Inr J Radumon Oncology Bml Phv~ Vol Pnnted ,n the U.S.A. All n&s reserved.
22. pp. 64X-647 Copyright
0360.3016/92 $5.00 + .OO 0 1992 Pergamon Press plc
??Session D: Bioreductive Mechanisms
THE ROLE OF HUMAN AND RODENT DT-DIAPHORASE IN THE REDUCTIVE METABOLISM OF HYPOXIC CELL CYTOTOXINS M.
I. WALTON;
MRC Clinical Oncology
PH.D.,
N.
SUGGET
and Radiotherapeutics
AND P. WORKMAN,
Unit, Hills Rd, Cambridge,
PH.D. CB2 2QH, UK
DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins E09, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, E09, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in singlestrand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than E09 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4s reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic le radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate ‘enzyme-directed’ analogue development. DT-diaphorase,
Enzymology,
Mitomycin C, E09, SR 4233, CB 1954.
INTRODUCTION
METHODS
A variety of enzymes can reductively bioactivate hypoxic cell cytotoxins to reactive species via one-electron (le) donation under hypoxia ( 1, 12, 19). In contrast to such reductases, DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase, EC 1.6.99.2) an obligate two-electron (2~) donating flavoenzyme (4, 6), may catalyse either bioactivation or bioprotection reactions with these drugs. DTdiaphorase also shows elevated expression in a variety of rodent and human tumor cell lines (4, 9, 16), and consequently represents a candidate enzyme for ‘enzyme-directed’ bioreductive drug development (22). However, the precise role of this enzyme in the activation or detoxification of hypoxic cell cytotoxins is unclear. Using a rodent and human tumor source of DT-diaphorase, we have therefore studied the reduction of examples of three classes of bioreductive prodrugs: 1) complex quinones such as mitomycin C (MMC) and the developmental indoloquinone E09, 2) the mixed-function dinitrophenylaziridine CB 1954 and 3) the benzotriazine di-N-oxide SR 4233 (see Fig. 1).
AND
MATERIALS
DT-diaphorase preparations were obtained from the UK strain of the rat Walker 256 mammary carcinoma and from human HT29 colon carcinoma cells, both of which are known to be a rich source of DT-diaphorase (9, 16, 17). Cells were grown as previously described under a humidified atmosphere of 8% COz (9, 16, 17). Walker cells were grown in suspension while HT29 cells formed an adherent monolayer requiring trypsinization. At confluence, cells were harvested, pooled, concentrated by centrifugation, and then sonicated on ice. Sonicates were centrifuged to remove cell debris and the supernatant recovered and used as a crude source of DT-diaphorase. Purified DT-diaphorase from rat Walker cells was a gift from Dr. Richard Knox (Institute Cancer Research, Sutton, UK). Purified human kidney DT-diaphorase was a gift from Garth Powis and John J. Schlager (Mayo Clinic, Rochester, MI). Enzyme preparations were stored at -70°C. Quinone metabolism by DT-diaphorase was assayed spectrophotometrically under optimal conditions by
Reprint requests to: Professor P. Workman, CRC Department of Medical Oncology, University of Glasgow, CRC Beatson Laboratories, Switchback Road, Bearsden, Glasgow, G6 1 1BD,
Accepted
UK. 643
for publication
26 July 199 1.
644
I. J. Radiation
Oncology
0 Biology 0 Physics
Volume
22, Number
4. I992
0
NO2
0 SR 4233
Menadione
CB 1954
0
c
0
N
H2N
H3C
0
0 Mitomycin C Fig. 1. Structures
of the bioreductive
CH3
E09 prodrugs
and of the benchmark
monitoring cytochrome c reduction at 550 nm (3). Standard aerobic reactions contained NADH (2 mM), BSA (0.14%) KCN (1 mM), menadione (25-50 PM) or bioreductive drug (OS-3 mM), and dicoumarol inhibitor (100 PM), as appropriate in a final volume of 1 ml of 50 mM Tris HCl buffer (pH 7.5). CB 1954 and SR 4233 metabolism was assayed by reverse-phase HPLC as previously described with modifications ( l&20). All incubations were carried out at 37°C. Reaction rates were derived from linear progress curves using 4 (HPLC assay) or 5-30 (spectral assay) time points. Enzyme activities were normalized to that for the simple bench-mark quinone menadione. Michaelis-Menten kinetics were established using standard criteria and constants were determined using at least six substrate concentrations (5). Drug induced DNA damage was assessed by monitoring the conversion of supercoiled (form I) plasmid pBR322 DNA to the relaxed (form II) and linearized (form III) configurations ( 14). Incubations contained purified rat Walker DT-diaphorase (12U), drug (2-2000 FM), DNA (0.8 pg), NADH (2 mM), and the inhibitors dicoumarol (l- 100 PM) and superoxide dismutase (SOD,25-200U) in a final volume of 100 ~10.1 M sodium phosphate buffer (pH 7.4). The incubation period was 60 min. RESULTS Table 1 summarizes the enzyme kinetic parameters for menadione and the four bioreductive hypoxic cell cyto-
DT-diaphorase
substrate
menadione.
toxins studied (Fig. 1). The simple quinone menadione was rapidly reduced by both human and rat tumor DTdiaphorase with substrate inhibition above 25-50 PM (not shown). Although rat tumor preparations were more active on a volumetric basis, both the rat and human tumor enzymes exhibited identical kinetic behaviour (Table 1). The novel indoloquinone E09 was readily reduced by rat DT-diaphorase (Table 1 and Fig. 2A). The Vmax was only 5-to 6-fold lower than for mendione and the Km about lo-fold higher. By comparison, the human tumor enzyme was 20% as efficient as the rat at 100 PM E09 (Table 1). Reduction of E09 by purified rat DT-diaphorase was shown to produce single-strand breaks in plasmid DNA (Fig. 3). The reaction was dependent on drug and enzyme concentration, as well as incubation time, with complete nicking occurring at 30-50 PM in the presence of 12 U after 30 min incubation. Dicoumarol completely inhibited the reaction at a concentration of 10 PM (Fig. 3) but SOD up to 2000 U ml-’ had no effect (not shown). There was no evidence of DNA linearization. The quinone anticancer drug MMC was shown to undergo reductive metabolism by the rat Walker tumor enzyme. but the reaction was very inefficient compared to E09 with a high Km of 1 mM and low Vmax (Table 1). There was no measurable reduction by human tumor or kidney DT-diaphorase, and in addition neither preparation caused DNA damage with MMC as substrate up to 2 mM (not shown).
DT-diaphorase
and hypox cell cytotoxins
Table 1. Michaelis-Menten kinetics for the reduction of menadione and several hyoxic cell cytotoxins by rat and human tumor DT-diaphorase Apparent Km (JLM)
Enzyme source
Drug
Rat Walker Tumor cells
Menadione ED9
Human HT29 Tumor cells
MMC CB 1954 SR 4233 Menadione E09 MMC CB 1954 SR 4233
vides a direct measure of cytotoxic metabolite formation. Formation and subsequent acetylation of the 4-hydroxylamine represents a potent bioactivation pathway (7). Similar studies with the benzotriazine di-N-oxide SR 4233 and rat Walker tumor DT-diaphorase showed preferential 4e reduction to the parent benzotriazine SR 4330 with minimal 2e product formation (Table 1 and Fig. 5). In contrast to CB 1954, this represents a potential bioprotection pathway since the cytotoxic radical formed by 1e reduction is effectively bypassed.
Apparent Vmax (nmol min-’ U-r)
2.0, 2.1 16.8, 22.1 1080, 983 1800, 1360 ND ND 2.4, 2.8 ND ND BLD BLD 1160, 820 ND ND
1000, 1000 187, 130 7.4, 6.6 0.085, 0.091 (0.077, 0.12) 1000, 1000 (24.2, 29.3) BLD BLD 0.034, 0.020 ND ND
DISCUSSION Simple quinones, such as menadione, can undergo either 1e reduction to the semi-quinone or 2e reduction to the hydroquinone (4). The former reaction is catalyzed by a variety of reductases such as NADPH:cytochrome P-450 reductase and under aerobic conditions results in toxic free radical formation (4, 11). On the other hand, 2e reduction by DT-diaphorase yields the relatively stable hydroquinone in a detoxification reaction that bypasses the le semi-quinone radical (3, 9). The present results confirm that menadione is an ideal substrate for 2e reduction by DT-diaphorase from both rat and human tumor sources. By contrast, for more complex quinones possessing alkylating moieties or reactive side groups, toxicity may arise from a variety of reactions. Aerobic reduction may generate both free radicals and DNA alkylating species (1, 12, 13) whereas under hypoxia the latter reaction should predominate (7, 12). We show here that the aziridine containing indoloquinone E09 is readily reduced by both rodent and human DT-diaphorase, though with different efficiencies. Moreover this was shown to represent a bioactivation pathway for the rat tumor enzyme to a species causing single-strand breaks in plasmid DNA.
Note: Values shown are from two independent experiments. Rates were normalized to that with menadione (= 1000). Values in brackets represent rates determined at a fixed drug concentration (2 mM SR 4233 and 100 p,M E09). BLD = Below limit of detection: LLD = Lower limit of reduction; for spectral assay this was 0.5 nmol cyt c red mini’ U-‘; ND Not determined. For SR 4233 and CB 1954, kinetics determined by HPLC analysis of metabolite formation.
Using an HPLC method capable of resolving all the known stable metabolites of CB 1954, it was shown to be reduced predominantly to the 4-hydroxylamine by DTdiaphorase from both rat (Walker tumor) and human (HT29 tumor and kidney) sources (Fig. 4). Rates of metabolite formation were relatively low for both preparations, and the Km remarkably high at l-2 mM (Table 1). The kinetics were highly comparable for the 2 enzymes. It should be noted that in contrast to the indirect spectrophotometric quinone assay, this HPLC technique pro-
O.lO-
A
6
80 /O
5-
P
0.08 -
645
0 M. I. WALTON et al.
4-
0
/
K0
P
0 0.06 -
,o
0
3-
0’
0
0’ 0’ 0.04 -
,o’
2-
/O
/ 0
.o
0,02 ON0 0.W
/ 0
10
20
Time (min)
30
0‘,/ 0
1
2 S
I 3
(mM)
Fig. 2. (A) A typical progress curve for the reduction of E09 (150 PM) by rat Walker tumor DT-diaphorase (10 ~1 of 1:40 dil of cell sonicate). Symbols: control 0 and 0 with 100 KM dicoumarol. (B) An s versus v plot for the reduction of mitomycin C by rat Walker tumor DT-diaphorase. Activity was measured by the direct transfer of electrons to cytochrome c (detected at 550 nm) following the reduction of quinone substrate by DT-diaphorase (see Methods and Materials).
I. J.
646
Radiation
Oncology
0 Biology
0 Physics
Volume
22. Number
4. 1992
Relaxed (Form II)
Supercoiled (Form I) Time I I 1234
I
I
I
I
56
I I I 7891011
I
I
Fig. 3. Effect of E09 and dicoumarol concentration on rat Walker tumor DT-diaphorase-catalyzed damage in pBR 322 plasmid DNA. Lanes l-6 contained increasing E09 concentrations (0, 2, 10, 50, 100 and 200 PM) and lanes 7-I I increasing dicoumarol concentrations (0, I, 5, IO and 100 PM) at a fixed E09 concentration of 100 PM. Reaction conditions as in Methods and Materials.
The precise nature of this species is unknown, but the lack of effect of SOD suggests it may be an alkylating intermediate. The antitumor quinone MMC, by comparison, was a much poorer substrate with rat tumor DT-diaphorase, exhibiting a remarkably high Km and a Vmax only 5%
;,
;
6
6
8
Time
lb
12 lb
16
li
2b
2;
(min)
Fig. 4. HPLC chromatogram of an aliquot of a 100 min aerobic reduction of CB 1954 by purified human kidney DT-diaphorase, without (-) and with 100 PM dicoumarol (----). Traces show the 4-hydroxylamine metabolite of CB 1954 (peak 1,O. 125 pmol for control and 0.042 pmol with dicoumarol), misonidazole internal standard (peak 2, 2.5 mot) and CB 1954 (peak 3, 51.2 pmol). Reactions contained 3 U enzyme and 10 mM CB 1954 substrate. For other conditions see Methods and Materials.
(min)
Fig. 5. Progress curves for the aerobic reduction of SR 4233 to its 2 and 4e reduction products, SR 43 17 and SR 4330. respectively, by rat Walker DT-diaphorase. Symbols: SR 4317 formation in the absence n and presence A of 100 FM dicoumarol. SR 4330 formation in the absence 0 and presence ??of dicoumarol. Reactions contained 14 U enzyme and 2 mM SR 4233. For other conditions see Materials and Methods.
of that for E09. The failure of the enzyme to elicit DNA damage suggests that this reaction may be a bioprotective pathway, but possibly the reaction rate is too low to generate sufficient active metabolites. Several investigators have proposed that MMC can undergo 2e reduction to alkylating species. though most work has relied on indirect evidence derived from the use of the potent DT-diaphorase inhibitor menadione (7, 2 1). However, studies using purified human kidney DT-diaphorase failed to show that MMC was a substrate for this enzyme at neutral pH (15). However, MMC is activated more efficiently to DNAdamaging species at acidic pH (17). Metabolism of CB 1954 by Walker tumor cell DT-diaphorase has previously been shown to be a potent bioactivation pathway (8, 9). Our results show that this reduction can be catalyzed by the human HT29 colon tumor enzyme and that the predominant product is the 4-hydroxylamine. As for E09, the rate of reduction was lower for the human compared to the rat tumor enzyme. The benzotriazine di-N-oxide SR 4233 was shown to be preferentially reduced to the 4e product by the rat tumor DT-diaphorase. In contrast to the reaction with E09 and CB 1954, this probably represents a detoxification pathway (22). In conclusion, the present results show that DT-diaphorase can function as either a bioactivating or bioprotective reductase, depending on the bioreductive drug involved. Tumors rich in DT-diaphorase should represent appropriate targets for E09 and CB 1954, as a result of selective activation by the enzyme, whereas they may be comparatively resistant to SR 4233. This information should not only facilitate clinical evaluation of bioreductive agents, but should also aid our “enzyme directed” approach to drug development (22).
0 M. 1. WALTON et al.
DT-diaphorase and hypox cell cytotoxins
647
REFERENCES 1. Bachur, N. R.; Gordon, S. L.; Gee, M. V.; Kon, H. NADPH: cytochrome P-450 reductase activation of quinone anticancer agents to free radicals. Proc. Natl. Sci. 76:954- 957; 1979. 2. Baker, M. A.; Zeman E. M.; Hirst, V. K.; Brown, J. M. Metabolism of SR 4233 by Chinese hamster ovary cells: The basis for selective hypoxic cell cytotoxicity. Cancer Res. 48: 5947-5952; 1988. 1. 3. Ernster, L.; Danielson, L.; Ljunggren, M. DT-diaphorase Purification from the soluble fraction of rat liver cytoplasm and properties. Biochem. Biophys. Acta. 58: 17 1- 188; 1962. 4. Ernster, L.; Estabrook, R. W.; Hochstein, P.; Orrenius, S. DT-diaphorase. Chemica Scripta 27A, 1987. P. J. F. Statistical analysis of enzyme kinetic 5. Henderson, data. In: Kornberg, H. L., Metcalfe, J. C., Nortcote, D. H., Pogson, C. I., Tipton, T. F., eds. Techniques in protein and enzyme biochemistry. Amsterdam: Elsevier Biomedical Press; 1978: Bl-ii, l-43. 6. lyanagi. T.; Yamazaki, I. One electron transfer reactions in bichemical systems. V Differences in the mechanism ofquinone reduction by the NADH dehydrogenase and the NAD(P)H dehydrogenase (DT-diaphorase). Biochem. Biophys. Acta. 2 16:282-290; 1970. I. Keyes, S. R.; Fracasso, P. M.; Heimbrook, D. C.; Rockwell, S.; Sliger, S. G.; Sartorelli, A. C. Role of NADPH: cytochrome c reductase and DT-diaphorase in the bio-transformation of mitomycin C. Cancer Res. 44:5633-5643; 1984. 8. Knox, R. J.; Friedlos, F.; Jarman, M.; Roberts, J. J. A new cytotoxic DNA interstrand crosslinking agent, 5-(aziridin1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4_dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. Biochem. Pharmacol. 37:466 l-4669; 1988. 9. Knox, J.; Boland, M. P.; Friedlos, F.; Coles, B.; Southan. C.; Roberts, J. J. The nitroreductase enzyme in Walker cells that activates 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide is a form of NAD(P)H dehydrogenase (quinone) (EC 1.6.99.2). Biochem. Pharmacol. 37:4671-4677; 1988. as a qui10. Lind, C.; Hochstein, P.; Ernster, L. DT-diaphorase none reductase: a cellular device against semiquinone and superoxide formation. Arch. Biochem. Biophys. 2 16: 178185; 1982. 11. Morrison, H.; Jernstrom, B.; Nordenskjold, M.; Thor, H.;
12. 13.
14.
15.
16.
17.
18.
Orrenius, S. Induction of DNA damage by menadione (2methyl- 1,4_naphthaquinone) in primary cultures of rat hepatocytes. Biochem. Pharmacol. 33: 1763-1769; 1984. Powis, G. Metabolism and reactions of quinoid anticancer agents. Pharmac. Ther. 35:57-162; 1987. Pritsos, C. A.; Sartorelli, A. C. Generation of reactive oxygen radicals through bioactivation of mitomycin C antibiotics. Cancer Res. 46:3528-3532; 1986. Seawell, P. C.; Ganesan, A. K. Measurement of strand breaks in supercoiled DNA by gel electrophoresis. In: Friedberg, E. C., Hanawalt, P. C., eds. DNA repair, Vol. 1 Part B. New York: Marcel Dekker; 1981: 425-445. Schlager, J. J.; Powis, G. Mitomycin C is not metabolised by but is an inhibitor of human kidney NAD(P)H:(quinoneacceptor) oxidoreductase. Cancer Chemother. Pharmacol. 22:126-130; 1988. Siegel, D.; Gibson, N. W.; Preusch, P. C.; Ross, D. Metabolism of diaziquone (AZQ) by NADP(H):(quinone-acceptor) oxidoreductase (DT-diaphorase):role in AZQ-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 50:7293-7300: 1990. Siegel, D.; Gibson, N. W.; Preusch, P. C. and Ross, D. Metabolism of mitomycin C by DT-diaphorase: role in mitomycin C-induced DNA damage and cytotoxicity in human colon carcinoma cells. Cancer Res. 50:7483-7489; 1990. Walton, M. I.; Workman, P. HPLC assay for the benzotriazine di-N-oxide (SR4233) and its reduced metabolites in biological material. J. Chromatogr. 430:429-437; 1988.
19. Walton, M. I.; Wolf, C. R.; Workman, P. Molecular enzymology of the reductive bioactivation of hypoxic cell cytotoxins. Int. J. Radiat. Oncol. Biol. Phys. 16:983-986; 1989. 20. Workman, P.; White, R. A. S.; Talbot, K. CB 1954 revisited 1. Disposition kinetics and metabolism. Cancer Chemother. Pharmacol. 16: l-8; 1986. 21. Workman, P.; Walton, M. 1.; Powis, G.; Schlager, J. J. DTdiaphorase: questionable role in mitomycin C resistance, but a target for novel bioreductive drugs? Br. J. Cancer 60: 800- 802; 1989. bioreductive 22. Workman, P.; Walton, M. I. Enzyme-directed drug development. In: Adams, G. E., Breccia, A., Fielden, E. M., Wardman, P., eds. Selective activation of drugs by redox processes. New York, NY: Plenum; 1990: 173-191.
22. pp. 64X-647 Copyright
0360.3016/92 $5.00 + .OO 0 1992 Pergamon Press plc
??Session D: Bioreductive Mechanisms
THE ROLE OF HUMAN AND RODENT DT-DIAPHORASE IN THE REDUCTIVE METABOLISM OF HYPOXIC CELL CYTOTOXINS M.
I. WALTON;
MRC Clinical Oncology
PH.D.,
N.
SUGGET
and Radiotherapeutics
AND P. WORKMAN,
Unit, Hills Rd, Cambridge,
PH.D. CB2 2QH, UK
DT-diaphorase is a unique two electron (2e) donating reductase catalyzing either bioactivation or bioprotection reactions. Using human and rodent DT-diaphorase preparations (cell extracts and purified enzyme) we have characterized the reductive metabolism of the hypoxic cell cytotoxins E09, mitomycin C (MMC), CB 1954, and SR 4233 in vitro. Drug metabolism was assayed spectrophotometrically or by HPLC, with dicoumarol as a selective inhibitor. DNA damage was measured using an agarose gel mobility technique with plasmid pBR322 DNA. The developmental indoloquinone, E09, was metabolized by both rat Walker and human HT29 tumor DT-diaphorases. Reduction proceeded 5-fold more efficiently with the rat than the human tumor enzyme and resulted in singlestrand breaks in plasmid DNA. The structurally related MMC was metabolized much more slowly than E09 by the rat Walker tumor enzyme and there was no detectable reaction with the human HT29 tumor DT-diaphorase. No DNA damage was seen with MMC for either enzyme. The dinitrophenylaziridine CB 1954 was reduced by both human and rat enzymes forming, preferentially, the highly toxic 4-hydroxylamine as a 4e reduction product. Rates were 3-fold lower than for the human tumor enzyme. SR 4233 was also reduced by the rat tumor enzyme predominantly via 4s reduction to the benzotriazine SR 4330, in a novel reaction mechanism. This appears to be a bioprotection pathway that bypasses the toxic le radical formed by other reductases. Such information may be valuable in the selection of hypoxic cell cytoxins to treat human tumors high or low in DT-diaphorase and should facilitate ‘enzyme-directed’ analogue development. DT-diaphorase,
Enzymology,
Mitomycin C, E09, SR 4233, CB 1954.
INTRODUCTION
METHODS
A variety of enzymes can reductively bioactivate hypoxic cell cytotoxins to reactive species via one-electron (le) donation under hypoxia ( 1, 12, 19). In contrast to such reductases, DT-diaphorase (NAD(P)H: quinone acceptor oxidoreductase, EC 1.6.99.2) an obligate two-electron (2~) donating flavoenzyme (4, 6), may catalyse either bioactivation or bioprotection reactions with these drugs. DTdiaphorase also shows elevated expression in a variety of rodent and human tumor cell lines (4, 9, 16), and consequently represents a candidate enzyme for ‘enzyme-directed’ bioreductive drug development (22). However, the precise role of this enzyme in the activation or detoxification of hypoxic cell cytotoxins is unclear. Using a rodent and human tumor source of DT-diaphorase, we have therefore studied the reduction of examples of three classes of bioreductive prodrugs: 1) complex quinones such as mitomycin C (MMC) and the developmental indoloquinone E09, 2) the mixed-function dinitrophenylaziridine CB 1954 and 3) the benzotriazine di-N-oxide SR 4233 (see Fig. 1).
AND
MATERIALS
DT-diaphorase preparations were obtained from the UK strain of the rat Walker 256 mammary carcinoma and from human HT29 colon carcinoma cells, both of which are known to be a rich source of DT-diaphorase (9, 16, 17). Cells were grown as previously described under a humidified atmosphere of 8% COz (9, 16, 17). Walker cells were grown in suspension while HT29 cells formed an adherent monolayer requiring trypsinization. At confluence, cells were harvested, pooled, concentrated by centrifugation, and then sonicated on ice. Sonicates were centrifuged to remove cell debris and the supernatant recovered and used as a crude source of DT-diaphorase. Purified DT-diaphorase from rat Walker cells was a gift from Dr. Richard Knox (Institute Cancer Research, Sutton, UK). Purified human kidney DT-diaphorase was a gift from Garth Powis and John J. Schlager (Mayo Clinic, Rochester, MI). Enzyme preparations were stored at -70°C. Quinone metabolism by DT-diaphorase was assayed spectrophotometrically under optimal conditions by
Reprint requests to: Professor P. Workman, CRC Department of Medical Oncology, University of Glasgow, CRC Beatson Laboratories, Switchback Road, Bearsden, Glasgow, G6 1 1BD,
Accepted
UK. 643
for publication
26 July 199 1.
644
I. J. Radiation
Oncology
0 Biology 0 Physics
Volume
22, Number
4. I992
0
NO2
0 SR 4233
Menadione
CB 1954
0
c
0
N
H2N
H3C
0
0 Mitomycin C Fig. 1. Structures
of the bioreductive
CH3
E09 prodrugs
and of the benchmark
monitoring cytochrome c reduction at 550 nm (3). Standard aerobic reactions contained NADH (2 mM), BSA (0.14%) KCN (1 mM), menadione (25-50 PM) or bioreductive drug (OS-3 mM), and dicoumarol inhibitor (100 PM), as appropriate in a final volume of 1 ml of 50 mM Tris HCl buffer (pH 7.5). CB 1954 and SR 4233 metabolism was assayed by reverse-phase HPLC as previously described with modifications ( l&20). All incubations were carried out at 37°C. Reaction rates were derived from linear progress curves using 4 (HPLC assay) or 5-30 (spectral assay) time points. Enzyme activities were normalized to that for the simple bench-mark quinone menadione. Michaelis-Menten kinetics were established using standard criteria and constants were determined using at least six substrate concentrations (5). Drug induced DNA damage was assessed by monitoring the conversion of supercoiled (form I) plasmid pBR322 DNA to the relaxed (form II) and linearized (form III) configurations ( 14). Incubations contained purified rat Walker DT-diaphorase (12U), drug (2-2000 FM), DNA (0.8 pg), NADH (2 mM), and the inhibitors dicoumarol (l- 100 PM) and superoxide dismutase (SOD,25-200U) in a final volume of 100 ~10.1 M sodium phosphate buffer (pH 7.4). The incubation period was 60 min. RESULTS Table 1 summarizes the enzyme kinetic parameters for menadione and the four bioreductive hypoxic cell cyto-
DT-diaphorase
substrate
menadione.
toxins studied (Fig. 1). The simple quinone menadione was rapidly reduced by both human and rat tumor DTdiaphorase with substrate inhibition above 25-50 PM (not shown). Although rat tumor preparations were more active on a volumetric basis, both the rat and human tumor enzymes exhibited identical kinetic behaviour (Table 1). The novel indoloquinone E09 was readily reduced by rat DT-diaphorase (Table 1 and Fig. 2A). The Vmax was only 5-to 6-fold lower than for mendione and the Km about lo-fold higher. By comparison, the human tumor enzyme was 20% as efficient as the rat at 100 PM E09 (Table 1). Reduction of E09 by purified rat DT-diaphorase was shown to produce single-strand breaks in plasmid DNA (Fig. 3). The reaction was dependent on drug and enzyme concentration, as well as incubation time, with complete nicking occurring at 30-50 PM in the presence of 12 U after 30 min incubation. Dicoumarol completely inhibited the reaction at a concentration of 10 PM (Fig. 3) but SOD up to 2000 U ml-’ had no effect (not shown). There was no evidence of DNA linearization. The quinone anticancer drug MMC was shown to undergo reductive metabolism by the rat Walker tumor enzyme. but the reaction was very inefficient compared to E09 with a high Km of 1 mM and low Vmax (Table 1). There was no measurable reduction by human tumor or kidney DT-diaphorase, and in addition neither preparation caused DNA damage with MMC as substrate up to 2 mM (not shown).
DT-diaphorase
and hypox cell cytotoxins
Table 1. Michaelis-Menten kinetics for the reduction of menadione and several hyoxic cell cytotoxins by rat and human tumor DT-diaphorase Apparent Km (JLM)
Enzyme source
Drug
Rat Walker Tumor cells
Menadione ED9
Human HT29 Tumor cells
MMC CB 1954 SR 4233 Menadione E09 MMC CB 1954 SR 4233
vides a direct measure of cytotoxic metabolite formation. Formation and subsequent acetylation of the 4-hydroxylamine represents a potent bioactivation pathway (7). Similar studies with the benzotriazine di-N-oxide SR 4233 and rat Walker tumor DT-diaphorase showed preferential 4e reduction to the parent benzotriazine SR 4330 with minimal 2e product formation (Table 1 and Fig. 5). In contrast to CB 1954, this represents a potential bioprotection pathway since the cytotoxic radical formed by 1e reduction is effectively bypassed.
Apparent Vmax (nmol min-’ U-r)
2.0, 2.1 16.8, 22.1 1080, 983 1800, 1360 ND ND 2.4, 2.8 ND ND BLD BLD 1160, 820 ND ND
1000, 1000 187, 130 7.4, 6.6 0.085, 0.091 (0.077, 0.12) 1000, 1000 (24.2, 29.3) BLD BLD 0.034, 0.020 ND ND
DISCUSSION Simple quinones, such as menadione, can undergo either 1e reduction to the semi-quinone or 2e reduction to the hydroquinone (4). The former reaction is catalyzed by a variety of reductases such as NADPH:cytochrome P-450 reductase and under aerobic conditions results in toxic free radical formation (4, 11). On the other hand, 2e reduction by DT-diaphorase yields the relatively stable hydroquinone in a detoxification reaction that bypasses the le semi-quinone radical (3, 9). The present results confirm that menadione is an ideal substrate for 2e reduction by DT-diaphorase from both rat and human tumor sources. By contrast, for more complex quinones possessing alkylating moieties or reactive side groups, toxicity may arise from a variety of reactions. Aerobic reduction may generate both free radicals and DNA alkylating species (1, 12, 13) whereas under hypoxia the latter reaction should predominate (7, 12). We show here that the aziridine containing indoloquinone E09 is readily reduced by both rodent and human DT-diaphorase, though with different efficiencies. Moreover this was shown to represent a bioactivation pathway for the rat tumor enzyme to a species causing single-strand breaks in plasmid DNA.
Note: Values shown are from two independent experiments. Rates were normalized to that with menadione (= 1000). Values in brackets represent rates determined at a fixed drug concentration (2 mM SR 4233 and 100 p,M E09). BLD = Below limit of detection: LLD = Lower limit of reduction; for spectral assay this was 0.5 nmol cyt c red mini’ U-‘; ND Not determined. For SR 4233 and CB 1954, kinetics determined by HPLC analysis of metabolite formation.
Using an HPLC method capable of resolving all the known stable metabolites of CB 1954, it was shown to be reduced predominantly to the 4-hydroxylamine by DTdiaphorase from both rat (Walker tumor) and human (HT29 tumor and kidney) sources (Fig. 4). Rates of metabolite formation were relatively low for both preparations, and the Km remarkably high at l-2 mM (Table 1). The kinetics were highly comparable for the 2 enzymes. It should be noted that in contrast to the indirect spectrophotometric quinone assay, this HPLC technique pro-
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Fig. 2. (A) A typical progress curve for the reduction of E09 (150 PM) by rat Walker tumor DT-diaphorase (10 ~1 of 1:40 dil of cell sonicate). Symbols: control 0 and 0 with 100 KM dicoumarol. (B) An s versus v plot for the reduction of mitomycin C by rat Walker tumor DT-diaphorase. Activity was measured by the direct transfer of electrons to cytochrome c (detected at 550 nm) following the reduction of quinone substrate by DT-diaphorase (see Methods and Materials).
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Fig. 3. Effect of E09 and dicoumarol concentration on rat Walker tumor DT-diaphorase-catalyzed damage in pBR 322 plasmid DNA. Lanes l-6 contained increasing E09 concentrations (0, 2, 10, 50, 100 and 200 PM) and lanes 7-I I increasing dicoumarol concentrations (0, I, 5, IO and 100 PM) at a fixed E09 concentration of 100 PM. Reaction conditions as in Methods and Materials.
The precise nature of this species is unknown, but the lack of effect of SOD suggests it may be an alkylating intermediate. The antitumor quinone MMC, by comparison, was a much poorer substrate with rat tumor DT-diaphorase, exhibiting a remarkably high Km and a Vmax only 5%
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Fig. 4. HPLC chromatogram of an aliquot of a 100 min aerobic reduction of CB 1954 by purified human kidney DT-diaphorase, without (-) and with 100 PM dicoumarol (----). Traces show the 4-hydroxylamine metabolite of CB 1954 (peak 1,O. 125 pmol for control and 0.042 pmol with dicoumarol), misonidazole internal standard (peak 2, 2.5 mot) and CB 1954 (peak 3, 51.2 pmol). Reactions contained 3 U enzyme and 10 mM CB 1954 substrate. For other conditions see Methods and Materials.
(min)
Fig. 5. Progress curves for the aerobic reduction of SR 4233 to its 2 and 4e reduction products, SR 43 17 and SR 4330. respectively, by rat Walker DT-diaphorase. Symbols: SR 4317 formation in the absence n and presence A of 100 FM dicoumarol. SR 4330 formation in the absence 0 and presence ??of dicoumarol. Reactions contained 14 U enzyme and 2 mM SR 4233. For other conditions see Materials and Methods.
of that for E09. The failure of the enzyme to elicit DNA damage suggests that this reaction may be a bioprotective pathway, but possibly the reaction rate is too low to generate sufficient active metabolites. Several investigators have proposed that MMC can undergo 2e reduction to alkylating species. though most work has relied on indirect evidence derived from the use of the potent DT-diaphorase inhibitor menadione (7, 2 1). However, studies using purified human kidney DT-diaphorase failed to show that MMC was a substrate for this enzyme at neutral pH (15). However, MMC is activated more efficiently to DNAdamaging species at acidic pH (17). Metabolism of CB 1954 by Walker tumor cell DT-diaphorase has previously been shown to be a potent bioactivation pathway (8, 9). Our results show that this reduction can be catalyzed by the human HT29 colon tumor enzyme and that the predominant product is the 4-hydroxylamine. As for E09, the rate of reduction was lower for the human compared to the rat tumor enzyme. The benzotriazine di-N-oxide SR 4233 was shown to be preferentially reduced to the 4e product by the rat tumor DT-diaphorase. In contrast to the reaction with E09 and CB 1954, this probably represents a detoxification pathway (22). In conclusion, the present results show that DT-diaphorase can function as either a bioactivating or bioprotective reductase, depending on the bioreductive drug involved. Tumors rich in DT-diaphorase should represent appropriate targets for E09 and CB 1954, as a result of selective activation by the enzyme, whereas they may be comparatively resistant to SR 4233. This information should not only facilitate clinical evaluation of bioreductive agents, but should also aid our “enzyme directed” approach to drug development (22).
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