(CANCER RESEARCH 50, 7483-7489. December 1. 1990]

Metabolism of Mitomycin C by DT-Diaphorase: Role in Mitomycin C-inducedDNA Damage and Cytotoxicity in Human Colon Carcinoma Cells1

David Siegel, Neil W. Gibson, Peter C. Preusch, and David Ross2

Molecular Toxicology and Environmental Health Sciences Program, School of Pharmacy, University of Colorado, Boulder, Colorado 80309 ¡D.S., D. RJ; Division ofPharmaceutics, School of Pharmacy and Comprehensive Cancer Center, University of Southern California, Los Angeles, California 90033 [N. W. (i.¡;and Departmentof Chemistry, University of Akron, Akron, Ohio 44325 [P. C. P.]

ABSTRACT

The role of DT-diaphorase in bioreductive activation of mitomycin Cwas examined using HT-29 and BE human carcinoma cells which havehigh and low levels of DT-diaphorase activity, respectively. HT-29 cellswere more sensitive to mitomycin C-induced cytotoxicity than the DT-diaphorase-deficient BE cell line. Mitomycin C induced DNA interstrandcross-linking in HT-29 cells but not in BE cells. Both mitomycin C-induced cytotoxicity and induction of DNA interstrand cross-links couldbe inhibited by pretreatment of HT-29 cells with dicoumarol. Metabolismof mitomycin C by HT-29 cell cytosol was pH dependent and increasedas the pH was lowered to 5.8, the lowest pH tested. Metabolism ofmitomycin C by HT-29 cytosol was inhibited by prior boiling of cytosolor by the inclusion of dicoumarol. Little metabolism was detected in BEcytosols. When purified rat hepatic DT-diaphorase was used, metabolismof mitomycin C increased as the pH was decreased and could be detectedat pH 5.8, 6.4, 7.0, 7.4, but not at 7.8. Metabolism of mitomycin C wasNADH dependent and inhibited by dicoumarol or by prior boiling ofenzyme. An approximate 1:1 stoichiometry between NADH and mitomycin C removal was demonstrated and no oxygen consumption could bedetected. Metabolism of mitomycin C by purified HT-29 DT-diaphorasewas also dicoumarol inhibitable and pH dependent. The major metaboliteformed during metabolism of mitomycin C by HT-29 cytosol, purifiedHT-29, and rat hepatic DT-diaphorase was characterized as 2,7-diami-nomitosene. These data suggest that two-electron reduction of mitomycinC by DT-diaphorase may be an important determinant of mitomycin C-induced genotoxicity and cytotoxicity.

INTRODUCTION

The mechanisms underlying the antitumor activity of mitomycin C, a clinically important antitumor quinone, have beenthe subject of intensive research but remain unclear. Bioreductive activation of mitomycin C to genotoxic metabolites hasbeen considered a critical determinant of its biological activity(1-5). Metabolic pathways for both one- and two-electron reduction of mitomycin C which results in the generation ofreactive intermediates which can alkylate DNA have been proposed (6-10). In addition, one-electron reduction of mitomycinC to its semiquinone derivative, followed by interaction withmolecular oxygen, may generate aggressive oxygen specieswhich have also been proposed to be responsible for the biological activity of mitomycin C ( 11-14). Thus, both one- and two-electron reductive processes may be involved in bioactivationof mitomycin C in biological systems.

Mitomycin C has been shown to be anaerobically reduced inpyridine nucleotide-dependent reactions by bacterial (1,3) andyeast ( 15) reductases, rat liver homogenate (16-19), cytochrome

Received 6/11/90: accepted 8/30/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by grant RO1CA 51210 from the National Cancer Institute. Parts

of this work have been presented in abstract form at the Annual Meeting of theAmerican Association for Cancer Research, Washington. DC. 1990 (Proc. Am.Assoc. Cancer Res., 31: 2367, 1990).

2To whom requests for reprints should be addressed, at School of Pharmacy,Campus Box 297, University of Colorado, Boulder. CO 80309-0297.

P-450 reducíase(20), and xanthine oxidase (20). Bioreductiveactivation of mitomycin C to species which were capable ofcross-linking DNA was first demonstrated in bacterial systems(1, 3). More recently, NADPH-fortified mouse liver micro-somes or nuclei were found to catalyze activation of mitomycinC to an intermediate which alkylated 4-(/7-nitrobenzyl)-pyridine

(18, 19). Metabolism of mitomycin C by rat liver microsomesin an NADPH-dependent reaction under anaerobic conditions

in phosphate buffer resulted in the formation of various phosphate alkylation products at the C-l aziridine position (17).Mitomycin C-nucleotide adducts have been isolated after chemical reduction or acid-mediated degradation of mitomycin C(17, 21-25), and recently, a DNA adduci, cross-linked via the1 and 10 positions of mitomycin C, has been characterizedwhich provides unequivocal evidence for bifunctional alkylation(25). The bis adduct was formed in Micrococcus luteus DNAafter chemical reduction of mitomycin C in vitro and was alsoisolated from rat hepatic DNA after injection of mitomycin C(25). Mitomycin C-induced DNA cross-linking in EMT6 cellsunder aerobic conditions was potentiated as the extracellularpH was lowered from 7.5 to 5.7 (26). Since these authors alsoshowed that the decreased extracellular pH was paralleled by adecrease in intracellular pH, this work suggested an importantrole for pH in the genotoxic and cytotoxic activity of mitomycinC. Interestingly, metabolite profiles observed during anaerobicbiotransformation of mitomycin C by various enzymes are alsoinfluenced by pH (15, 20). To date no evidence exists formetabolism of mitomycin C under aerobic conditions, sinceboth the one- and two-electron reduced forms of mitomycin C

are considered to be unstable to oxygen (14, 15).A major two-electron reducíase in biological systems is

NADPH-quinone oxidoreductase or DT-diaphorase (EC1.6.99.2). This enzyme is an obligate two-electron reducíasewhich can utilize either NADH or NADPH as an electrondonor and is inhibited by dicoumarol (27). The role of DT-diaphorase in bioreductive activation of mitomycin C is controversial. Dicoumarol has been found to inhibit the cytotoxicityof mitomycin C in EMT6 cells under aerobic conditions (28).This would support a role for DT-diaphorase in bioreductiveactivation, but in hypoxic cells mitomycin C-induced cytotoxicity was enhanced by dicoumarol (28). A detailed study of themetabolism of mitomycin C by purified human kidney DT-diaphorase concluded that mitomycin C was not a substrate forthis enzyme and was actually a weak inhibitor (29). Previouswork had claimed that mitomycin C was a substrate for partiallypurified beef liver DT-diaphorase, although detailed experimental methodologies were not presented (30). Recently, dicoumarol has been found to inhibit the cytotoxicity and cross-linking induced by mitomycin C under aerobic conditions inL5178Y cells made resistant to hydrolyzed benzoquinone mustard (31). The mustard-resistant cell line had 24-fold higherDT-diaphorase activity than the parent L5178Y line and was4-fold more sensitive to mitomycin C. Similarly, recent work

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BIOREDUCTIVE ACTIVATION OF MITOMYC1N C BY DT-DIAPHORASK

in human fibroblast lines (32) and Chinese hamster ovary cells(33), sensitive and resistant to mitomycin C, suggested a rolefor DT-diaphorase in mitomycin C-induced cytotoxicity.

Cellular data implicating DT-diaphorase in bioreductive activation of mitomycin C have been based, to some extent, onthe use of inhibitors of DT-diaphorase such as dicoumarol. Theproblems inherent in the use of inhibitors in cellular systemswithout direct evidence of metabolism of mitomycin C havebeen discussed recently (34, 35). Considering that attempts todemonstrate metabolism of mitomycin C by purified DT-dia-phorases from rat liver (unpublished, cited in Ref. 3), humankidney (29), and DT-diaphorase-rich preparations from bothHT-29 human colon carcinoma cells and Walker cells (unpublished, in 34) have failed, these authors reasonably concludedthat the role of DT-diaphorase in mitomycin C resistance wasquestionable (34). In this study we demonstrate that mitomycinC is a substrate for DT-diaphorases purified from both rat liverand HT-29 human colon carcinoma cells but that metabolismof mitomycin C is pH dependent. In addition we present datawhich suggest that DT-diaphorase plays a critical role in activation of mitomycin C to genotoxic and cytotoxic species inHT-29 human colon carcinoma cells.

MATERIALS AND METHODS

Chemicals. Mitomycin C was a generous gift from the Pharmaceutical Research and Development Division, Bristol-Myers Company,Syracuse, NY. NADH (grade IV), DCPIP,' and reactive blue (Cibacron

blue) 2-Sepharose CI-6B were obtained from Sigma Chemical Company, St. Louis, MO. Dicoumarol and 5-chloro-2-pyridinol were purchased from Aldrich Chemical Company, Milwaukee, WI. All otherreagents were of analytical grade.

Cell Lines. HT-29 and BE human colon carcinoma cells were maintained by growing cells at 37°Cin monolayers in Eagle's minimum

essential media supplemented with 10% bovine calf serum, gentamicin(0.05 mg/ml), glutamine (0.03 mg/ml), 0.1 mM nonessential aminoacids, 0.1 mM sodium pyruvate, and 0.02 M 4-(2-hydroxyethyl)-l-

piperazineethanesulfonic acid.Drug Treatment. Mitomycin C and dicoumarol, dissolved in water

and dimethyl sulfoxide, respectively, were prepared immediately beforetreatments. Cells were pretreated with 20 ¿tMdicoumarol 30 min priorto the addition of mitomycin C. Control cells received dimethyl sulfoxide, and dimethyl sulfoxide concentrations were never >1%. Cellswere exposed to mitomycin C for 2 h at 37°C,and the treatments were

terminated by aspiration of the drug-containing media and replacementwith fresh media.

In Vitro Cytotoxicity Assays. Inhibition of colony-forming ability ofHT-29 and BE human colon carcinoma cells was assayed as describedpreviously (36). Cells were exposed to various concentrations of mitomycin C (2-10 (¿M)in the presence or absence of 20 ^M dicoumarol for2 h at 37°Cas described above.

Alkaline Elution Experiments. DNA interstrand cross-linking wasmeasured using alkaline elution techniques as described previously (37).In all experiments, internal standards were [MC]thymidine-labeledLI210 cells irradiated with 3 Gy of 137Cs 7-rays in the cold. For allcross-linking assays, control and drug-treated cells were also irradiatedwith 3 Gy. Drug treatments were for 2 h at 37°Cas described above.

Cell Cytosol Preparations. HT-29 and BE human colon carcinomacells were grown to confluency under standard conditions. The mediumwas removed and the cells washed with Hanks' balanced salt solution

and then scraped into 1 ml of 0.25 M sucrose. The cell suspension wassonicated for 30 s and then centrifuged at 100,000 x g for l h at 4°C

'The abbreviations used are: DCPIP, 2,6-dichlorophenolindophenol; HPLC,high-performance liquid chromatography, NMR, nuclear magnetic resonance;GC-MS, gas chromalography-mass spectrometry: BSA, bovine scrum albumin:t,, retention time: metabolite I, major metabolite formed during DT-diaphorase-

mediated metabolism of mitomycin C.

to yield a clear cytosolic fraction. Cell cytosol was concentrated byultrafiltration using membrane filters (Amicon, Danvers, MA, cutoffM, 10,000). Protein determinations were made using the method ofLowry el al. (38).

DT-Diaphorase Activity. DT-diaphorase activity was assayed essentially according to the method of Ernster (27) as modified by Bensonet al. (39). The reaction mixture contained 0.025 M Tris HCl (pH 7.4),BSA (0.7 mg/ml), 0.2 mM NADH, and 0.04 mM DCPIP. Reactions (1ml) were performed at 25°Cin the presence and absence of 20 ¿IM

dicoumarol. DT-diaphorase activity was measured as the dicoumarol-sensitive reduction of DCPIP (t 21,000 M ' cm"1). DCPIP was meas

ured as 600 nm using a Hewlett-Packard HP8452 diode array spectro-photometer.

Purification of HT-29 DT-Diaphorase. HT-29 DT-diaphorase waspurified from cell cytosol using Cibacron blue affinity chromatographyas described previously (40, 41). The purified protein was resolved as asingle band on sodium dodecyl sulfate-polyacrylamide gel electropho-resis and had a specific activity of 84 nmol/min/^g protein. All reducíaseactivity was dicoumarol inhibitable.

Purification of Rat Hepatic DT-Diaphorase. Rat hepatic DT-diaphorase was purified according to the method of Hojberg el a!. (42) fromuninduced rats, yielding a protein with a specific activity of 660 nmol/min/Vg protein.

HPLC Analysis of Mitomycin C Metabolism. HPLC methods weremodified from the methods of Pan et al. (20). HPLC analysis of themetabolism of mitomycin C by DT-diaphorase was performed using aShimadzu SIL 6A gradient system with UV/Visible detection. Reactions were analyzed on a Supelco C-18 reverse phase column (5 firn) ata detection wavelength of 314 nm. The elution system consisted of alinear gradient of 5% solution II to 75% solution II over 20 min(solution I, 10 mM potassium phosphate, pH 7.2; solution II, 10 mMpotassium phosphate, pH 7.2:100% methanol; 1:1). The flow rate was1.5 ml/min. Reactions were stopped with an equal volume of ice-coldmethanol containing 5-chloro-2-pyridinol (final concentration, 10 ng/ml). Aliquots ( 100 ^1)were immediately analyzed by H PLC. PreparativeHPLC for the isolation of 2,7-diaminomitosene was performed as aboveexcept that 10 mM ammonium bicarbonate, pH 7.2, replaced potassiumphosphate and the gradient was modified to 20-90% B over 20 min.Aliquots (1 ml) were injected onto a EM Scientific Hibar RP-18 (250

x 10 mm) preparative column at a flow rate of 3 ml/min. Collectionsof the major metabolite (/, = 27.2 min) were pooled, diluted with anequal volume of H2O, and applied to a Sep-Pak cartridge (C-18, WatersAssociates). The cartridge was washed with 60 ml H2O to remove buffersalts. 2,7-Diaminomitosene was eluted off with 3 ml methanol (100%)and evaporated to dryness under vacuum for NMR and GC-MS analy

sis.NMR Spectroscopy. 'H NMR spectra (64 acquisitions) of syntheti

cally prepared 2,7-diaminomitosene ( 17) and metabolite I were obtainedusing a Varian Gemini 300-MHz spectrometer (Department of Chemistry, University of Colorado, Boulder, CO). The samples were dissolvedin Me2SO-d6 for analysis.

Derivalization and GC-MS. The putative 2,7-diaminomitoseneformed during DT-diaphorase-catalyzed metabolism of mitomycin Cwas derivatized using Tri-Sil/BSA (Pierce, Rockford, IL.). The metabolite was then dissolved in 2 drops of dry pyridine plus 0.1 ml of Tri-Sil/BSA (in pyridine). The mixture was heated for 20 min at 60°Cand

then subjected to combined GC-MS using an HP5988A GC-MS systemwith a 12 m x 0.22 mm x 0.33 /im HP-1 column. The columntemperature was 220°Cand was increased 4°C/minto 300°C;electron

impact mass spectrometry (70 eV) showed four products with retentiontimes of 6.26, 7.27, 7.81, and 8.07 min. The molecular ions (m/z =378, t, = 6.36, 7.27, 8.07 min; m/z = 450, t, = 7.81 min) andfragmentation patterns were consistent with mono- and ditrimethylsi-lylated derivatives of reduced 2,7-diaminomitosene.

HPLC Analysis of NADH Oxidation. NADH oxidation was measured by HPLC as described (43).

Oxygen Uptake Experiments. Measurements for oxygen consumption were performed using a Clark-type electrode (Yellow SpringsInstrument Company) as described previously (41). Reactions were

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BIOREDUCTÕVE ACTIVATION OF MITOMYCIN C BY DT-D1APHORASK

monitored for 30 min in air-saturated 0.1 M potassium phosphatebuffer (pH 5.8 and 7.8) at 37°C.

RESULTS

Mitomycin C-induced cytotoxicity and DNA damage wasexamined in HT-29 and BE human colon carcinoma cells. HT-29 cells have exceedingly high levels of DT-diaphorase (1740nmol/mg •protein/min), whereas BE cells have very low levels(<5 nmol/mg •protein/min). Cell-killing assays, based on colony-forming ability, showed HT-29 cells to be 6-fold moresensitive to mitomycin C than BE cells at a 1 log cell kill (Fig.1). Consistent with a role for DT-diaphorase in mitomycin C-induced cytotoxicity was the ability of dicoumarol, an inhibitorof DT-diaphorase (27), to protect HT-29 cells from mitomycinC-induced cytotoxicity. A 2-fold higher concentration of mitomycin C in the presence of dicoumarol was required to producethe same degree of cell killing as observed with mitomycin Calone (Fig. 1). Pretreatment of BE cells with dicoumarol hadlittle effect on the toxicity of mitomycin C. The extent of DNAdamage induced by mitomycin C was examined using alkalineelution methodology to measure the formation of single-strandbreaks and interstrand cross-links. Mitomycin C was found toinduce DNA interstrand cross-links in HT-29 cells, and cross-linking could be markedly reduced by inclusion of dicoumarol(Fig. 2). In contrast, little DNA interstrand cross-linking wasobserved in the DT-diaphorase-deficient BE cell line either inthe presence or absence of dicoumarol (Fig. 2). No single-strandbreaks were detected in either cell line after exposure to mitomycin C (not shown).

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Fig. 1. Inhibition of colony-forming ability of HT-29 and BE human coloncarcinoma cells exposed to mitomycin C. Colony-forming ability was measuredin HT-29 (triangles) and BE (circles) human colon carcinoma cells exposed tomitomycin C (0-6.4 UM) in the presence (closed symhols) and absence (opensymbols) of 20 »JMdicoumarol. Cells were exposed to dicoumarol 30 min prior toaddition of mitomycin C. Points, means of three separate treatments; bars, SD.

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Fig. 2. DNA interstrand cross-link indices (Gy equivalents) in HT-29 and BEhuman colon carcinoma cells exposed to mitomycin C. DNA interstrand cross-linking was measured using alkaline elution techniques in HT-29 and BE humancolon carcinoma cells exposed to mitomycin C (HT-29. 0.625-2.5 ^M; BE, 2.5-10 JIM)either in the presence (D. HT-29; •BE) or absence (S. HT-29; D, BE)of 20 iiM dicoumarol. Elution experiments were performed 4 h after a 2-h drugtreatment. Dicoumarol was present 30 min prior to the addition of mitomycin C.Columns, means of three independent experiments; bars, SD.

In an attempt to provide evidence for mitomycin C metabolism in HT-29 cells, we examined removal of mitomycin C andmetabolite generation in HT-29 and BE cell cytosols using

HPLC. Metabolism of mitomycin C and metabolite generationwas pH dependent and was observed at pH 5.8 but not at pH7.8. (Fig. 3). Metabolism of mitomycin C increased as the pHwas lowered from 7.8 to 5.8 and was effectively inhibited(>90%) by both prior boiling of HT-29 cytosol and inclusionof dicoumarol (0.02 mM) in incubations. The ability of HT-29cytosol to metabolize mitomycin C in the absence of addedNADH (Fig. 3) presumably reflects endogenous reduced pyri-dine nucleotides in the cytosolic preparation. The major metabolite formed in these incubations eluted at a slightly longerretention time than mitomycin C (mitomycin C, 20.1 min;metabolite, 22.8 min). Although mitomycin C can undergoacid-catalyzed degradation, we observed negligible non-enzymatic removal of mitomycin C and metabolite formation underour conditions. Acid-catalyzed degradation has been demonstrated to be marked at pH values <5 (21). In contrast to theresults obtained with HT-29 cytosol, little removal of mitomycin C or metabolite generation could be demonstrated using BEcytosol at either pH 5.8 or 7.8 (Fig. 3).

These data suggested that mitomycin C could be metabolized

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Fig. 3. Mitomycin C removal and metabolite formation by HT-29 and BE cellcytosol. HPLC was used to measure pH-dependent removal of mitomycin C (A)and metabolite formation (B) by HT-29 and BE cell cytosol. Complete system:100-j/M mitomycin C, 200 /IM NADH, and 0.5 mg/ml cell cytosol in 0.1 Mpotassium phosphate buffer (pH 5.8 or 7.8). Reactions were performed in thepresence and absence of 20 ¡IMdicoumarol in a tola! volume of 1 ml a( 25"C for1 h. Cell cytosol preparation and HPLC conditions as described in "Materialsand Methods." Columns, means from three separate determinations; bars, SD.

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BIOREDUCTIVE ACTIVATION OF MITOMYCIN C BY DT-DIAPHORASE

in a pH-dependent reaction by DT-diaphorase contained inHT-29 cells. In order to confirm that mitomycin C was asubstrate for DT-diaphorase and to examine the mechanism inmore detail, we utilized purified rat hepatic DT-diaphorase. Inagreement with the results using HT-29 cytosol, metabolism ofmitomycin C was observed at pH 5.8 but not at pH 7.8 andcould be inhibited by dicoumarol (0.02 mivi)and by prior boilingof enzyme (Fig. 4). This is in accord with previous work whichshowed no metabolism of mitomycin C by purified rat hepaticDT-diaphorase when the reaction was examined at pH 7.8 (29).In contrast to the data obtained using HT-29 cytosol, metabolism of mitomycin C by DT-diaphorase at pH 5.8 was depend

ent on the addition of NADH. The major metabolite formed(I) cochromatographed with the product formed in HT-29cytosol. Product I had a chromophore typical of 7-aminomito-senes (15) and demonstrated absorbance maxima at 314 and550 nm. Metabolite I cochromatographed with the productobtained during xanthine oxidase-catalyzed metabolism of mitomycin C at pH 6.5 under anaerobic conditions (not shown),which has previously been characterized as 2,7-diaminomito-sene (20).

Metabolite I cochromatographed with a synthetic standardof 2,7-diaminomitosene, prepared according to Ref. 17. When

examined using diode array detection, the single peak corré

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Fig. 4. HPLC analysis of mitomycin C metabolism by purified rat hepaticDT-diaphorase. Reaction conditions: complete system, 100 ¡IMmitomycin C, 200pM NADH, and 5.2 ng/ml rat hepatic DT-diaphorase in 0.1 M potassiumphosphate buffer (pH 5.8 or 7.8). Reactions were performed in a total volume of0.5 ml at 25°Cfor 30 min. HPLC conditions as described in "Materials andMethods." A, complete system pH 7.8; B. complete system pH 5.8; C, complete

system pH 5.8 in the presence of 20 pM dicoumarol; D, complete system pH 5.8with boiled rat hepatic DT-diaphorase; IS, internal standard; MC, mitomycin C.Peak (I, 5.8 min) corresponds to NADH.

sponding to the mixed chemical and enzymatic productsshowed identical spectra across the peak, indicating peak purity.Metabolite I was collected using preparative scale HPLC, re-chromatographed using analytical HPLC to ensure peak purity,and subjected to NMR spectroscopy. The NMR spectrum (notshown) was consistent with that previously published for 2,7-diaminomitosene (17) and with the synthetic standard of 2,7-diaminomitosene. A sample of metabolite I was also derivatizedwith Tri-Sil/BSA and analyzed by GC-MS. Four products wereobserved (see "Materials and Methods") and their mass spectrawere consistent with mono- and ditrimethylsilyl derivatives ofreduced 2,7-diaminomitosene (m/z 378 and 450). Other minormetabolites were detected during metabolism of mitomycin Cby purified rat hepatic DT-diaphorase (Fig. 3). Product IIprobably represents a phosphate alkylation product (17) sincethis metabolite was not observed when incubations were performed in citrate buffer at pH 5.8. From previous work (20),the retention time of product III would be consistent with theformation of m-2,7-diamino-l-hydroxyaminomitosene.

A more detailed examination of the pH dependence of mitomycin C metabolism by purified rat hepatic DT-diaphorasewas performed at pH values between 5.8 and 7.8 (Fig. 5). Thesedata show that both removal of mitomycin C and metabolite

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rat hepatic DT-diaphorase. HPLC was used to measure pH-dependent mitomycinC removal and metabolite formation catalyzed by rat hepatic DT-diaphorase.Reaction conditions: 100 /ÌMmitomycin C. 200 /ÕMNADH, and 5.2 ^g/ml rathepatic DT-diaphorase in 0.1 M potassium phosphate buffer (pH 5.8-7.8). Reactions were performed in a total volume of 0.5 ml at 25°Cfor 30 min. HPLCconditions as described in "Materials and Methods." Columns, means from three

separate determinations: bars, ±SD. A, mitomycin C removal; B, metaboliteformation.

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BIOREDUCTIVE ACTIVATION OF MITOMYCIN C BY DT-D1APHORASK

formation increase as the pH is lowered. Control experimentsusing menadione as substrate showed that the activity of DT-diaphorase did not decrease appreciably over this pH range.The stoichiometry of the reaction with respect to mitomycin Cand NADH removal was also examined at different pH values.The results (Table 1) show that the ratio of mitomycin Cremoved to NADH removed is approximately 1:1 at the different pH values examined. When the amount of NADH wasdecreased to 0.5 and 0.25 equivalents with respect to mitomycinC (0.1 ITIM),mitomycin C removal and metabolite formationdecreased markedly, indicating the absence of an autocatalyticmechanism (15). In addition a lag phase for mitosene formationat 550 nm which was observed during autocatalytic reductionof mitomycin C (15) was not detected during DT-diaphorase-mediated metabolism of mitomycin C at pH 5.8.

Oxygen uptake during NADH-dependent (0.2 mM) metabolism of mitomycin C (0.1 mivi) by rat hepatic DT-diaphorase(5.2 Mg/ml) could not be detected at either pH 5.8 or pH 7.8.Oxygen consumption during metabolism of the antitumor qui-none diaziquone by purified rat hepatic DT-diaphorase (41)was utilized as a positive control. When DT-diaphorase-me-diated metabolism of mitomycin C was examined using UV/VIS spectrophotometry under either aerobic or anaerobic conditions, essentially identical absorption changes were observed.Difference spectra (final —initial) of the reactions after 30 minunder aerobic and anaerobic conditions were superimposable.These data show that oxygen does not interact to any significantextent with the metabolites generated during DT-diaphorase-mediated metabolism of mitomycin C.

In order to confirm metabolism of mitomycin C by HT-29DT-diaphorase the enzyme was purified from HT-29 cells aspreviously described (41). HPLC analyses demonstrated pH-dependent and dicoumarol-inhibitable metabolism of mitomycin C with formation of 2,7-diaminomitosene as the majorproduct (Fig. 6).

DISCUSSION

In this manuscript we report that mitomycin C can be metabolized by both purified rat hepatic and HT-29 human colon cellDT-diaphorase in a pH-dependent manner under aerobic conditions. In agreement with other workers (29, 34), metabolismcould not be detected at pH 7.8 using either removal of mitomycin C, removal of pyridine nucleotide, or metabolite formation as indicators of metabolism. In addition, oxygen uptakewas not observed at this pH. The lack of detectable NADHremoval and oxygen consumption rules out a cycling mechanism by which the hydroquinone is reoxidized to the quinone.The degree of metabolism of mitomycin C, however, increased

Table 1 Stoichiometry of NADH oxidation and mitomycin C removal duringpurified rat hepatic DT-diaphorase-cataly:ed metabolism of mitomycin C

Values represent means ±SD of separate experiments. Number of experimentsare shown in parentheses. Reaction conditons: 0.1 M potassium phosphate buffer(pH 7.8-5.8). 100 fiM mitomycin C, 200 «IMNADH, and 5.2 /ig/ml rat hepaticDT-diaphorase. Reactions were performed in a total volume of 0.5 ml at 25'C

for 30 min. NADH oxidation and mitomycin C removal were assayed by HPLCas described in "Materials and Methods." NADH oxidation was corrected fornon-enzymatic removal.

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Fig. 6. HPLC analysis of mitomycin C metabolism by purified human HT-29DT-diaphorase. Reaction conditions: Complete system, 200 /JM NADH, 100 ^Mmitomycin C and 8.0 jig/ml human HT-29 DT-diaphorase in 0.1 M potassiumphosphate buffer (pH 5.8 or 7.8). Reactions were performed in a total volume of0.5 ml at 25'C for 30 min. HPLC conditions as described in "Materials andMethods" except that a gradient of 20-80% solution II over 25 min was used. A,

complete system pH 7.8; B, complete system pH 5.8; C, complete system pH 5.8in the presence of 20 nM dicoumarol; IS, internal standard; MC, mitomycin C; I,2,7-diaminomitosene.

as the pH was decreased from 7.8 to 5.8. The major metabolite(metabolite I) had a characteristic 7-aminomitosene chromo-phore and cochromatographed with the product of anaerobicxanthine oxidase-mediated metabolism of mitomycin C whichhas previously been characterized as 2,7-diaminomitosene (20).Metabolite I was characterized as 2,7-diaminomitosene by coch-romatography with a synthetic standard which indicated a highdegree of peak purity when analyzed using diode array spectrophotometry, by NMR spectroscopy, and by GC-MS after deri-vatization. In accord with mitomycin C removal, the formationof 2,7-diaminomitosene increased as the pH was decreased.Metabolism of mitomycin C and formation of 2,7-diaminomitosene by purified rat and HT-29 DT-diaphorase was NADHdependent and inhibited by prior boiling of the enzymes or byinclusion of dicoumarol.

Increased 2,7-diaminomitosene formation under acidic conditions has previously been observed during chemical reduction(17) and during anaerobic metabolism of mitomycin C by OldYellow enzyme (15), NADPH cytochrome P-450 reducíase,and xanthine oxidase (20). Previous attempts to demonstratemetabolism of mitomycin C by purified DT-diaphorase havebeen performed at pH 7.8 using human kidney DT-diaphorase(29) and rat liver DT-diaphorase.4 In collaborative experiments

with these workers we have demonstrated that, although metabolism of mitomycin C by human kidney DT-diaphorasecannot be detected at pH 7.8, mitomycin C removal and 2,7-diaminomitosene formation can be detected at pH 5.8. To whatextent this behavior extends to other mitomycins is unclear, butwe have observed a similar pH-dependent metabolism of por-firomycin by purified rat hepatic DT-diaphorase.

The role of DT-diaphorase-mediated metabolism in mitomycin C-induced cytotoxicity and genotoxicity was exploredusing two human colon carcinoma cell lines which have verydifferent levels of DT-diaphorase activity. HT-29 cells havevery high levels of activity, whereas the BE cell line has a verylow activity (41). Mitomycin C was 6-fold more toxic to HT-29 cells than to BE cells, and cytotoxicity was inhibited bydicoumarol in HT-29 cells but not in BE cells. Mitomycin Cinduced extensive DNA cross-linking in HT-29 cells but not inBE cells, and consistent with the role of DT-diaphorase in

*G. Powis, personal communication.

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BIOREDUCnVE ACTIVATION OF MITOMYCIN C BY DT-DIAPHORASK

activation, this could be effectively inhibited by dicoumarol.Single-strand breaks were not detected in either cell line, suggesting the absence of oxygen radical-mediated DNA damage(44). In agreement with these cellular data we found that HT-29 cell cytosol was capable of metabolizing mitomycin C,whereas BE cell cytosol was not. Metabolism of mitomycin Cand formation of 2,7-diaminomitosene in HT-29 cell cytosolwas pH dependent and inhibited by dicoumarol, with the greatest extent of enzymatic metabolism occurring at pH 5.8 (thelowest pH tested). The pH requirements for metabolism ofmitomycin C using HT-29 cell cytosol are therefore analogousto those observed using either purified HT-29 or rat hepaticDT-diaphorases. Metabolism of mitomycin C and formation of2,7-diaminomitosene was not detected using BE cell cytosol atany pH tested. The cellular data taken together with the evidence showing metabolism of mitomycin C by both HT-29 cellcytosol and DT-diaphorase purified from HT-29 cells suggestedthat metabolism of mitomycin C by DT-diaphorase was involved in production of cytotoxic and genotoxic metabolites inthis cell type.

Reaction mechanisms have been proposed for 2,7-diaminomitosene formation following two-electron reduction of mitomycin C and elimination of methanol leading to the formationof a reactive quinone methide (15, 17). The latter can beprotonated under acidic conditions leading to 2,7-diaminomitosene formation (15). In pyridine the hydroquinone derivativeof mitomycin C can reoxidize to the parent quinone, whichargues against automatic elimination (45). It has been suggested, however, that in aqueous systems proton assistance tothe methoxy-leaving group may render elimination reactionsmore facile (15). Alternatively, after two-electron reduction atacidic pH, protonation of the hydroquinone form of mitomycinC may occur, leading to 2,7-diaminomitosene formation viarearrangement reactions (46). Our data cannot address theintermediates involved in this reaction but show a lack ofoxygen consumption during metabolism, virtually identicalspectrophotometric changes during metabolism under aerobicor anaerobic conditions, and an approximate 1:1 stoichiometrybetween NADH oxidized and mitomycin C removed. Thissuggests that the reactions leading to formation of 2,7-diaminomitosene at acidic pH after two-electron reduction of mitomycin C by DT-diaphorase occur faster than reoxidation bymolecular oxygen to generate quinone. The absence of detectable metabolism of mitomycin C at pH 7.8 when compared topH 5.8 suggests that mitomycin C is unable to act as a substratefor DT-diaphorase at pH 7.8. Other substrates for DT-diaphorase such as DCPIP (27), menadione (27), and the antitu-mor quinone diaziquone (41) can all be metabolized by DT-

diaphorase at pH 7.8.Peterson and Fisher (15) have described an autocatalytic

mechanism for anaerobic reduction of mitomycin C by yeastOld Yellow enzyme. The cis- and /ra/«-l-hydroxy-2,7-diami-nomitosenes are major products of this reaction at pH 8 andarise from rearrangement of the hydroquinone form of mitomycin C to form a quinone methide which acts as an electro-phile toward water. The resultant mitosene hydroquinones areoxidized to their quinone form by mitomycin C, providing anautocatalytic cycle. An autocatalytic mechanism would be unlikely in the case of metabolism of mitomycin C by DT-diaphorase since the major product is 2,7-diaminomitosene whichis a chain-breaking metabolite (15). The absence of a lag phasefor mitosene product formation, the 1:1 stoichiometry betweenNADH and mitomycin C removal, and the observation that

metabolism decreased markedly during incubation with DT-diaphorase as the NADH concentration was decreased to 0.5and 0.25 equivalents (relative to mitomycin C) confirmed theabsence of an autocatalytic mechanism (15).

The observation of pH-dependent metabolism of mitomycinC by DT-diaphorase may have some relevance for the increasedmitomycin C-induced DNA cross-linking observed in EMT6cells at pH 5.7 as opposed to pH 7.5 (26). These cells havesignificant DT-diaphorase activity (47) and increased bioreduc-tion of mitomycin C at acidic pH may contribute to the cross-linking observed. Our data may also explain the inhibitoryeffect of dicoumarol on mitomycin C-induced cytotoxicity andgenotoxicity under aerobic conditions in various cellular systems (28, 31-33). These data should, however, be interpretedwith some caution in view of the lack of evidence to confirmmetabolism of mitomycin C by cellular fractions or by DT-diaphorase purified from these cells (34). Under hypoxic conditions, dicoumarol does not inhibit but potentiates mitomycinC-induced cytotoxicity (28, 48), suggesting that this effect maybe mediated through other mechanisms than inhibition of DT-diaphorase. This view is supported by the observation thatdicoumarol also potentiates mitomycin C cytotoxicity underhypoxic conditions in LI 210 cells which do not have detectableDT-diaphorase activity (49). Under aerobic conditions LI210cells are not protected against mitomycin C-induced cytotoxicity by dicoumarol and similar results were obtained in thisstudy using the DT-diaphorase-deficient BE cell line. Interestingly, Tomasz et al. (25) have shown that chemical reductionof mitomycin C by either dithionite or by catalytic hydrogénation in the presence of DNA greatly influences the type andquantity of DNA adducts identified. The adduci profile formedafter enzymatic reduction of mitomycin C by DT-diaphorase atacidic pH values remains to be determined.

The influence of pH on the bioreduction of mitomycin C mayalso have therapeutic implications. Tumor cells in general andhypoxic cells in particular have lower intracellular pH valuesthan normal cells (50). This may indicate a greater propensityfor DT-diaphorase-mediated reduction of mitomycin C in these

cells as opposed to normal cells and the greater sensitivity ofhypoxic cells to mitomycin C is well characterized (28, 51).Hoey et al. (46) have shown that activation of mitomycin Carises from formation of the hydroquinone and have proposedthat the protonated hydroquinone could be the reactive metabolite which induces cross-linking of DNA. Although, our resultssuggest that DT-diaphorase is involved in the biological activityof mitomycin C in HT-29 cells, unequivocal evidence relatingreductive metabolism of mitomycin C at acidic pH to DNAdamage must await experiments using purified DT-diaphorasein cell-free systems.

ACKNOWLEDGMENTS

The authors would like to thank Dr. T. Koch and E. Frank for aninitial supply of 2,7-diaminomitosene and for use of the diode arrayHPLC detector. Dr. J. A. Ruth for assistance in synthesis of 2,7-diaminomitosene, Dr. J. L. Bollón for recording NMR spectra, andDr. G. Powis for helpful discussions.

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1990;50:7483-7489. Cancer Res   David Siegel, Neil W. Gibson, Peter C. Preusch, et al.   Colon Carcinoma CellsMitomycin C-induced DNA Damage and Cytotoxicity in Human Metabolism of Mitomycin C by DT-Diaphorase: Role in

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