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[CANCER RESEARCH 38, 1745-1750, June 1978]

A General Mechanism for Microsomal Activation of Quinone AnticancerAgents to Free Radicals1

Nicholas R. Bachur, Sandra L. Gordon, and Malcolm V. Gee

Laboratory of Clinical Biochemistry, Baltimore Cancer Research Center, Division of Cancer Treatment, National Cancer Institute, Baltimore, Maryland21211

ABSTRACT

The highly active, quinone-containing anticancer drugs,Adriamycin, daunorubicin, carminomycin, rubidazone, no-galamycin, aclacinomycin A, and steffimycin (benzan-thraquinones); mitomycin C and streptonigrin (/V-hetero-cyclic quiÃ±ones);and lapacho! (naphthoquinone) interactwith mammalian microsomes and function as free radicalcarriers. These quinone drugs augment the flow of electrons from reduced nicotinamide adenine dinucleotidephosphate to molecular oxygen as measured by enhanced reduced nicotinamide adenine dinucleotide phosphate oxidation and oxygen consumption. This reaction iscatalyzed by microsomal protein and produces a freeradical intermediate form of the drugs as determined byelectron paramagnetic resonance spectroscopy. Micro-somes from mouse and rat liver, heart, lung, and spleenand mouse L1210 and P388 tumors all catalyze the augmented oxygen consumption. Apparent Kmvalues determined with normal rat liver microsomes range from 0.49x 10~"Mfor steffimycin to 13.4 x 10~"Mfor lapacho!. Since

SKF 525A and carbon monoxide have little effect on thisreaction, cytochrome P-450 is probably not involved. Several nonquinone anticancer agents were tested and werefound inactive in the system. Since quinone anticancerdrugs are associated with chromosomal damage thatappears to be dependent on metabolic activation of thesedrugs, we propose that the intracellular activation ofthese drugs to a free radical state may be primary to theircytotoxic activity. As free radicals, these drugs, becauseof their high affinity and selective binding to nucleic acids,have the potential to be "site-specific free radicals" that

bind to DMA or RNA and either react directly or generateoxygen-dependent free radicals such as Superoxide radical or hydroxyl radical to cause the damage associatedwith their cytotoxic actions.

INTRODUCTION

Although naturally occurring substances with anticanceractivity range widely in molecular size and structural complexity, a significant number of these agents are quiÃ±ones.Quinone-containing anticancer drugs include Adriamycin,daunorubicin, mitomycin C, streptonigrin, lapachol, andanalogs of these agents. Investigations on the mechanismof action of these agents that have anticancer as well asmutagenic and carcinogenic activity show that they interfere with DMA and RNA replication. However, other re-

1 Presented in part at the 10th International Congress of Chemotherapy.

September 18 to 23, 1977, Zurich, Switzerland (2).Received July 13, 1977; accepted March 1, 1978.

search suggests that the quinone of anthracycline antibiotics interferes with mitochondrial oxidative pathways (14),that streptonigrin and mitomycin C require biological reduction for activity (7, 27), or that streptonigrin and mitomycin C form Superoxide, peroxide, and hydroxide radicalsas toxic products in the cell (5, 12, 29, 30). However, nounifying mechanism encompassing all of these data exists.

In our studies of the microsomal metabolism of theanthracycline antibiotics, we observe that Adriamycin, daunorubicin, and other anthracycline analogs stimulate aNADPH-dependent oxygen utilization in microsomes (3).This phenomenon results from augmented transfer of electrons from NADPH to molecular oxygen that is catalyzed bya phenobarbital-inducible microsomal enzyme system thatapparently does not involve cytochrome P-450. During thiselectron transfer no visible irreversible biotransformation ofthe anthracycline molecule occurs. The anthracycline antibiotic participates as a reversible shuttle of electrons fromNADPH through a microsomal protein to oxygen. As theelectron carrier intermediate, the anthracycline molecule isa free radical, a semiquinone.

We have extended those studies of the anthracyclineantibiotics to include the /V-heterocyclic quinone and thenaphthoquinone anticancer agents. By using the microsomal system to ascertain the activities of quinone-containing and other cytotoxic drugs as stimulators of freeradical formation, we demonstrated a pattern of activitiesfor the quinone agents for free radical formation. Wepropose that the quinone anticancer agents act pharmacologically as "site-specific free radicals" following their acti

vation intracellularly.

MATERIALS AND METHODS

Adriamycin HCI was obtained from Adria Laboratories,Inc., Wilmington, Del. Carminomycin HCI was provided byProfessor G. Cause, Antibiotic Institute, Moscow, USSR.Steffimycin and nogalamycin were supplied by Dr. G. Neil,Upjohn Co., Kalamazoo, Mich. Rubidazone HCI was the giftof Dr. R. Maral, RhÃ´ne-Poulenc Co., Paris, France. Thefollowing drugs were supplied by the Drug Synthesis andChemistry Branch and the Natural Products Branch, Division of Cancer Treatment, National Cancer Institute: daunorubicin HCI, aclacinomycin A, mitomycin C, streptonigrin, lapachol, cyclophosphamide, camptothecin sodiumsalt, chlorozotocin, 1,3-bis-(2-chloroethyl)-1-nitrosourea,c/s-diaminodichloroplatinum, methotrexate, dibromoman-nitol, mithramycin, and procarbazine HCI. 1-/3-D-Arabino-furanosylcytosine, Â«-tocopherol, Triton N-101, and super-oxide dismutase were purchased from Sigma Chemical Co.,St. Louis, Mo. 1,4-Diazabicyclo-2.2.2-octane was obtained

JUNE 1978 1745

Research. on August 17, 2020. © 1978 American Association for Cancercancerres.aacrjournals.org Downloaded from

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N. R. Bachur et al.

from Aldrich Chemical Co., Milwaukee, Wis. SKF 525A wasthe gift of Smith Klein & French Laboratories, Philadelphia,Pa.

Microsomes were prepared from liver, lung, spleen, andheart tissues of male Sprague-Dawley rats (150 to 200 g)and adult male DBA/2 mice and from L1210 and P388murine tumors grown in DBA/2 mice as previously described (18, 22). The final microsomal sediments weresuspended in 0.15 M KCI-0.1 M potassium phosphate buffer,pH7.4.

L1210 and P388 cells were harvested 5 and 7 days,respectively, after i.p. implantation of 1 x 106 cells into

recipient DBA/2 mice. The tumor cells were suspended in0.15 M KCI-0.1 M potassium phosphate buffer, pH 7.4, andwere quick-frozen to -20Â°and thawed 4 times. The resulting suspension was homogenized with a Potter-Elvehjem-type homogenizer, and the microsomes were isolated asdescribed for the normal tissues above.

Triton treatment of microsomes, a means to lower endogenous oxygen consumption and not affect augmentation ofoxygen consumption (3), was done daily by making microsomal suspensions 2% (v/v) Triton N-101 and allowing 30min exposure at 0Â°prior to use.

Protein content of these preparations was determined bythe method of Lowry ef al. (17). NADPH oxidation wasmeasured at 340 nm on a Gary Model 14 spectrophotome-ter.

Oxygen Measurements. Oxygen content of reaction mixtures was determined with a Yellow Springs Instrument Co.Model 53 Clark electrode prepared for measurement ofmicrosamples as previously described (3). The 1.0-ml reaction mixture contained 0.2 M potassium phosphate buffer,pH 8.0, 0.5 to 4.0 mg microsomal protein and 6 x 10~3 M

NADPH. Buffer and water were aerated by air bubbling for3 min at 37Â°.Microsomes were added, the system was

closed by insertion of the electrode, and the mixture wasequilibrated for 2 min. NADPH was added, and endogenousoxygen consumption was monitored for 2 min. Finally, drugwas added at 5 x 10 " M, and total oxygen consumption

was measured. Oxygen consumption evaluation was basedon a 100% value of 1.58 x 10~7mol dissolved oxygen. When

used, effectors were added with the microsomes; drugswere added with microsomes during Kmand Vmaxdeterminations.

EPR2 Spectroscopy. EPR spectroscopy was conductedwith a VarÃanE-9 spectrometer at room temperature, 100-KH, field modulation, and 9.453 GHz frequency. Thereaction mixtures were the same as for oxygen measurements.

RESULTS

As described by Orrenius ef al. (23), microsomes consume oxygen in the presence of NADPH (Chart 1). Theendogenous, NADPH-dependent oxygen consumption isstimulated severalfold by the quinone-containing antican-cer drugs Adriamycin, mitomycin C, and lapachol (Chart 1,A to C). This augmentation of oxygen consumption dependson all 3 components: NADPH, microsomal protein, and the

quinone, anticancer drug, and the augmentation dependslinearly on microsomal protein (Chart 2). Boiling the microsomes renders them inactive.

A survey of anticancer agents from different structuraland mechanistic classes shows that all quinone-containingagents tested augment microsomal oxygen consumption(Table 1). Drugs of the benzanthraquinone class (anthracy-cline antibiotics), the W-heterocyclic quinone class (strep-tonigrin and mitomycin C), and the naphthoquinone class(lapachol) all stimulate the system but to different degrees.The augmentation reaction is saturable by the anthracyclineantibiotics and follows classical enzyme kinetics of a substrate-enzyme relationship (3). These saturation kinetics

ii5

100

90

80

70

80

SO

40

30

20

NADPH (6x10 3 M) NADPH (6x10 3 M) NADPH (6x10 3 M) NADPH (6x10 3 M)

| ADRIAMYCIN | MITOMYCIN C | | METHOTREXATE

Chart 1. Stimulation of microsomal oxygen consumption by quinonecytotoxic agents. Conditions for the reactions are in "Materials and Methods." Washed rat liver microsomes (2 to 4 mg protein) were used in these

assays.

100

90

80

.Â£ 70

Ã• 60c

UJI50O 40

30

20

10

'The abbreviation used is: EPR, electron paramagnetic resonance.

1 0.1 0.2 0.3 0.4 0.5 1.0

mg PROTEIN/mlMICROSOMAL PROTEIN

Chart 2. Dependence of augmented oxygen uptake on microsomal protein. The assay conditions are described in "Materials and Methods."Adriamycin (5 x 10~4M) was used as the augmenter.

1746 CANCER RESEARCH VOL. 38



Microsomal Activation of Quinone Anticancer Agents

Table 1

Anticancer agents as augmentors of microsomal electrontransport

Assay conditions are described in "Materials and Methods."

Apparent kinetic constants foroxygen consumption

Apparent Km(M x 10-4)

Apparent Vmax(10 7mol/

min)

Active

Benzanthraquinones

AdriamycinDaunorubicinCarminomycinRubidazoneNogalamycinSteffimycinAclacinomycin A

/V-Heterocyclic quiÃ±onesMitomycin CStreptonigrin

NaphthoquinoneLapachol

Inactive

ProcarbazineDibromomannitolc/s-Diaminodichloroplat-

inum1-/3-D-Arabinofuranosyl-

cytosineCyclophosphamideMethotrexateCamptothecinMithramycin

2.61.91.16.53.90.492.2

8.00.78

13.4

2.44.07.70.854.14.50.75

2.66.7

0.3

hold true for the other quinone anticancer agents studiedhere. The data fit classical enzyme kinetic equations, andthe resultant data determine the kinetic constants for thequinone substrates. The apparent Km's for the quiÃ±onesas

substrates for augmented oxygen consumption by rat livermicrosomes fall within a relatively specific range of 0.5 x10 4 M for steffimycin to 13 x 10 4 M for lapachol (Table 1).Apparent Vmaxvalues show a range from 0.3 x 10 7 mol/min for lapachol to 7.7 x 10 7 mol/min for carminomycin.

Several class-representative cytotoxic agents that do notcontain quinone groups (e.g., cyclophosphamide, metho-trexate, 1-/3-D-arabinofuranosylcytosine, etc.) do not affectmicrosomal oxygen consumption (Table 1).

Another parameter of the microsomal reaction affectedby the quinone agents is microsomal NADPH oxidation(Table 2). Triton-treated microsomes show an inhibitedendogenous NADPH oxidation (3) but demonstrate enhanced NADPH oxidation in the presence of each class ofquinone agents. The cytotoxic agents that are inactive asaugmentors of microsomal oxygen consumption are alsoinactive in the microsomal NADPH oxidation assay (e.g.,cyclophosphamide, methotrexate) (Table 2). The nitrosou-reas, 1,3-bis-(2-chloroethyl)-1-nitrosourea and chlorozoto-cin, which cannot be assayed for oxygen consumptionbecause of technical difficulties, do not support micro

somal NADPH oxidation (Table 2).Microsomes from rat liver, lung, and heart were com

pared for activity with Adriamycin as the substrate (Table3). All supported the enhanced oxygen consumption, although the heart sarcosomes showed a higher apparent Kmthan did other tissues. Mouse liver, lung, spleen, and heartmicrosomes also catalyzed the augmented oxygen consumption (Table 3). Whereas microsomes from malignantcells, murine leukemia L1210, and P388 cells catalyzed theaugmented oxygen consumption, their apparent Km's (4.4and 0.6 x 10^" M, respectively) were lower than was theapparent Kmof normal splenic microsomes (28 x 10 4 M)

(Table3).Since we previously reported that anthracycline agents

participate in the microsomal catalysis of free radical formation (3), we compared several agents for free radicalformation by electron paramagnetic resonance spectrome-

Table 2Ouinone-affected microsomal NADPH oxidation

Triton N-101-treated microsomes were used to reduce endogenous NADPH oxidation. The reaction mixtures (1.0 ml) contained 1x 10 4 M drug, 2 x 10~" M NADPH, Triton N-101-treated microsomes (0.05 mg protein), and 2 x 10"' M potassium phosphate

buffer, pH 8.0. The reaction was started by addition of microsomes.Reaction mixtures in a carbon monoxide atmosphere were bubbledwith 100% carbon monoxide for 1 min in the dark, capped, andassayed. Control reaction mixtures did not contain drug.

NADPHoxidation(nmol/mgprotein/min)ControlAdriamycinMitomycin

CStreptonigrinLapacholCyclophosphamideChlorozotocinMethotrexate1,3-Bis(2-chloroethyl)-1-nitrosoureaAir07384249150000Carbon

monoxide0848120911

Table 3Kinetic constants for quinone-augmented oxygen consumption by

various microsomesThe assays were carried out as described in "Materials and

Methods." Adriamycin was used as the quinone augmentor.

MicrosomesRatLiverLungHeartMouseLiverLungHeartSpleenL1210P388Apparent

kineticApparent

Km(MxIO"4)3.63.1186.08.02.43.128.04.40.6constants

foroxygenuseApparent

Vma,(molx10~7/min)0.920.390.892.60.440.060.140.200.04

JUNE 1978 1747



N. R. Bachur et al.

try. Pronounced free radical signals were observed in reaction mixtures containing the quinone drugs (Chart 3). Thefree radical signals depended on the presence of NADPH,microsomes, and the quinone drugs since the absence ofany 1 of these 3 constituents resulted in no free radicalsignal (control). Although mitomycin C is an excellentsubstrate for stimulation of microsomal oxygen consumption (Table 1) and of NADPH oxidation (Table 2) and although several different experimental conditions were tried,mitomycin C gave a suggestive but not a definitive EPRsignal (Chart 2).

Several effectors of microsomal metabolism and freeradical formation were tested on the augmented oxygenconsumption produced by Adriamycin, mitomycin C, andstreptonigrin. The free radical scavenger, 1,4-diazabicyclo-2.2.2-octane, had little effect on Adriamycin- and streptoni-grin-augmented oxygen consumption, but it inhibited themitomycin C reaction slightly (Table 4). a-Tocopherol, anaturally occurring free radical scavenger which has beenreported to protect mice from Adriamycin toxicity (21),inhibited the Adriamycin reaction approximately 60%, themitomycin C reaction 20%, and the streptonigrin reaction10%. SKF 525A did not affect Adriamycin action but didinhibit both mitomycin C and streptonigrin action moderately. Because of technical problems, carbon monoxidewas not used in the oxygen consumption assay but wastested for its effect on NADPH oxidation (Table 2). Althoughstreptonigrin activity was reduced slightly (15%), this inhibitor of cytochrome P-450 activity had no pronounced inhibitory effect on the activity of Adriamycin or mitomycin C.The effect of carbon monoxide on lapachol activity wasquestionable.

DISCUSSION

In earlier studies, mitomycin C and streptonigrin wereconsidered to function through the metabolic activation oftheir quinone groups. One of the earliest observations bySchwartz ef al. (27) showed that microsomal reduction ofthe quinone agent mitomycin C to a hydroquinone wasnecessary for its cytotoxic activity. Later, Iyer and Szybalski

Mitomycm

^^/lJHA/vv^*\A^^

Chart 3. EPR spectra obtained from microsomal reaction mixture containing the quinone antibiotics. Conditions are described in "Materials andMethods," and the scans are over a 50-gauss range. Control spectra are

obtained from reaction mixtures devoid of either NADPH or drug.

Table 4Effectors of quinone-augmented microsomal oxygen consumption

The assays were carried out as described in "Materials andMethods." The Adriamycin, mitomycin C, and streptonigrin concentrations were 5x10"" M.

Oxygen consumption(nmol/min)

AdriamycinMitomycin

CStreptoni

grin

Control 52.7 54.4 98.9

+ effector (1 xlfr3M)1,4-Diazabicyclo-2.2.2-octanea-TocopherolSKF

525A52.323.147.446.643.833.192.989.776.1

(16) verified the need for reduction of mitomycin C to effectDNA cross-linking. Since those studies, Tomasz (29) reported that chemical or biological reduction of mitomycin Cresulted in the generation of peroxide. Tomasz concludedthat the end product peroxide inflicted the damage to DNA.

When Hochstein ef al. (13) found that streptonigrin stimulated mitochondrial NADPH oxidation, they suggested arole for this agent in oxidative-reductive reactions. Whiteand White (30) and Gregory and Fridovitch (11) reportedthat streptonigrin caused Superoxide production in bacterial systems and required oxygen for its activity as anantibacterial agent. Recently, Cone ef al. (5) have implicated Superoxide ion and hydroxyl radical as the activecomponents of streptonigrin action.

Of the quinone agents in question, streptonigrin and theanthracycline antibiotics have a clearly identified ability toinhibit nucleic acid replication or transcription by interference with DNA template for DNA or RNA polymerase (6,10,20, 25). The anthracycline antibiotics have a high affinityand tight binding characteristics for DNA by virtue of theirplanar ring systems and amino sugar moieties (6). Mitomycin C and streptonigrin also bind to DNA (19, 20). However,the binding to DNA and the inhibition of nucleic acidmetabolism cannot account for the lethal effects of thesesubstances on nondividing tumor cells. The DNA bindingalone does not explain how these drug molecules inducethe well-documented damage to DNA such as the chromosome aberrations, breakage and fragmentation with resultant increased frequency of sister chromatid exchange, andinduction of miotic recombination, etc., associated with theaction of the drugs in vivo on cells and tissues (8, 28).These agents do not break DNA strands when only drugand DNA are incubated together (5, 16, 24, 25). DNA strandbreakage requires cellular activity as shown by the fragmentation of nuclear DNA caused by Adriamycin and daunoru-bicin and detected in alkaline sucrose gradient analysis (26)or in the increased rate of DNA double-strand unwinding(24).

Handa and Sato (12) reported that mitomycin C and otherquinone agents enhanced microsomal sulfite oxidation andpostulated that this occurred through Superoxide formation. They postulated that the Superoxide product formedfrom mitomycin C and that streptonigrin was the activeagent for producing peroxide that caused intracellular cytotoxic damage. Our experimental procedure and data




Microsomal Activation of Quinone Anticancer Agents

showed that sulfite was not required in the reaction andthat a direct transfer occurred of a single electron fromNADPH to the semiquinone carrier and subsequently tooxygen (3). Microsomes catalyzed the transfer of singleelectrons from NADPH through flavoproteins to quinonereceptors to form free radicals (15). Our studies showedthat the cytotoxic quinone drugs also functioned in themicrosomal system to accelerate NADPH oxidation andoxygen consumption and to form free radicals.

Since other quiÃ±onessuch as menadione and coenzymeQ are reduced by microsomes to free radical forms (4) andsupport the formation of Superoxide radicals just as thequinone cytotoxic drugs do (15), they may be toxic throughSuperoxide production (2). However, cells have extensiveand fully developed protective mechanisms against super-oxide, which is a normal component of the cellular enzymecomplement (9). Therefore, it is difficult to implicate thegeneralized production of Superoxide as the active agent ofcytotoxic quinone drugs. The major difference between themolecular forms of cytotoxic quinone drugs and otherquiÃ±ones rests in the specificity of the cytotoxic drugmolecules for DNA as the end target.

We propose that the cytotoxic quinone anticancer agentsare converted intracellularly to site-specific free radicals(Chart 4). These semiquinone free radical drug intermediates may be sufficiently stable to enter the nucleus and bindthrough intercalation or other means to nuclear DNA as aconsequence of the structural specificity and affinity characteristics of each agent. By having a high affinity for and aresultant close proximity to DNA, the drug-free radicals maydirect their reactive energy to the complexed DNA or maygenerate reactive oxygen radicals such as Superoxide orhydroxyl radical to react with proximal DNA and therebyproduce the specific destructive effects to DNA that havebeen reported. Through this mechanism, cross-linkingreactivity (mitomycin C) and strand breakage (Adriamycin,daunorubicin, streptonigrin) are plausible.

In this study we have demonstrated that these quinonecytotoxic drugs form free radicals that give characteristicEPR signals upon microsomal activation. Mitomycin C,however, did not yield an overly convincing EPR signal.This may be related to a very short half-life and unstablenature of that free radical. The signal position and peak-to-peak width of each derived free radical is characteristic foreach drug and proves that the EPR signal is not an artifactof microsomal derivation.

All of the agents that are active in the microsomal electrontransport and oxygen consumption possess the quinonestructure planar to an adjacent resonating ring system thatmay be heterocyclic. Although cytotoxic agents may possess planar ring systems with aromatic keto groups (mith-ramycin, camptothecin) or other planar W-heterocyclicresonating rings (1-ÃŸ-D-arabinofuranosylcytosine, camptothecin), apparently these are inadequate for free radicalactivation.

Our data show that microsomes from both normal andmalignant tissues catalyze the quinone cytotoxic agentaugmentation of oxygen consumption. However, the comparison of normal mouse spleen cells with malignant he-matological tumors (L1210 and P388) shows a higher activity for the tumor cells, which may account for the therapeu-

Fipiox;

Flplredl

CLASSES

BENZANTHRAQUINONE

N-HETEROCYCLIC QUINONE

oNAPHTHOQUINONE

Chart 4. Proposed mechanism of microsomal activation of quinone anti-cancer agents to free radicals. The hypothetical reaction of anticanceragents derived site-specific free radicals, and nucleic acid targets areindicated. In the lower half of the chart, the classes of quinone anticancerdrugs are represented as nuclear structures.

tic advantage of these agents against the murine leukemias.Through our use of specific substrates (hexobarbital,

aspirin) and inhibitors (SKF 525A, carbon monoxide) (3), wefeel that this electron carrier process does not include thecytochrome P-450 system in the microsomes but presumably occurs before that and involves microsomal flavoproteins, probably NADPH cytochrome P-450 reducÃase.

The inhibitory action of a-tocopherol on free radicalgeneration caused by Adriamycin may be responsible forlowering Adriamycin toxicity in mice (21). It will be important to view these cytotoxic agents from a new perspectivewith the potential of a new mechanism of action.

ACKNOWLEDGMENTS

The authors appreciate the help of Dr. Hideo Kon, Laboratory of ChemicalPhysics, National Institute of Arthritis, Metabolism, and Digestive Diseases,in obtaining electron paramagnetic resonance spectra.

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1978;38:1745-1750. Cancer Res Nicholas R. Bachur, Sandra L. Gordon and Malcolm V. Gee Anticancer Agents to Free RadicalsA General Mechanism for Microsomal Activation of Quinone

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