Journal of Photochemistry
B: Biology, 4 (1990)
371 - 378
DIFFERENT LETHAL EFFECTS BY ENZYME-GENERATED TRIPLET INDOLE-3-ALDEHYDE IN DIFFERENT Escherichia coli STRAINS NELSON
Instituto de Quimica, Biological Chemistry Laboratory, Universidade Estadual de Campinas, C. P. 6154, Campinas, S. P., CEP 13.081 (Bmzil) MARIA
S. V. GATT1
Instituto de Biologia, Universidade Estadual de Campinas, C. P. 6154, Campinas, S. P., CEP 13.081 (Brazil) LUCIANA
C. C. LEITE
lnstituto de Quimica, Universidade de Sao Paul0 (Brazil) (Received
May 12, 1969)
Keywords. DNA, Escherichia Coli, 4.thiouridine, indole-&aldehyde, triplet state, peroxidase.
Summary Strains of Escherichia coli which lack 4-thiouridine (S4U) exhibit a higher survival rate than their wild-type parents which contain S4U after treatment with enzyme-generated triplet indole-3aldehyde. In a similar manner to results obtained with monochromatic 334 nm UV light, the survival is related to single-strand breakage of DNA in E. coli containing the pBR 322 plasmid. The effects of the excited states generated by an enzymatic system suggest that S4U is an important chromophore in the lethal effects observed. The results also suggest that the energy transferred from triplet indole-3aldehyde to S4U may also be passed from S4U of t-RNA to DNA, possibly through a singlet oxygen intermediate generated by excited S4U, resulting in a decrease in the survival rate of E. coli containing S4U. These results emphasize the importance of excited states in biological systems.
1. Introduction 4.Thiouridine has been shown to be involved in near-UV-induced growth delay and inhibition of tryptophanase reduction in bacterial capacity *Author
should be addressed. Elsevier
in The Netherlands
for phage development. It has also been shown to play a possible role in nearUV lethality. It has been suggested that the excitation energy from 334 nm UV light may be transferred from RNA to DNA resulting in single-strand breaks [l]. Indole-3aldehyde (IAl), formed in the horseradish peroxidase (HRP) catalysed oxidation of indole-3-acetic acid (IAA), is generated in an electronically excited state [2 - 41. This energized species reacts with free thiouridine and with t-RNA; in the latter case, energy transfer to the 4-thiouridine (S4U) groups occurs [ 5, 61. In this process the S4U is transformed to uridine  and to S4U-cytidine photoadduct . Escherichia coli containing PAT 153 plasmid undergoes DNA strand scission when exposed to the enzymatic system [ 91. Similar results with X-phage DNA by enzyme-generated triplet acetone have been observed [lo]. Thus the possibility of DNA lesions induced under physiological conditions, without the stimulus of agents external to the cell, may be related to the phenomenon of spontaneous mutations and carcinogenesis, and to the evolution of cellular DNA repair mechanisms. In a preliminary study we found an increased mortality of E. coli when exposed to enzyme-generated triplet IAI. DNA damage was followed by Tb3+-DNA complex fluorescence [ 111. The same effect was also observed with triplet acetone through the induction of the SOS function sfiA (repair system) in E. coli . In the IA1 system it was suggested that this process could involve energy transfer from triplet IA1 to S4U in t-RNA [ 13, 141. We now confirm the influence of S4U on the survival of S4U(-) and S4U(+) strains exposed to enzyme-generated triplet IA1 and its influence on the accumulation of DNA single-strand breaks in different strains.
2. Materials and methods 2.1. Enzyme-generated excited state system Triplet IA1 was generated by the peroxidase (HRP, type VI, Sigma) catalysed oxidation of IAA in the presence of EDTA in 0.05 M acetate buffer at pH 5.6 and room temperature . 2.2. Plasmid preparation E. coli (HB 101) containing pBR 322 plasmid were grown at 37 “C in LB medium (5 g NaCl, 5 g yeast extract and 10 g tryptons per litre of water solution) and were prepared as described previously .
2.3. Cells E. coli B/r NC 32 (S4U(+)) and B/r NC 32 RJ (S4U(-)) were kindly supplied by Dr. M. J. Peak (Argonne National Laboratory). E. coli 248 WT, 1088 WT, 871 recA, 1089 uvrB and 877 polA were kindly supplied by Prof. K. C. Smith (Department of Radiology, Stanford University School of Medicine).
E. coli B/r cells were grown as described by Peak et al. [l]. E. coli cultures were prepared as follows. Cell suspension (0.2 ml) in 0.15 M NaCl (McFarland scale 3) was added to a Khan tube with 50 mM acetate buffer (pH 5.6) (0.4 ml) containing 30 FM EDTA and 0.1 M NaCl, 0.1 mM IAA (0.2 ml) and 1.0 PM HRP (0.2 ml). This solution was submitted to various incubation times. To stop the reaction after the determined time interval, 0.1 M PBS buffer (pH 7.2) (1.0 ml) was added. The samples were centrifuged and bacteria were resuspended in 0.15 M NaCl (0.5 ml). The suspensions (0.1 ml) were cultured in a cell dilution of low8 on DIFCO nutrient agar plates in triplicate and colonies were counter after 48 h incubation at 30 “C. All the experiments were carried out in triplicate. 2.4. Determination of breaks in DNA The reaction mixture, with a final volume of 1.0 ml, contained 30 PM EDTA, 500 (uM IAA and 2 optical density (OD) units (600 nm) of E. coli cells in a 0.05 M acetate buffer (pH 5.6). The reaction was initiated by the addition-of 10 1.t1HRP (0.25 PM) and incubated at 25 “C for 15 min. The cells were pelleted and submitted to plasmid preparation under neutral conditions as described previously [ 151. The plasmid preparation was analysed by electrophoresis carried out on 1% agarose slab gels in 40 mM TRIS buffer (pH 7.7), 50 mM EDTA and 80 mM sodium acetate at 50 mA for 16 h.
3. Results and discussion In a previous study, pAT 153 plasmid DNA in E. coli was observed to undergo single-strand breaks when exposed to the enzyme-generated triplet IA1 molecules produced during HRP catalysed oxidation of IAA . In ref. 9 it was not possible to conclude whether the observed effect was due to direct DNA strand breakage, or to the formation of alkali-labile sites because of the alkaline extraction method used for the PAT 153 plasmid. Under the neutral conditions used in this study in the extraction of pBR 322 plasmid, the same results are observed as in the alkaline extraction of PAT 153 plasmid, demonstrating that the triplet IA1 induces direct DNA strand breaks. Control experiments have shown that IAA, HRP and the final stable products (IAl, methylenoxindole) [ 31 are not responsible for the effect observed. That singlet oxygen  (generated in the energy transfer from triplet IA1 to oxygen) is one of the effecters of DNA scission in E. coli is indicated by the partial inhibition of this effect by 10 mM guanosine (62% protection), 0.1 mM histidine (40% protection) and 10 mM tryptophan (75% protection) observed using a slightly modified ethidium bromide technique [ 171. In a survival curve experiment on E. coli exposed to the energized process, the mutant strain (1089 uvrB), which differs in its repair capability, was very sensitive to the enzymatic system compared with the wild-type E. coli strain (Fig. 1). A similar behaviour was observed by Hass and Webb
( MN 1
Fig. 1. A comparison of the survival of E. coli strains lacking excission repair (1089 uurB) (a) with that of the wild-type strain (1088 WT) (0). Filled symbols represent the same strains after interaction with the IAA-HRP-02 system for various periods of incubation.
 on illumination in the presence of acridine orange. This study indicated that although DNA lesions produced by 460 or 500 nm light in the presence of acridine orange were rendered non-lethal by rec+lex+-dependent repair processes, such repair was more efficient for the lesions produced by 500~nm light. Several other E. coli strains are sensitive to the excited IA1 interaction. The lethal action of the enzymatic system in three strains of E. coli, which differ in repair capability, is shown in Fig. 2. The controls for 877 polA and 1089 uurB strains showed some environmental effects under these conditions probably due to EDTA-pH effects. The significance of these results is still under study. The important point is that the wild type is again more resistant than any other strain deficient in repair capability. S4U plays an important role in energy transfer processes such as the generation of singlet oxygen [ 7, 191, DNA cleavage , near-UV lethality [20, 211, accumulation of DNA single-strand breaks in different strains [l] and the induction of SOS response by near-UV light . Since this probably occurs through sensitization with S4U  or S2U [ 23,241, we decided to study the differential sensitivity of S4U(-) and S4U(+) strains in the observed DNA breakage. Figure 3 confirms (as in the photoenergized method [l]) that the mutant lacking S4U is less sensitive than its corresponding wild-type S4U(+) parent to the lethal effect of excited IA1 molecules. The Peak et al. [l] experiments with E. coli mutants demonstrate a correlation between DNA
TIME OF INCUBATION IN IAAIHRPWN) Fig. 2. A comparison of the survival of E. coli strains which differ in repair capacity (871 recA (*), 1089 uurE (A) and 877 polA (A)) with that of the wild-type (248 WT) strain (0) after interaction (various periods of incubation) with the IAA-HRP-02 system. Controls were not treated with the IAA -HRP-O2 system.
strand breakage and lethality. Their explanation is consistent with the following facts: (i) a greater killing differential is observed at 334 nm (near the maximum for S4U) than at 313 nm; (ii) photosensitization appears to be characteristic of near-UV lethal effects; (iii) the oxygen enhancement ratio for survival after exposure to 334 nm UV light is greater for the E. coli B/r S4U(+) strain than for the S4U(-) strain. All of these observations are consistent with a photosensitized role of oxygen in the S4U effect. The possibility that S4U may somehow inhibit the fast repair of single-strand breaks in DNA  seems less likely. Our results with pBR 322 plasmid suggest that singlet molecular oxygen is involved in the observed DNA breakage induced by the enzyme-generated triplet indole-3-aldehyde. This possibility is supported by our previous results on singlet oxygen production in this enzymatic system [3, 4, 6, 7, 161. This indicates that DNA damage may be respon-
comparison of the survival of the E. coli strain lacking t-RNA (S4U(-)) with that of the wild-type (S4U(+)) strain after different incubation times with the IAA-HRPO2 system. The controls of both strains were not treated with the IAA-HRP-02 system. .
sible for the observed lethality through S4U sensitization of singlet oxygen
Since the production of excited states may occur under physiological conditions without stimulation by external agents (by the organism’s own internal enzymatic system), it could produce DNA modifications and be responsible for the evolution of cellular DNA repair mechanisms . This emphasizes the importance of excited states in biological systems [ 4,271. Acknowledgments The support of Prof. K. C. Smith (Stanford University) in the preliminary experiments on the survival factor is gratefully acknowledged. We thank Dr. S. M. De Toledo for the preliminary results on this subject and Dr. M. J. Peak for interesting discussions in this field. We also thank the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), the Conselho NacionaI de Pesquisa (CNPq), the Financiadora de Estudos e Projetos (FINEP), the Organization of American States and UNESCO programmes for financial support.
References 1 M. J. Peak, J. G. Peak and L. Nerad, The role of 4-thiouridine in lethal effects and in DNA backbone breakage caused by 334 nm ultraviolet light in Escherichia coli, Photochem. Photobiol., 37 (1983) 169 - 172. 2 M. P. De Mello, S. M. De Toledo and N. Duran, Photo- and biophotoenergixed cycloaddition of indole-3-aldehyde to uridine, Acta Sud Am. Quim., 1 (1981) 135 141. 3 N. Duran, J. E. Brunet and H. Gallardo, An experiment in photochemistry. (YOxidation of indole-3-acetic acid catalyzed by peroxidase, Biochem. Ed., 12 (1984) 173 - 178. 4 N. Duriin and E. Cadenas, The role of singlet oxygen and triplet carbonyls in biological systems, Rev. Chem. Intermediates, 8 (1987) 147 - 187. 5 M. P. De Mello, S. M. De Toledo, M. Haun, G. Cilento and N. Duran, Excited indole3-aldehyde from peroxidase catalyzed aerobic oxidation of indole-3-acetic acid. Reaction with and energy transfer to transfer-ribonucleic acid, Biochemistry, I9 (1980) 5270 - 5275. 6 M. P. De Mello, S. M. de Toledo, H. Aoyama, H. K. Sarkar, G. Cilento and N. Duran, Peroxidase-generated triplet iridole-3-aldehyde adds to uridine bases and excites the 4-thiouridine group in t-RNA(Phe), Photochem. Photobiol., 36 (1982) 21 - 24. 7 N. Duran, M. C. Marcucci, M. P. De Mello and A. Faljoni-Alario, Enzymatically generated electronically excited molecules induce transformation of I-thiouridine to uridine, Biochem. Biophys. Res. Commun., 117 (1983) 923 - 929. 8 N. Duran and M. P. De Mello, Photobiochemistry of 4-thiouridine in E. coli t-RNA, Photochem. Photobiol., 37 (1983) Sll. 9 S. M. De Toledo, A. Zaha and N. Duran, DNA strand scission in E. coli by electronically excites state molecules generated by enzymatic systems, Biochem. Biophys. Res. Commun., 104 (1982) 990 - 995. 10 J. W. Menck, J. B. Cabral Neto, A. Faljoni-Alario and R. Alcantara-Gomes, Damage induced in X-phase DNA by enzyme-generated triplet acetone, Mutat. Res., 165 (1985) 9 - 14. 11 L’ A. Guillo, S. M. De Toledo and N. Duran, Attemptet detection by fluorescence probes of DNA modifications produced by bioenergized triplet acetone, Photobiochem. Photobiophys., 6 (1983) 177 - 186, 12 L. Nassi, D. Schiffmann, A. Fabre, W. Adam and R. Fuchs, Induction of the SOS function sfiA in E. coli by systems which generate triplet ketones, Mutat. Res., 198 (1988) 53 -.60. 13 N. Durfin, M. C. Marcucci, M. P. De Mello, L. C. C. Leite and A. Faljoni-Alario, DNA modification through biophotoenergized processes on t-RNA. Photochem. Photobiol., 39 (1984) 80s. 14 N. Duran, M. C. Marcucci, M. S. V. Gatti and L. C. C. Leite, Lethal effect and DNA breakage caused by biophotoenergized indole-3aldehyde in E. coli, Znt. Symp. on Free Radicals and Excited State in Biological Systems, Buenos Aires, 1986, Abstract 16. 15 H. C. Birnboim and J. Doty, A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nuclear Acid Res., 2 (1979) 1513 - 1520. 16 N. Durfin, S. T. Faria-Furtado, A. Faljoni-Alario, A. Campa, J. E. Brunet and J. Freer, Singlet oxygen generation from the peroxidase-catalyzed aerobic oxidation of an activated -CH substrate, J. Photochem., 25 (1984) 285 - 295. 17 J. M. Hardwick, R. S. Von Sprecken, K. L. Yielding and L. W. Yielding, Ethidium binding sites in plasmid DNA determined by photoaffinity labeling, J. Biol. Chem., I59 (1984) 11090 - 11097. 18 B. S. Hass and R. B. Webb, Photodynamic effects of dyes on bacteria. I. V. Lethal effects of acridine orange and 460 or 500 nm monochromatic light in strains of Escherichia coli that differ in repair capability, Mutat. Res., 81 (198 1) 277 _ 285.
378 19 C. Salet, M. Bazin, G. Moreno and A. Favre, 4-Thiouridine as photodynamic agent, Photo&em. Photobiol., 41 (1985) 617 - 619. 20 G. Thomas and A. Favre, 4-Thiouridine triggers both growth delay induced by near-ultraviolet light and photoprotection, Eur. J. Biochem., 113 (1980) 825 - 834. 21 S. C. Tsai and J. Jagger, The roles of the Rel(+) gene and of 4-thiouridine in killing and photoprotection of Escherichiu coli by near ultraviolet radiation, Phofochem. Phofobiol., 33 (1981) 825 - 834. 22 A. Caldeira de Araujo and A. Favre, Near ultraviolet DNA damage induces the SOS response in Escherichia coli, EMBO J., 5 (1986) 175 - 179. 23 J. G. Peak, M. J. Peak and M. MacCoss, DNA breakage caused by 334 nm ultraviolet light enhanced by naturally occurring nucleic acid components and nucleotide coenzymes,Phofochem. Phofobiol., 39 (1984) 713 - 716. 24 J. C. Peak, M. J. Peak and C. S. Foote, Observation on the photosensitized breakage of DNA by 2-thiouracil and 334 nm ultraviolet radiation, Phofochem. Phofobiol., 44 (1986) 111 - 116. 25 C. D. Town, K. C. Smith and H. S. Kaplan, DNA polymerase required for rapid repair of X-rays-induced DNA strand breaks in uiuo, Science, 171 (1971) 851 - 854. 26 K. C. Smith and N. J. Sargentini, Metabolically produced UV-like DNA damage and its role in spontaneous mutagenesis, Phofochem. Phofobiol., 42 (1985) 801 - 803. 27 N. Duran, A. Campa, L. C. C. Leite, G. Cilento and E. Cadenas, Microsomal lipid peroxidation concomitant to peroxidase-catalyzed aerobic oxidation of indole-3acetate, Phofobiochem. Phofobiophys., 11 (1986) 281 - 292.