Chem.-Biol. Interactions, 13 (1976) 0 Elsevier Scientific

Publishing

89-97 Company, Amsterdam

- Printed in The Netherlands

H202 GENERATION DURING THE REDOX CYCLE OF MITOMYCIN AND DNA-BOUND MITOMYCIN C

C

MARIA TOMASZ

Department of Chemistry, Hunter College. City University, New York, N.Y. (U.S.A.) (Received March 24th, 1975) (Revision received September 10th. 1975) (Accepted September 18th. 1975)

SUMMARY

Reduction of mitomycin C by NaBi& or by NADPH in the presence of a cell extract followed by exposure to air results in the generation of Hz02. This phenomenon occurs not only with free mitomycin but also with mitomycin irreversibly bound to DNA. In view of these findings, the antibiotic activity of mitomycin was tested in two bacterial systems: a facultative aerobic bacterium grown in the presence or absence of oxygen and an obligate anaerobic bacterium. No oxygen effect could be demonstrated in either case in the growth-inhibitory and bactericidal activity of the drug. Nevertheless, the H202 generating capacity of mitomycin-DNA complexes inside the nucleus may play a role in the drug-induced biological damage to the genetic material of cells. __.

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INTRODUCTION

Intracellular reduction followed by oxidation by O2 to a lethal species has been suggested as the mode of action for several antibiotics and other inhibitory agents. Thus, toxoflavin, a potent antibiotic and poison, was shown to act in various microorganisms and cell extracts as an electron carrier between NADH (or NADPH) and O2 with concomitant production of H202. The drug proved completely inactive under anaerobic conditions, i.e. when H201 generation was prevented, and this was taken as strong evidence that H202 formation is the direct cause of the antibiotic effect of toxoflavin [l]. Streptonigrin, another antibiotic and antitumor agent, was shown to possess similar HzOz-generating and electron carrier properties in mitochondrial suspensions or in the presence of purified diaphorase [ 21. Furthermore, a cellular reduction/oxidation cycle was shown to be required for the lethality of streptonigrin in bacteria [3,4]. Some simple quinones (e.g. benzo-

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quinone and 1,Cnaphthoquinone) behaved similarly to streptonigrin in these systems [ 31. Mitomycin C, the potent antibiotic and antitumour drug, possesses an aminoquinone moiety similar in structure and redox potential properties to the redox system of streptonigrin [3]. It is also known to be reduced intracellularly [ 61 and in cell free systems [ 51 in complete analogy to the above drugs. Its bactericidal and lysogenic inducer effects as well as its response to redox type synergizing and antagonizing agents are similar to those of streptonigrin [ ‘7] . Both drugs selectively inhibit DNA synthesis and subsequently induce massive degradation of bacterial DNA. The molecular effects on DNA, however, are different for the two antibiotics [8]. In view of the above similarities, it appeared possible that a reduction reoxidation cycle similar to that of toxoflavin, streptonigrin, and other quinones plays a role in the antibiotic action of the mitomycins. We tested this idea and report here our results. MATERIALS

Mitomycin C was purchased from Kiowa Hakko Kogyo Ltd., Tokyo, Japan. Helium gas (research grade, > 99.9999% pure) was obtained from Matheson Company. RESULTS

H202 generation in chemically reduced mitonzycin solution (Table I)

Mitomycin reduced by NaBHa becomes rapidly autoxidized upon exposure to air, as seen by the color change (pale yellow to purple) and known from previous work [6,9]. Table I shows that a variable amount of H202 is produced in such autoxidized mixtures. The reoxidized quinone (II) which is somewhat different from mitomycin 6, due to secondary changes in the reduced state [5,6,9] generates HIOl in comparable amounts when resubmitted to a second reduction-oxidation cycle. Para-benzoquinone gave a negative HzOz test after a similar reduction/air exposure cycle. The conditions during the reduction step (aerobic or anaerobic) did not appreciably influence the amount of Hz02 detected. This is as expected, for the following reason: Although under the aerobic reduction conditions some reduced mitomycin is undoubtedly autoxidized, then reduced again by excess NaBH9 (repeatedly), any H202 resulting from these “extra” cycles would be destroyed by excess NaBH,,, as indicated by our control experiment (H202 as “substrate”, in Table I). It follows that the HaOz detected in our analysis is only that which is formed after the termination of the reduction step of the excess NaBHJ with CH,COOH, followed by bubbling air through the mixtures, as described in Table I. The quantity of H202 thus obtained should be more or less the same in both cases since both the aerobic and the anaerobic mixtures appear completely reduced (pale yellow color) at the time of termination.

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TABLE I FORMATION OF Hz& IN THE REDUCTION-AUTOXIDATION CYCLE OF M;TOMYCIN C USING NaBH4 AS THE REDUCING AGENT a . _ . ~___. _ Substrate reduced Conditions of Amount of HzO: the reduction step (molequiv. ) -Mitomycin C aerobic 0.9-1.: Mitomycin C + CiltZdXX ” aerobic 0.01 Mitomycin C anaerobic 0.87 None aerobic e. 0.01 p-Benzoquinone aerobic c 0.01 aerobic *.O.Ol H202 Reduction-oxidation product ol’ MC (II) aerobic 1.1 Mitomycin--DNA complex ( anaerobic 2:13 Same + 18 units cakllase per 0.2 I.tmole substrate anaerobic O.(J:! -_ a

The substrate (5 fimole in 0.8 ml aqueous solution) was reduced aerobically or anaerobically by the addition of 25 /.d 1 IM NaBH4 solution. After 5 min. the NaBHa was dcstroyed by SO /.d CHjCOOH and the mixture (pH 5) was stirred open to air for 5 min. then applied onto a 1 X 4.5 cm cation-exchange column (AG-.?O\V~‘-s.4. ‘LOO-400 mesh. H’-form). On washing with water Hz02 appeared in the effluent and was ;tnalyzed by lhr TiS04 method [ 101. Anaerobic conditions during the reduction were achirvr~tl by bu bbling He gas through the component solutions for 10 min before mixing and continuing the bubbling during and after the mixing. b Added to a!iquots prior to their analysis for HzO:. c The complex of mitomycin and DNA (denatured, E. coli K-12) possessing a binding ratio = 2 (mole nucleotide unit per mole mitomycin) was prepared and characterlLed a.z described elsewhere. An aliquot of the complex in 3 ml SSC corresponding to O.titi9 pmole of complexed mitomycin was reduced with 50 ~1 0.25 M NaBHa solution itllilcrobically. The purple color of the complex disappeared rapidly. Excess NaBI was tfeslroyc*d by addition of 5 /.d CH3COOH and the mixture was exposed to air. The color return4 almost immediately. The complex precipitated upon cooling and was removed by cc.ntri. fugation. Hz02 was detected in the supernatant by the ferrous thiocyanatr method [IQ 1 No Hz02 was present in the control non-reduced complex solution.

IJ202 generation 6y mitomycirz-DNA complex (Table 1) Reduction of DNA-bound mitomycin by NaHHj proceeds smoothly, as seen by the disappearance of the purple color. Upon exposure to air the color returns, in complete analogy LOthe behavior of free mitomycin. The amount of H20z formed by this cycle appears to be greater (2.42 mole per mole mitomycin), however, than that formed by free mitomysin. H202 gensratiotl irz cell-free extracts (Table II) In anaerobic cell extracts of pneumococcus containing added NADPH, reduction of mitomycin took place as evidenced by the characteristic fainting of its blue color into yellowish brown. On exposure to air, Hz02 could be detected immediately, in somewhat less than stoichiometric quantity. No Hz02 was formed in the absence of either the cell extract or NADPH. 91

TABLE II FORMATION OF H202 IN THE REDUCTION-AUTOXIDATION CYCLE OF MITOMYCIN C IN A CELL-FREE EXTRACT OF DIPLOCOCCUS PNEUMONZAE a __~________-_--_-._-_-_------.__--.-. ._._.___.--_.___ .._.__ ...-___ Amc,unt of H202 Components (molequiv.) .- ---I___-----.-__-. -__--..__ . .~...__-__-_.-_-~_-~ ___ ____ _.---.---.. 0.70 Mitomycin + extract + NADPH < 0.01 Extract + NADPH co.01 Mitomycin + extract

H2O2 generation during the redox cycle of mitomycin C and dna-bound mitomycin C.

Chem.-Biol. Interactions, 13 (1976) 0 Elsevier Scientific Publishing 89-97 Company, Amsterdam - Printed in The Netherlands H202 GENERATION DURING...
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