Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.

The mitomycin antibiotics, produced by various Streptomyces, give rise to electrophiles capable of cross-linking DNA following either one-electron or two-electron reduction by enzymatic or chemical systems (1). The one-electron reduction product, the mitomycin C semiquinone radical anion (MC . ), 1 reacts with molecular oxygen (itself a stable diradical) at close to the diffusion controlled rate (10 9 to 10 10 M Ϫ1 s Ϫ1 ) to give the parent mitomycin C (MC) quinone and superoxide (O 2 . ) (2).
Since very few cross-links are required to give rise to a lethal event (3), the regeneration of MC and the production of a superoxide anion, a species of low toxicity relative to a DNA cross-link, represents a detoxification step. Under physiological oxygen concentrations, the half-life of MC . would be expected to be less than 0.1 ms. Therefore, in the presence of physiological concentrations of oxygen, only an extremely small proportion of the MC . produced could be involved in the direct alkylation of biomolecules, and the yields of DNA cross-links via this pathway would be negligible. The two-electron reduced species, mitomycin C hydroquinone (MCH 2 ), does not react rapidly with oxygen and, therefore, cross-links DNA in a manner largely independent of the concentration of oxygen. Thus, the degree of initial DNA damage by MC under aerobic conditions almost exclusively depends upon a two-electron reduction to MCH 2 . Under very low oxygen concentrations, greater cellular damage would be expected, reflecting the rate of production of both MCH 2 and MC . . There is also evidence that most of the damage resulting from the production of MC . under hypoxic conditions is due to disproportionation (4) or further reduction of MC . to MCH 2 and subsequent formation of an alkylating species therefrom, and not due to the direct interaction of MC . with DNA. Such processes may also play a role in aerobic alkylations following one-electron reduction, but competition from the reaction of MC . with O 2 is likely to greatly reduce the interaction of MC . with further reducing molecules. In biological systems MC can be reduced by a variety of enzymes, some favoring one-and some two-electron activation (5). Solid tumors show resistance to therapeutic modalities such as radiation due to areas of hypoxia arising from poor tumor vascularization (6). Mitomycin antibiotics have been used as an adjunct to x-irradiation to selectively target the radiation-resistant hypoxic fraction of human tumors (7,8). Variations in the toxicity of MC to cells have been attributed to differing levels of oxygenation, activating enzymes (and their localization), export pumps, and DNA repair (9 -12).
A number of genes coding for resistance proteins in Streptomyces lavendulae have been cloned and expressed. One of these genes, mcrA, codes for a 54-kDa protein (MCRA) that appears to be a mitomycin C hydroquinone oxidase (13,14). Expression of the cDNA for the bacterial resistance protein MCRA in CHO-K1/dhfr Ϫ cells resulted in profound resistance to MC in these cells under aerobic conditions, with little change in sensitivity to MC under hypoxia (14). The marked resistance to MC under aerobic conditions observed in MCRA-expressing CHO-K1/dhfr Ϫ cells resembles that produced in cell lines selected for resistance to MC under aerobic conditions (14). This finding suggests that a mechanism of resistance based upon the oxidation of MCH 2 by a functional homologue of the MCRA protein may be operative. Since many peroxidases have high * This work was supported in part by United States Public Health Service Grant CA-80845 from the NCI, National Institutes of Health. 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" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Current address: Vion Pharmaceuticals Inc., New Haven, CT 06511. ** To whom correspondence should be addressed. Tel.: 203-785-4533; Fax: 203-737-2045. 1 The abbreviations used are: MC . , mitomycin C semiquinone radical anion; H33258, Hoechst 33258; MC, mitomycin C; MCH 2 , mitomycin C hydroquinone; HRP, horseradish peroxidase; LP, lactoperoxidase; MP, myeloperoxidase; MCRA, mitomycin resistance protein A; QH 2 , hydroquinone; Rz, Reinheitszahl ratio, the ratio of the absorbance at the Soret band max to that of the max of the protein band; HPLC, high performance liquid chromatography. affinities for hydroxylated aromatic compounds and hydroquinones as oxidizable substrates, we compared the ability of various peroxidases to that of MCRA to oxidize MCH 2 and to inhibit DNA cross-linking. In this report we demonstrate that horseradish peroxidase (HRP), myeloperoxidase (MP), and lactoperoxidase (LP) oxidize MCH 2 in the presence of a source of hydrogen peroxide and prevents to various degrees the crosslinking of T7 DNA by MCH 2 .

EXPERIMENTAL PROCEDURES
Chemicals-T7 DNA and other chemicals and enzymes were purchased from Sigma, except where specified. Peroxidase purity was accessed by determining the Rz value (the Rz value equals the ratio of the absorbance at the heme prosthetic group Soret band max to that of the protein band max ) for the various enzyme preparations; for HRP (affinity-purified) the Rz (A 403 nm /A 275 nm ) value was equal to that reported for the pure protein (15), for LP the Rz value (A 412 nm /A 280 nm ) was 89% of that reported for the pure protein (16), and for MP the Rz value (A 430 nm /A 275 nm ) was 60% of that reported for the pure protein (17). Hoechst 33258 was obtained from Molecular Probes, Inc. (Eugene, OR). Purified MCRA was prepared by David H. Sherman, and MC was supplied by Bristol Myers-Squibb, Inc. (Wallingford, CT). Stock solutions of MC (2.8 mM) and sodium borohydride (NaBH 4 ) (20 mM) were made in isopropanol, which was used because it is an excellent hydroxide radical scavenger. Theoretically, the indirect generation of hydroxide radicals as a consequence of the redox cycling of MC, the reduction of oxygen by NaBH 4 , or the action of potentially protective peroxidases could interfere with the DNA cross-linking assay by introducing strand breaks. Therefore, since the objective was to examine DNA-DNA cross-linking by MC/NaBH 4 and enzymatic inhibition of cross-linking, the prevention of radical nicking was essential to these meas-urements. The final concentration of isopropanol in the reaction mixture was 2.4%, which was more than sufficient to prevent radical nicking in this system (18).
DNA Cross-linking and Nicking-DNA cross-linking kinetics and the extent of DNA cross-linking were determined using a DNA renaturation assay (14,19). The assay is based upon the observation that, under conditions of neutral pH, upon snap cooling thermally denatured covalently cross-linked T7 DNA rapidly renatures since the strands are held in register, yielding a highly fluorescent complex with H33258, whereas T7 DNA containing no cross-links does not. T7 DNA at a concentration of 100 g/ml in 20 mM Tris-HCl buffer containing 1 mM EDTA and 40 M MC at pH 7.4 was reacted with NaBH 4 . The crosslinking reaction was initiated by the addition of 1% by volume of freshly prepared 20 mM NaBH 4 solution in isopropanol, which was prepared by a 25-fold dilution of a 0.5 M solution of NaBH 4 in 2-methoxyethyl ether with dry isopropanol. The final concentration of NaBH 4 in the reaction mixture was 200 M. Small (15 l) aliquots were removed at various time intervals, diluted 100-fold with 5 mM Tris-HCl, 0.5 mM EDTA buffer, pH 8.0, containing 0.1 g/ml H33258, and assayed for DNA cross-links. The 100-fold dilution of DNA and alkylating agent effectively prevents significant further reaction. The concentrations of reactants were chosen to give a maximum of ϳ30 -50% of the DNA being cross-linked (i.e. at least one cross-link in 30 -50% of the molecules). The potential nicking by radicals or other species under the reaction conditions employed was ascertained by measuring the decrease in the fluorescence of the complex of H33258 and the stably pre-cross-linked DNA following a heat/chill cycle (19,20). The pre-cross-linked T7 DNA was prepared by treating 0.5 mg/ml DNA with 0.2 mM 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine in 10 mM Tris-HCl, 1 mM EDTA buffer, pH 8.0, for 24 h at 37°C. Fluorescence measurements were performed using a Hoefer Scientific Instruments TKO 100 fluorometer.
Determination of MC by HPLC-HPLC measurements of the loss and regeneration of MC were performed in 20 mM Tris-HCl buffer containing 1 mM EDTA, 40 M MC, and 200 M NaBH 4 at pH 7.4 using a modification of the method of Kumar et al. (4).
Spectroscopic Measurement of the Formation of Mitosene-All spectroscopic determinations were performed using a Beckman model 25 spectrophotometer at 575 nm. In these studies, MC and NaBH 4 were used at increased final concentrations of 200 and 500 M, respectively, because of the low absorbances of MC and the MC-derived species being measured at 575 nm. Stock solutions of 10 mM MC and 50 mM NaBH 4 in isopropanol were employed for these studies. All cuvettes were freshly acid-washed (HCl/HNO 3 ) and stored submerged in distilled H 2 O prior to use. This procedure minimized problems due to bubble formation on and adhesion to the faces of the cuvettes.
Decomposition Kinetics of NaBH 4 -The decomposition kinetics of NaBH 4 in 20 mM Tris-HCl buffer containing 1 mM EDTA and 20 g/ml phenol red at pH 7.4 were determined by following the change in absorbance of phenol red at 560 nm, which varies linearly with very small molar changes in the consumption or generation of hydrogen ions (21). The measurements were based upon the alkalinization of the medium from the consumption of 1 mol of protons/mol of NaBH 4 during its decomposition at pH values close to neutrality, as represented in the following reaction.
The alkalinization occurs because at pH values close to neutrality boric acid (pK a 10.2) is essentially undissociated. The reaction was FIG. 1. Comparison of the decay kinetics of NaBH 4 and the post-reduction spectral changes produced in MC solutions under identical conditions (pH 7.4, 37°C). A, decomposition kinetics of NaBH 4 as measured by medium alkalinization at 560 nm. B, the initial dashed portion of this trace largely represents the kinetics of the reduction of MC to MCH 2 by NaBH 4 (⌬ absorbance at 575 nm), whereas the latter continuous portion largely represents the kinetics of the progression of MCH 2 to high absorbing mitosene species (⌬ absorbance at 575 nm). Determination of Hydrogen Peroxide-The quantity of H 2 O 2 generated during the decomposition of NaBH 4 in air saturated buffer was estimated using a modification of the method of Pick and Keisari (22), which is based upon the oxidation of phenol red by H 2 O 2 /HRP to a compound that absorbs strongly at 610 nm at high pH values. The quantity of H 2 O 2 generated was determined by comparing the absorption values to a standard curve.
Spectroscopic Measurement of Hydroquinone Oxidation-The oxidation of hydroquinone (QH 2 ) by HRP, LP, or MP was evaluated by following the decrease in absorption of QH 2 at 288 nm in a 20 mM Tris-HCl, 1 mM EDTA buffer, pH 7.4, at 37°C.

Kinetics of NaBH 4 Decomposition, MC Reduction, and MCH 2
Decomposition-The decomposition kinetics of NaBH 4 in 20 mM Tris-HCl, 1 mM EDTA buffer, pH 7.4, using a protometric assay at 37°C appeared to follow first order or pseudo first order kinetics with a short t1 ⁄2 value of 4.7 Ϯ 0.9 s (Fig. 1). If the rate of reduction of MC by NaBH 4 is assumed to be proportional to the concentration of NaBH 4 , the rate of reduction of MC would be expected to follow the disappearance of NaBH 4 and occur rapidly over a short temporal window, resulting in the bolus production of MCH 2 . Reduction of MC to form MCH 2 should result in a disruption of the extended conjugation of the double bonds, resulting in a shift in the absorption maxima to lower wavelengths and in a bleaching of absorbance at higher wavelengths. However, upon the subsequent formation of high absorbing mitosene products, the absorbance at these wavelengths should then increase beyond the initial level of MC.
Visible spectral scans of MC before and at various times after the addition of NaBH 4 were performed, and a rapid bleaching followed by a rise in absorbance beyond the original level was observed, centered around 575 nm. At 37°C, the bleaching at 575 nm was maximal within 3-5 s (Fig. 1). This bleaching was followed by a progressive increase in absorption at this wavelength to approximately twice the absorption of the initial MC solution and appeared to follow first order or pseudo first order kinetics with a t1 ⁄2 value of 14.8 Ϯ 2.8 s at 37°C and pH 7.4. This finding is consistent with the "pulse-like" generation of MCH 2 . HPLC studies indicated that only about one half of the initial amount of MC was reduced under these conditions despite the 5-fold molar excess of NaBH 4 . The short t1 ⁄2 of NaBH 4 due to its rapid reaction with the vast molar excess of H 2 O provides a possible explanation for the limited reduction of the available MC. Since only ϳ50% of the MC was reduced, the initial reduction product(s) has little absorption at 575 nm compared with MC and the mitosene products subsequently generated have ϳ2-3-fold greater absorption at 575 nm than MC.
The rate of production of mitosenes was highly temperaturesensitive with the t1 ⁄2 at 30°C being ϳ30 s compared to ϳ15 s at 37°C (Fig. 2A). Addition of MCRA prior to the reduction of MC by NaBH 4 blocked these spectral changes under aerobic conditions (Fig. 2B). MC is believed to react following reduction as shown in Fig. 3; the methoxy group at position 9a is lost subsequent to hydroquinone formation to give leuco-aziridinomitosene. Oxidation after the formation of leuco-aziridinomitosene cannot result in the regeneration of MC, which can only occur if enzymatic oxidation occurs at the level of MCH 2 . Ad-

by various oxidizing components as determined by HPLC
The data have been normalized such that the % yield of MC from MCH 2 has been calculated relative to the assumed level of MCH 2 formation. The addition of NaBH 4 to aerobic solutions of MC under the conditions described resulted in a 50 -60% net loss of MC in all experiments. It is assumed that this net loss of MC represents the quantity of MCH 2 initially formed and that, in the absence of added oxidizing components, which increase the level of recovery of MC, essential all the MCH 2 spontaneously gives rise to products other than MC. The values presented are the average of two experiments each conducted in duplicate. dition of MCRA at the point of maximum bleaching (3-5 s after the NaBH 4 ) gives results almost equivalent to those obtained by the addition of MCRA prior to NaBH 4 . The spectroscopic absorption is restored to close to the original MC level without the production of high absorbing mitosenes; the bleaching dip is reduced slightly in magnitude when MCRA is added prior to the NaBH 4 , probably due to some MCRA mediated re-oxidation occurring during the reduction phase. The late addition of MCRA, i.e. at the maximum MCH 2 concentration, served to combat the argument that the MCRA merely prevented the initial reduction of MC. HPLC analysis indicated that, when MCRA was initially present in the reaction mixture, essentially all of the MCH 2 was back-oxidized to MC and there was little or no net MC loss or mitosene formed (Table I). Addition of MCRA or HRP/H 2 O 2 at a time point equivalent to the half-reaction point (ϳ30 s at 30°C and pH 7.4), as judged from the spectroscopic studies ( Fig. 2A), reduced the quantity of MC being restored by one half, indicating that one half of the total MCH 2 generated must still be present at this time. This finding implies that the t1 ⁄2 values determined spectroscopically and by DNA cross-linking reflect those for the loss of MCH 2 itself and not a subsequent low absorbing species, i.e. the conversion of MCH 2 to leucoaziridinomitosene, which appears to be the rate-determining step. The kinetics do not represent a rate-determining reduction of MC by NaBH 4 since the t1 ⁄2 of NaBH 4 is much shorter than that observed for the 575 nm spectral changes, nor can it represent the reaction of a low absorbing component formed subsequent to the loss of the methoxy group since MC regeneration would not be possible after this point (Fig. 3).
Peroxidase Oxidation of MCH 2 -Spectroscopic experiments similar to those described above were performed in which peroxidases were substituted for MCRA. Addition of HRP at equivalent time points gave visible spectral traces that were surprisingly similar to those obtained with MCRA (Fig. 4A). Surprisingly, the addition of H 2 O 2 was not required for HRP to block the production of the high absorbing species (Fig. 4A), to prevent the net loss of MC as determined by HPLC, or to block the cross-linking of T7 DNA by MC (Tables I and II). Thus, like MCRA, HRP catalyzed the regeneration of MC from the bleached material, although the yield was somewhat less. When these experiments were repeated using LP and MP, both of these peroxidases were able to decrease the generation of mitosenes and affect the regeneration of MC from the bleached material to various degrees, but were much less effective than HRP and MCRA ( Fig. 5 and Table I). The order of effectiveness was MCRA Ͼ HRP Ͼ LP Ͼ MP.
The absence of a requirement for H 2 O 2 to manifest the inhibitory effects of HRP coupled with the observation that catalase antagonized the effects of HRP (Fig. 4B and Tables I and  II)  If a self-propagating chain reaction occurred to any significant extent, as shown in Scheme 1, a greater ratio of QH 2 oxidation to H 2 O 2 addition would be expected. Since this did not occur, we examined H 2 O 2 production via mechanism (c), the partial-reduction of O 2 by NaBH 4 . Solutions in which NaBH 4 was allowed to decompose were assayed under aerobic and anaerobic (purged N 2 ) conditions for H 2 O 2 . H 2 O 2 was detected under aerobic conditions only when the peroxidase/phenol red system was present during the decomposition of the NaBH 4 . The quantity of H 2 O 2 trapped by HRP increased with increasing quantities of HRP within the range examined (10 -50 units/ml) (Table III). This effect was not observed when standard H 2 O 2 calibration curves were generated, where the absorbance at 610 nm was dependent upon the concentration of H 2 O 2 only and independent of the concentration of HRP in the range of the enzyme concentration employed. This was due to the fact that the 10 units/ml HRP used was vastly more than sufficient to complete the oxidation of the phenol red indicator by the available H 2 O 2 present during the assay period. The dependence upon the concentration of HRP suggested that some other component was competing with the HRP for the generated H 2 O 2 . Since NaBH 4 is a powerful reductant and H 2 O 2 a powerful oxidant, one would expect the NaBH 4 to readily reduce the H 2 O 2 . To test this possibility, the addition of HRP was delayed until after the NaBH 4 had decomposed; this procedure resulted in no H 2 O 2 being trapped, even when the mixture was spiked with as much as 40 M H 2 O 2 before the addition of NaBH 4 . When spiked with 80 M H 2 O 2 , only ϳ5% of the H 2 O 2 was found to remain after the decomposition of the NaBH 4 (Table III). Thus, H 2 O 2 has a dynamic existence in this system, with H 2 O 2 being generated from the reduction of O 2 by NaBH 4 and then consumed by further reduction. These results explain the lack of a need for an external source of H 2 O 2 for the peroxidases to oxidize the MCH 2 while still being sensitive to inhibition by catalase.
Effects of Peroxidases on DNA Cross-linking-Enzymes with the ability to rapidly oxidize MCH 2 back to MC before a significant proportion of the MCH 2 had undergone subsequent reactions would be expected to protect DNA against cross-linking in model systems. For this reason we compared the ability of HRP, LP, and MP to that of MCRA to protect T7 DNA from cross-linking by NaBH 4 reduced MC. All of the peroxidases were effective in blocking the cross-linking of T7 DNA by re-duced MC, and relatively high levels of protection were produced by relatively low concentrations of some of these enzymes (Table II). Smaller differences were observed in the ability of peroxidases to block T7 DNA cross-linking by reduced MC than in their ability to inhibit the production of high absorbing mitosenes and the yields of MC as a result of the back oxidation of the reduced species. Matching kinetics were observed for DNA cross-linking by reduced MC and for the postreduction spectral changes in MC. Half-maximal effects were found to occur for both parameters in ϳ15 s at 37°C and pH values of 7.4 (Fig. 7) and in ϳ4 min at room temperature (ϳ23°C) and pH 8.0.
The primary reason isopropanol was used as the vehicle for both MC and NaBH 4 was to provide protection for the T7 DNA from potential oxygen-derived radical-mediated nicking. Previously, it had been shown that the 2.4% final concentration of isopropanol used in these experiments was more than sufficient to block radical nicking of DNA (18). However, to verify that the T7 DNA was not being nicked significantly relative to the level of cross-linking, we measured the net level of crosslinking of T7 DNA stably pre-cross-linked to ϳ30% (30% of the population of DNA molecules contained one or more crosslinks) with another agent, 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine, after which the DNA was subjected to further cross-linking in the MC/NaBH 4 /peroxidase systems. If an intact T7 DNA molecule contained just one cross-link, the strands would be held in register and the entire molecule after thermal denaturation would renature upon snap cooling. If just one strand break was introduced into the DNA molecule, a minimum of two cross-links, depending upon their position relative to the nick, would be required to fully renature the DNA after snap cooling. Therefore, the presence of significant nicking in relation to the number of cross-links would greatly attenuate the measured cross-linking signal. If a pre-crosslinked population with an X mole fraction of the molecules containing one or more cross-links was subjected to further cross-linking by a second agent to the extent that this would give a Y mole fraction of cross-links when used as a single agent, the net mole fraction cross-linked in the resultant population if no nicking occurred would be X ϩ (1 Ϫ X)Y. This formula approximates the sum of X and Y only if X and Y are small. The true value is somewhat less than the sum because additional cross-links added to previously cross-linked molecules would not give rise to an increase in signal. If nicking was the primary reason, for example in the Ͼ90% decrease in the cross-linking signal observed when HRP/H 2 O 2 was added to the MC/NaBH 4 system, a comparable reduction would be expected in the pre-existing cross-linking signal and the overall level of cross-linking would be greatly reduced. In our dual  cross-linking experiments, the measured net cross-linking closely matched the calculated value for the combined crosslinking signal in the absence of significant nicking (Table IV). Therefore, the protection seen by the peroxidases was largely due to a decrease in the number of cross-links and not to an artifactually reduced signal due to the introduction of a large number of nicks.

DISCUSSION
The instability of MCH 2 under normal physiological conditions complicates the study of enzymes that interact with this species. Ideally, to study the oxidation of MCH 2 by enzymatic systems, it would be optimum to be able to add MCH 2 directly to reaction systems or to be able to "pulse" generate the MCH 2 in solution. NaBH 4 appears to reduce MC to MCH 2 as the major product over a short temporal window under the chosen conditions ( Fig. 1) relative to the rate of subsequent MCH 2 reactions and, therefore, appears to be a suitable reductant for studies of this kind. Since the kinetics and by-products of the reactions of NaBH 4 with MC, water, and oxygen are relevant to studies on the possible role of peroxidases in the mechanism of resistance to MC, we have examined these reactions. Of particular relevance is the transient generation of H 2 O 2 during the aerobic decomposition of NaBH 4 as a result of the reduction of molecular oxygen and the subsequent further reduction of the generated H 2 O 2 , obviating the addition of exogenous H 2 O 2 for the peroxidases to be operative, thereby explaining the sensitivity to inhibition of peroxidase protection by catalase without the addition of exogenous H 2 O 2 .
HPLC analysis confirmed the regeneration of MC from the initial reduced species (MCH 2 ) by MCRA and the various peroxidases. Addition of MCRA at the half-reaction point, based upon the time course of the spectroscopic changes (ϳ30 s at 30°C and pH 7.4), resulted in the regeneration of MC being decreased by approximately one half, indicating that one half of the total MCH 2 generated must still be present at this time. This finding implies that the t1 ⁄2 values determined spectroscopically and by DNA cross-linking reflect those for the loss of MCH 2 itself and not a subsequent low absorbing species, i.e. the conversion of MCH 2 to leuco-aziridinomitosene, which appears to be the rate-determining step. The kinetics do not represent a rate-determining reduction of MC by NaBH 4 since the t1 ⁄2 of NaBH 4 is much shorter than that observed for the spectral changes, nor can it represent the reaction of a low absorbing component formed subsequent to the loss of the methoxy group since MC regeneration would not be possible   after this point. The cross-linking of T7 DNA by NaBH 4 reduced MC appeared to follow first order kinetics and was extremely rapid under these conditions, with half maximal cross-linking occurring in ϳ15 s at 37°C and pH 7.4, matching the kinetics seen for the production of high absorbing mitosenes in the spectroscopic studies (Fig. 7). This finding implies that the same rate-determining step of MCH 2 to leuco-aziridinomitosene was limiting in both of these processes. If the enzymes were oxidizing MCH 2 , their inclusion should block T7 DNA cross-linking in the MC/NaBH 4 system; this indeed occurred, with MCRA, HRP, LP, and MP strongly blocking the measured cross-linking signal (Table II).
Single-strand breaks could arise from radicals that theoretically could be generated by the interaction of components in the reaction mixture and appear to reduce the level of crosslinking of DNA that was measured. However, experiments involving the additional cross-linking of stably pre-cross-linked DNA in the presence and absence of protective systems were consistent with the absence of significant nicking and the inhibition of cross-link formation. Unlike MCRA, concentrations of HRP, MP, and LP that largely blocked the cross-linking of T7 DNA were not as effective in preventing the net loss of MC or the spectral changes in the cases of MP and LP. Therefore, significant quantities of products other than MC must be produced by the HRP-, LP-, and MP-mediated oxidation of MCH 2 , or a large fraction of the protection from the cross-linking of DNA via these peroxidases arose from the preferred oxidation of species subsequent to MCH 2 formation but prior to cross-link formation. Preferential oxidation of the species formed after the loss of the methoxy group could restrict the molecule to monofunctional alkylation that would not be detected in our system. Oxidation to species that may not be able to be reductively reactivated to a cross-linking or alkylating species may result in superior resistance to that resulting from the regeneration of MC. Thus, it seems feasible that plant peroxidases such as HRP may offer plant roots some protection from the toxic products of Streptomyces and other soil bacteria.
Variations in the sensitivity of neoplastic cells to MC have been attributed to differing activities of reductive activating enzymes, export pumps, and DNA repair enzymes (9 -12). The existence of enzymes capable of oxidizing MCH 2 back to the MC prodrug or other inactive forms gives rise to another possible mechanism by which resistance to the mitomycin antibiotics and toxicity differentials between oxygenated and hypoxic tumor cells could arise. Recently, work from our laboratory has shown that MCRA expressed in CHO-K1/dhfr Ϫ cells conferred profound resistance to MC under aerobic conditions only, resulting in a phenotype with an extreme oxic/hypoxic differential (14). High levels of resistance only under aerobic conditions resembles that produced in cell lines selected aerobically for MC resistance (see Ref. 14 for the appropriate references). These findings suggest that a mechanism of resistance based upon the oxidation of MCH 2 by proteins that are functional homologues of MCRA could be a mechanism contributing to resistance to MC.
It is of interest to note that one of a group of proteins cloned because of their ability to block the hypersensitivity of Fanconi anemia lymphoblastoid cells to MC was tentatively identified as a peroxidase based upon sequence analysis (23); moreover, this protein showed the appropriate induction after H 2 O 2 exposure (24). It should be noted that the selective loss of twoelectron reducing pathways responsible for the toxicity under aerobic conditions could result in a similar resistance profile if the total two-electron flux was small compared with the total one-electron flux, resulting in an insignificant contribution of two-electron reducing mechanisms to the total toxicity under hypoxia. If some form of peroxidase activity were involved in the oxidative detoxification of MCH 2 , a source of H 2 O 2 would also be required. MC could supply this source of H 2 O 2 under aerobic conditions as a consequence of one-electron reduction and redox cycling. If such a system were operative, the ratio of one-to two-electron reducing systems, and possibly their relative locations in the cell, could influence toxicity. Increasing the level of one-electron reduction, thereby fueling a MCH 2 peroxidase with H 2 O 2 , could be expected to alleviate toxicity under aerobic conditions. Consistent with this hypothesis is the observation that increased resistance to MC under aerobic conditions was found when NADH:cytochrome b 5 reductase was over expressed in the mitochondria of Chinese hamster ovary cells (11).

TABLE IV
Comparison of calculated net and measured net cross-linking of DNA stably pre-cross-linked with 1,2-bis(methylsulfonyl)-1- (2chloroethyl)hydrazine and subjected to further cross-linking by NaBH 4 reduced MC T7 DNA and T7 DNA pre-cross-linked by treatment with 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)hydrazine to a level of 31.6 Ϯ 1.1% were each subjected to cross-linking by various reaction mixtures containing protective systems. If the decrease in the observed cross-linking of DNA as a result of the inclusion of a protective system was solely due to the prevention of cross-link formation, then the measured net % cross-linking should match the calculated value. The % cross-linking values are the arithmetic means of three determinations Ϯ the standard deviation. Heme peroxidases occur in a variety of mammalian tissues and secretions, with very high levels being found in some of the lymphocyte cell lineages. Neutrophils contain the highest levels of any normal mammalian tissue (25) containing 2% (dry weight) myeloperoxidase sufficient to impart a faint green color to these cells. Chloromas, an extramedullary tumor of granulocytic lineage, contain extremely high levels of myeloperoxidase (6% dry weight) giving the tumor a characteristic green coloration (26). This level of myeloperoxidase equates to several hundred times the concentration of MP used in these model studies.