB/psod + strain). Anaerobic experiments should be performed in an anaerobic chamber. In the absence of an anaerobic chamber, however, microaerobic conditions can be obtained by purging a filled 5-ml flask with nitrogen for 10 min, capping the flask, and allowing the cells to grow at 37° without shaking. One percent glucose should be added to the medium for anaerobic growth. In any case, successive precultures should be grown anaerobically for 48 hr before beginning the experiment to ensure anaerobic conditions. A set of isogenic strains can be used to test the effect of the xthA mutation. They include BW35, the parental strain (wild type); BW295, as BW35 but carrying the xthA mutation (the two former strains were provided by B. Weiss); QC910, as BW35 but sodA sodB; QC915, as BW295 but sodA sodB. Using these strains in the assay, one would expect to find enhanced mutagenicity in QC910 versus wild type but not (or slightly enhanced) in QC915. Both strains have approximately the same generation time. Strains bearing plasmids expressing Fe-SOD (pHS1-8) or Mn-SOD (pDTI-5) are also available: QC1093 is QC779/pHS1-8 and QC1094 is QC779/pDT1-5. In these strains mutagenicity should be the same as the ts S. G. Rogers and B. Weiss, this series, Vol. 65, p. 201.
[69]
ASSAY O F X A N T H I N E O X l D A S E A C T I V I T Y
651
TABLEI T h y + ~ Thy-FORWARDMOTATtON" Plus
Strain QC4468 QC779 QC1093 QC1094
Relevant genotype Wild sodA sodA sodA
type sodB sodB/sodB ÷ sodB/sodA ÷
Aerobic
Plus paraquat 50 tzM, 200 p.M
plumbagin (500/zM)
Oxygenated
Anaerobi~
20 81 21 19
21,19 105,160 NT NT
32 382 NT 25
31 1321 NT NT
15 13 NT NT
Data are numbers of Thy- mutants per 107 cells (as determined by viable counts) plated on LBq plus trimethoprim. A Forma Scientific Chamber, Model 1024, was used for anaerobic experi ments. Hyperoxygenation was achieved by bubbling oxygen constantly through the culture. Th~ efficiency of plating was 90% for oxygen-treated, wild type cells, 50% for sodA sodB; for para quat-treated cells it was 100% for 50/zM, 70% for 200/zM in wild type, 50% for 50/zM and 35~ for 200/zM in sodA sodB, for plumbagin-treated cells it was 70% for wild type, 30% for soda sodB. NT, Not tested.
wild-type levels. Generation times of sodA s o d B / p s o d ÷ strains are approximately that of the wild type (see Table I). Acknowledgments This work was supported by a grant from the Association pour la Recherche sur le Cancer (No. 6791). We thank A. Eisenstark for careful critical reading of the manuscript.
[69] M e a s u r e m e n t o f X a n t h i n e Oxidase in Biological Tissues
By LANCE S. TEgnDA, JONATHANA. LEFF, and JOHN E. REPINE Xanthine oxidase (EC 1.1.3.22) is found in many tissues and a variety of species ranging from bacteria to humans. ~,2 Despite the ubiquity of xanthine oxidase, its exact role in cellular physiology is unclear; however, one of its functions is thought to involve purine metabolism, and this forms the basis for most standard assays which follow the rate of formation of uric acid from xanthine. Additionally, xanthine oxidase has a i D. A. Parks and D. N. Granger, Acta Physiol. Scand. Suppl. $~18, 87 (1986). 2 T. A. Krenitsky, J. V. Tuttle, E. L. Cattau, and P. Wang, Comp. Biochem. Physiol. 49B, 687 (1974).
METHODS IN ENZYMOLOGY, VOL. 186
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.