Differential Enzymatic Reductions Governing the Differential Hypoxia

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Differential Enzymatic Reductions Governing the Differential Hypoxia-Selective Cytotoxicities of Phenazine 5,10-Dioxides Marıá Laura Lavaggi, Mauricio Cabrera, Mercedes Gonza´lez,* and Hugo Cerecetto* Departamento de Quı´mica Orgańica, Facultad de Quı´mica-Facultad de Ciencias, UniVersidad de la Repu´blica, Igua´ 4225, 11400 MonteVideo, Uruguay ReceiVed June 1, 2008

Some derivatives of phenazine 5,10-dioxide are selectively toxic to hypoxic cells commonly found in solid tumors. Previous studies of the phenazine 5,10-dioxide mechanism of action indicated that a bioreduction process could be involved in its selective toxicities, maybe as result of its potential HO•releasing capability in hypoxia. The major unresolved aspect of the mechanism of phenazine 5,10-dioxides is the identity of the reductase(s) in the cell responsible for activating the drug to its toxic form and metabolites. We have studied the metabolism in both hypoxia and oxia of some selected 2-amino and 2-hydroxyphenazine 5,10-dioxides, 1-5, using rat liver microsomal and cytosol fractions. Differential hypoxic/oxic metabolism was found to be correlated to a compound’s cytotoxic selectivity but, in general, without metabolic differences between liver microsomal or cytosolic enzymes. Dicoumarol and ketoconazole were found to inhibit the hypoxic metabolism of the most selective phenazine 5,10-dioxide, 1, inferring a role for DT-diaphorase and cytochrome P450. The least hypoxic selective agents, 4 and 5, possess different hypoxia-metabolic profiles as compared to derivative 1, explaining the differential cytotoxic biological behavior. The nonselective derivative, 2, suffered bioreduction in both conditions and, according to the inhibition studies with dicoumarol and ketoconazole, involves both DT-diaphorase and cytochrome P450. The nontoxic derivative, 3, showed poor bioreductive behavior. Introduction In contrast to normal tissues, which are well-perfused, the microenvironment of human solid tumors is heterogeneous with abnormal vascularization and contains regions that are poorly oxygenated (1). As a result, most primary tumors have lower pO2 levels than do normal tissue at the site of growth and contain regions of cells that are severely hypoxic. Hypoxic tumor cells are resistant to radiation and chemotherapy, which may lead to increased metastasis and accelerated malignant progression (2). However, the hypoxic environment of the tumor offers an attractive difference between normal and tumor cells that may be exploited with the use of bioreductive cytotoxins. These compounds are prodrugs that undergo reductive activation to yield cytotoxic metabolites or cytotoxic events (3). The reduction is facilitated by the appropriate reductases under the environment of low oxygen tension. Many compounds of diverse chemical structures including quinones, nitroimidazoles, nitrobenzyl derivatives, and N-oxide containing heterocycles have been designed as hypoxia-selective cytotoxins. The mechanism of cytotoxicity has been suggested to involve one- or two-electron reductive activation, via an enzymatic or no enzymatic process, or both, which could produce different kinds of cytotoxic events (4). Belonging to the N-oxide-containing heterocycles group, tirapazamine (TPZ,1 3-amino-1,2,4-benzotriazine-1,4-di-Noxide, Figure 1) is an agent that in combination with radiation * To whom correspondence should be addressed. Tel: +598 25258618, ext. 216. Fax: +598 25250749. E-mail: [email protected] (M.G.) and [email protected]. 1 Abbreviations: PDO, phenazine 5,10-dioxide; TPZ, tirapazamine; DTD, DT-diaphorase; AO, aldehyde oxidase; XO, xanthine oxidase; CYP, cytochrome P450; HCR, hypoxic cytotoxic ratio; HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography; EtOAc, ethyl acetate; MeOH, methanol; DMSO, dimethylsulfoxide; Dic, dicoumarol; Men, menadione; Ket, ketoconazole.

and with cisplatin in phase II and phase III clinical trials shows significant promise for both head and neck cancers and for nonsmall-cell lung cancer (5, 6). In hypoxic conditions, TPZ undergoes a one-electron activation, producing DNA single strand breaks, double strand breaks, and chromosome aberrations (7) with the concomitant formation of the two-electron reduction metabolite (SR4317, Figure 1). The neutral radical intermediate (NRI, Figure 1) undergoes homolytic fragmentation to release the well-known DNA-damaging agent, hydroxyl radical (HO•) (8). Under aerobic conditions, the TPZ radical preferentially reacts with oxygen to form a superoxide radical and the parent compound; this reaction is the source of TPZ-selective toxicity to hypoxic cells (9). Enzymes like NADPH:cytochrome P450 reductase, cytochrome P450 (CYP), xanthine oxidase (XO), and DT-diaphorase (DTD) are able to metabolize TPZ in vitro under hypoxic conditions (10–15). Recently, the mechanisms of cytotoxicity of quinoxaline 1,4-dioxide derivatives (QDO, Figure 1) were studied, as mixtures of positional isomers, by Monge and co-workers (16). These in vitro studies revealed that QDO generates reactive oxygen species, via redox cycling in the presence of the NADPH/cytochrome P450 enzyme system, which is responsible for aerobic cytotoxicity. On the other hand, it was hypothesized that hybrid compounds that possess an N-oxide and a π DNA-stacking moiety would be good bioreductive compounds. On this basis, several aliphatic amine-Noxides have been identified, including N-oxide derivatives of anthraquinones (AQ4N, Figure 1). AQ4N has a poor affinity for DNA and is poorly cytotoxic to cells in culture (17–19). In contrast, AQ4, the reduction metabolite, is at least 100-fold more cytotoxic than AQ4N. The reduction of AQ4N was shown to occur in the rat microsomal fraction but not in the cytosol and was dependent on both the anaerobic conditions and the presence of NADPH involving CYP as a reductase (20). In human liver,

10.1021/tx800199v CCC: $40.75  2008 American Chemical Society Published on Web 07/29/2008

Hypoxic Metabolism of Phenazine 5,10-Dioxides

Chem. Res. Toxicol., Vol. 21, No. 9, 2008 1901

Figure 1. Mechanism of bioreduction of TPZ, quinoxaline 1,4-dioxides, and AQ4N.

Figure 2. Studied PDO, 1-5, and expected metabolites of the bioreduction processes, 6-15.

the metabolism of AQ4N correlates with the CYP3A subfamily of enzymes (21). Our interest in the development of new hypoxia-selective cytotoxic agents (22–29) has encouraged us to synthesize phenazine 5,10-dioxide (PDO) derivatives, possessing Noxide and potential π DNA-stacking moieties. In our approaches, we have identified promising PDO derivatives with excellent behavior in hypoxic conditions, with derivative 1 having the best selective cytotoxic profile (Figure 2) (30–32). For this PDO, the hypoxic cytotoxic ratio (HCR), defined as the ratio of drug concentrations under aerobic to hypoxic conditions that give equal cell killing, is greater than 10 for V-79 cells (31). Potency (P), defined as the dose that gives 1% of control cell survival under hypoxia, is 10 µM for this cellular system (31). From these results, PDO 1 could be

selected to perform further in vivo antitumoral studies. The amino analogue, 2, was nonselective for the hypoxic conditions at the assayed concentrations, while the amino derivatives 4 and 5 were partially selective in these conditions. The methyl analogue of 1, PDO 3 (Figure 2), was completely inactive in the assayed conditions. The mechanism of action studies indicated that bioreduction could be involved in the observed selective cytotoxicity, maybe as result of its potential HO•-releasing capability, finding that PDO-DNA interaction does not participate in the toxic events (32). Testing the isolated 7- or 8-isomer, no preference on the DNA interaction was observed (data not published). In general, the hypothetical two- and four-electron metabolites, the corresponding phenazine monoxides or phenazines previously synthesized by us (31) (6-15, Figure 2), are nontoxic in hypoxia and normoxia. Additionally, some PDOs, as mixtures of 7(8)-isomers, were in vitro tested against human colorectal adenocarcinoma cell line Caco-2, studying the cell growth inhibition capacity, the capacity of altering the cell cycle, and the possible induction of apoptosis, DNA fragmentation, and genotoxic damage (33). The results confirmed the promising capabilities of PDOs as antitumoral agents. However, the major unresolved question is to identify the existence of reductive enzyme(s) in the bioactivation of phenazine 5,10-dioxides. Until now, the bioreductive profile of PDO has not been determined. In this sense, identification of relevant reductase(s) not only would aid in further drug development but also would be useful in identifying which tumors are sensitive to these agents. In this report, we show the metabolic profile of selected phenazine-5,10-dioxide derivatives, 1-5, in hypoxia and normoxia, identifying some of the involved enzymes. We have shown that DTD and CYP can metabolize derivative 1 in hypoxia to the end product 11, while the other derivatives 2-5 possess a different bioreductive metabolic profile correlated to its cytotoxic biological behavior.

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Figure 3. Synthesis of potential metabolic end products, 7 and 9-15, used as chromatographic standards.

Experimental Procedures Chemicals. All starting materials were commercially available research-grade chemicals and used without further purification. All solvents were dried and distilled prior to use. All of the reactions were carried out in a nitrogen atmosphere. The typical workup included washing with brine and drying the organic layer with sodium sulfate before concentration. 1H NMR and 13C NMR spectra and HSQC and HMBC experiments were recorded on a Bruker DPX-400 (at 400 and 100 MHz) instrument, with tetramethylsilane as the internal reference and in the indicated solvent. Mass spectra were recorded on a Shimadzu GC-MS QP 1100 EX instrument using electron impact ionization at 70 eV. The studied PDO, derivatives 1-5 (summarized in Figure 2) were synthesized using published methods (31). They were obtained and studied as mixture of 7- and 8-substituted positional isomers (30). In chemical reduction, potential bioreductive metabolic end products to be used as chromatographic standards were prepared using sodium dithionite (34) as a reductive enzyme analogue (35), to obtain compounds 7 and 9-15 (Figure 3). Using this procedure with 2-hydroxyphenazine 5,10-dioxides as reactants, 1 and 3, it was not possible to evidence the generation of the corresponding monoxide intermediates, 6 and 8. Only the final products, phenazines 11 and 13, were isolated in the chemical reduction assayed conditions. As an example, the synthesis and characterization of derivate 7 are presented. PDO 2 (0.5 mmol) was dissolved in concentrated hydrochloric acid (0.1 mL) and methanol (10.0 mL) at room temperature. A solution of sodium dithionite (1.0 mmol in 0.8 mL of water) was added in five portions to the stirred solution. The mixture was stirred at room temperature for 24 h. The methanol was evaporated in vacuo, and the residue was treated with aqueous saturated sodium bicarbonate (5.0 mL) and extracted with ethyl acetate (EtOAc) (3 × 10.0 mL). After the workup, the organic layer was eliminated by distillation at reduced pressure. The residue was purified by column chromatography [SiO2, petroleum ether:EtOAc: methanol (MeOH) (7:2:1)]. In this procedure was obtained derivative 12 (31) as the first fraction. The second chromatographic fraction corresponded to derivative 7, which was an orange solid (14%) (proportion 7:8 isomers ) 5.5:4.5). 7-Isomer: 1H NMR (CD3OD) δ: 7.27 (1H, d, H1, J ) 2.2 Hz), 7.29 (1H, dd, H3 J1 ) 8.4 Hz, J2 ) 2.1 Hz), 7.56 (1H, dd, H8, J1 ) 8.9 Hz, J2 ) 2.0 Hz), 7.89 (1H, d, H4, J ) 8.4 Hz), 7.93 (1H, d, H9, J ) 8.8 Hz), 8.26 (1H, s, H6). 13C NMR (HSQC-HMBC experiments) (CD3OD) δ: 96.20, 102.93, 120.41, 123.74, 127.38, 130.66, 131.70, 133.31, 141.29, 142.83, 146.73, 151.37. 8-Isomer: 1H NMR (CD3OD) δ: 7.27 (1H, d, H1, J ) 2.2 Hz), 7.29 (1H, dd, H3, J1 ) 8.4 Hz, J2 ) 2.1 Hz), 7.76 (1H, dd, H7, J1 ) 8.9 Hz, J2 ) 2.0 Hz), 7.91 (1H, d, H4, J ) 8.4 Hz), 7.93 (1H, s, H9), 8.29 (1H, d, H6, J ) 8.9 Hz). 13 C NMR (HSQC-HMBC experiments) (CD3OD) δ: 96.00, 103.11, 120.41, 123.74, 127.04, 130.87, 131.08, 133.31, 140.54, 144.30, 146.80, 151.69. EM (m/z), (%): 291/289 (M+•, 6.9/7.9), 275/273 (M+• - 16, 100.0/99.5). High-performance liquid chromatography (HPLC) grade EtOAc and MeOH were purchased from Fisher Scientific. Xanthine oxidase was obtained and purified from cow milk (36), and xanthine and catalase were purchased by Aldrich Co. All chemicals were used without further purifications.

Preparation of the Rat Liver Microsomal and Cytosolic Fractions. Livers were obtained from male Sprague-Dawley rats (198-202 g) from “Centro de Investigaciones Nucleares” (UdelaR, Montevideo, Uruguay). The animals were allowed food and water ad libitum. The experimental protocols with animals adhered to the Principles of Laboratory Animal Care (37). The animals were sacrificed by cervical dislocation, and the livers, maintained in a ice bath, were perfused in situ with an ice-cold KCl (0.9%) solution and washed with 3 volumes of Tris-HCl (0.05 M)-sucrose (0.25 M), pH 7.4, in a Potter-Elvehjem glass-Teflon homogenizer. The homogenates were centrifuged for 30 min at 900g at 4 °C, and the supernatant fraction was centrifuged at 10000g for 1 h at 4 °C. The pellet was discarded, and the supernatant fraction was further centrifuged at 100000g for 1 h at 4 °C. Metabolic assays were carried out with microsomes and cytosol either fresh or frozen in Tris-HCl buffer and stored at -80 °C. DTD activities of the microsomal and cytosolic fractions were determined spectrophotometrically, 600 nm, using 2,6-dichlorophenolindolphenol as a substrate (38). CYP activities of the microsomal and cytosolic fractions were determined using O-dealkylation of the 7-ethoxyresorufine assay (39). The aldehyde oxidase (AO) activity of the microsomal and cytosolic fractions was determined spectrophotometrically, 600 nm, using benzaldehyde as a reductive component and 2,6-dichlorophenolindolphenol as an electronic donor (40). Incubation of PDO 1-5 with Rat Microsomal and Rat Cytosolic Fractions in Normoxia and Hypoxia. Our procedure was adapted from published methods (41). The standard incubation mixture, in nitrogen or an air-purged flask, contained MgCl2 (1.3 mM), a NADPH-generating system (0.4 mM NADP+, 3.5 mM glucose 6-phosphate, and 0.5 U/mL glucose 6-phosphate dehydrogenase) in a 0.1 M potassium phosphate buffer (pH 7.4) containing EDTA (1.5 mM), and the corresponding PDO (40 µM) dissolved in dimethylsulfoxide (DMSO). Nitrogen gas was passed through a silica gel trap. The experiments were performed in triplicate. After pre-equilibration of the mixture at 37 °C and gassing with nitrogen or air, an appropriate volume of microsomal or cytosolic suspension was added to give a final protein concentration of 1 mg/mL. The mixtures were incubated for 30-120 min at 37 °C. Three control incubations were used as follows: (i) 0.1 M potassium phosphate buffer (pH 7.4) (C1); (ii) (C1) + NADPH-generating system (C2); and (iii) (C2) + heat-inactivated cytosolic or microsomal fractions (C3). Identification of the Involved Bioreductive Enzymes. The identification of the involved bioreductive enzymes was made by preincubating, for 30 min, the corresponding fraction with previously described enzymatic inhibitors (40 µM). The employed inhibitors were dicoumarol (Dic) for DTD, menadione (Men) for AO, and ketoconazole (Ket) for CYP. Enhanced Aldehyde Oxidase Bioreduction Study. Parallel to the inhibition assays with Men (section 2.4), the possible participation of AO (EC 1.2.3.1) was studied, enhancing its activity with an electron donor compound. PDO 1 and 4 were dissolved in DMSO (40 µM) and were incubated, in hypoxic conditions, for 30 min at 37 °C in 0.1 M potassium phosphate buffer (pH 7.4) containing EDTA (1.5 mM) with microsomal or cytosolic fractions (1 mg protein/mL) and benzaldehyde (40 µM, dissolved in DMSO)



Figure 4. HPLC chromatograms (see the Experimental Procedures for experimental conditions) taken after 60 (A) and 120 min (B) of incubation of PDO 4 (as 7- and 8-substituted positional isomers) with rat cytosolic fraction in hypoxia (according to the Experimental Procedures description). Inset: HPLC chromatogram for standards (PDO 4, phenazine 5-monoxide 9, and phenazine 14).

Table 1. Bioreductive Metabolites of PDO 1-5 Identified after Incubation with Rat Cytosolic and Microsomal Fractions in Hypoxia

Table 3. Bioreductive profile of selective cytotoxic PDO Hypoxia products detected after 30 min of incubation

products detected after 30 min of incubationa PDO 1 2 3 4 5

cytosolic fraction 11 7 + 12 othersb 9 + 14 10 + 15

cytosolic fraction pretreated with

microsomal fraction 11 7 + 12 NMc 9 + 14 10 + 15

PDO 1 2 4

a In the assays with negative controls, C1, C2, and C3 (see the Experimental Procedures), no presence of bioreductive metabolites was evidenced. b Others are metabolism products different from phenazine monoxides or phenazines. Elucidation of the chemical structures was not done. c NM, no metabolism.

Table 2. Bioreductive Metabolites of PDO 1-5 Identified after Incubation with Rat Cytosolic and Microsomal Fractions in Normoxia products detected after 30 min of incubation PDO 1 2 3 4 5

cytosolic fraction NMb 7 + 12 others NM NM

a

microsomal fraction othersc 7 + 12 NM NM NM

a In the assays with negative controls, C1, C2, and C3 (see Experimental Procedures), no presence of bioreductive metabolites was evidenced. b NM, no metabolism. c Others are metabolism products different from phenazine monoxides or phenazines. Elucidation of the chemical structures was not done.

as an electron donor. Bioreduction under hypoxic simulated conditions was made by gassing the reaction mixture with nitrogen for 20 min. Xanthine Oxidase Bioreduction Study. PDO 1-5 were dissolved in DMSO (40 µM) and were incubated for 30 min and 24 h at 37 °C in 0.1 M potassium phosphate buffer (pH 7.0) with the reaction mixture (0.4 U/mL XO, 500 µM xanthine, and 0.2 mg/ mL catalase) (42). Bioreduction in simulated hypoxic conditions was made by gassing the reaction mixture with nitrogen for 20 min. HPLC and Thin-Layer Chromatography (TLC) Monitoring of Metabolites. The incubated mixtures were extracted with EtOAc (3 × 400 µL), and the organic layer was evaporated to dryness in vacuo. The residue was treated twice with EtOAc, 500

microsomal fraction pretreated with

Dic

Men

Ket

Dic

Men

Ket

NMa 7 9 + othersc

11 7 + 12 9 + 14

11 7 9

11

11 7 + 12

NM

b

b

b

9

b

Normoxia products detected after 30 min of incubation cytosolic fraction pretreated with

microsomal fraction pretreated with

PDO

Dic

Ket

Ket

2

7

7

7 + 12

a NM, no metabolism. b Blank cells, not studied. c Others are metabolism products different from phenazine monoxides or phenazines. Elucidation of the chemical structures was not done.

µL each, and the combined organic layer was filtered through RC regenerated cellulose filters, 0.45 µm pore size (Sartorius). The 100 µL aliquot of the obtained EtOAc solution was analyzed by SiO2-HPLC, using a Hibar II Lichrosorb SI-60 column, 25 cm × 0.46 cm, 10 µm particle size (EM Reagents, Cincinnati, OH) and a Perkin-Elmer LC-135C/LC-235C HPLC system equipped with a diode array detector, series 410 LC BIO PUMP. HPLC analyses were carried out at a flow rate of 1 mL min-1 with EtOAc:MeOH (99.5:0.5) as a mobile phase and room temperature as the working temperature. Detection was at 275 nm, being the λmax, penazine monoxide near to 275 nm and λmax, phenazine near to 280 nm. Along with HPLC experiments, TLC was performed as a qualitative study. These studies were done in SiO2 as the solid phase and EtOAc:hexane (50:50). The spots were visualized using UV light (240 nm) and directly for its characteristic colors (PDO, redviolet; phenazine monoxides, orange; and phenazines, yellow). Because the PDO 1-5 are a mixture of 7- and 8-substituted derivatives in some chromatographic conditions, it was possible to visualize both positional isomers in the starting material and in the metabolic products (see the examples in Figure 4).

Results Hypoxic Bioreductive Metabolism. PDO hypoxic bioreductions were performed in nitrogen atmosphere and were

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Table 4. Summary of Chemical and Biological Findings Related to the Bioreduction of the Studied PDO enzyme g

bioreduction PDO 1 2 3 4 5

SFaira,b

SFhypoxb,c

80 12 93 61 87

0 0 100 11 23

biological behaviord S NS I PS PS

Epc vs SCE

e,f

(V)

-1.14 -0.92 -0.78 -1.00 -1.03

normoxia

hypoxia

N Y NM N N

Y Y NM Y Y

normoxia cytosol

hypoxia

microsome

h

h i

cytosol

microsome

DTD DTD/CYP

CYP NI h

DTD/CYP

NI

h

h

h

h

h

DTD/CYP

DTD

h

h

h

h

a SFair ) survival fraction in air at 20 µM. b SFhypox ) survival fraction in hypoxia at 20 µM. c Values from ref 31. d S, selective; NS, nonselective; I, inactive; and PS, partial selectivity. e Peaks potentials (∼(0.01 V) measured at a scan rate of 2.00 V/s; eV. f Values from ref 32. g N, bioreduction was not detected; Y, bioreduction was detected; and NM, no bioreductive metabolic products were evidenced. h Blank cells, not studied. i NI, not identified.

Figure 5. Pathways for PDO reductive enzymatic transformations in hepatic cytosol, a proposal based on the identified end products and the enzymatic inhibition assays.

monitored by HPLC and TLC. The different runs were monitored for 30-120 min. Representative HPLC chromatograms obtained for PDO 4 hypoxic bioreduction, at different times, are presented in Figure 4. The PDO 1, 2, 4, and 5 showed similar bioreductive behavior in both protein fractions (Table 1) identifying the corresponding phenazines, 11, 12, 14, and 15 as the end metabolic products since the first monitored time in all of the cases. Except for 2-hydroxyphenazine derivatives, 1 and 3, the phenazine monoxides, 7, 9, and 10, were also identified since the first 30 min of incubation. This could indicate either the higher capability of 2-hydroxy phenazine monoxides to suffer bioreduction as compared with 2-amino phenazine monoxides that did not allow us to evidence chromatographically the presence of 6 and 8 potential intermediates or the differential enzymatic processes that transform 2-hydroxy derivatives directly to phenazines. The consumption of PDO 1-5 through time came with the increment of the phenazine 11-15 amount. Concomitantly, the amount of phenazine monoxides, 7, 9, and 10, increased in the first hour of metabolism with a decrease in the following 60 min (see the example in Figure 4). The absence of bioreduction products in the assayed controls (C1, C2, and C3) confirmed that the processes are the results of biological metabolism, not of chemical reduction. Normoxia Bioreductive Metabolism. PDO bioreductions in normoxia were performed in air atmosphere and were monitored by HPLC and TLC. The different runs were monitored for 30-120 min. In general, the studied PDO 1-5 did not show bioreductive metabolism in both protein fractions (Table 2), evidencing other metabolic products that do not correspond to the reductive metabolic products (6-15), in the case of 2-hydroxyphenazine derivatives 1 and 3 in microsomal and cytosolic fractions, respectively. The chemical structures of these unexpected metabolic products were not elucidated. The nonselective cytotoxic PDO 2 was the unique derivative that yielded

bioreductive metabolic end products 7 and 12, explaining the differential biological behavior of this PDO in both conditions (see below). Enzymes Involved in the Bioreductive Mechanism of PDO. By means of enzymatic inhibitors, the proteins involved in the bioreductions in hypoxia and normoxia were identified using the selective cytotoxins 1 and 4 and the nonselective cytotoxin 2, both in hypoxia and in normoxia. For these purposes, the metabolic products profile after 30 min was studied (Table 3). Independent assays in hypoxia were performed with purified XO and enhancing AO activity, with benzaldehyde as an electron donor (43), showing nonparticipation of these enzymes in the bioreductions of any of the studied PDO (1–5).

Discussion In an attempt to determine if the designed hypoxia-selective cytotoxins, PDO derivatives, were selectively bioreduced under anaerobic conditions, a series of bioreductive experiments were performed. It was found that compounds 1, 4, and 5 that show the best selective hypoxic cytotoxicity profiles are bioreduced selectively under anaerobic conditions. This could indicate that these PDOs are selectively hypoxic and cytotoxic as a result of its bioreduction pathways (Table 4). Previously, it was observed (32) that the bioreductive capabilities of the PDO derivatives were related to the first cathodic potential, determined by cyclic voltammetry, showing for the hypoxic-selective cytotoxins values between -1.00 and -1.14 V (Table 4). Consequently, this bioreductive behavior was also found in the sodium dithionite chemical reduction. 2-Amino derivatives, 2, 4, and 5, were found to yield the monooxidized, 7, 9, and 10, and completely reduced products, 12, 14, and 15, during both chemical and biological procedures, while the 2-hydroxy derivatives, 1 and 3, were found to produce the complete reduced products, phenazines 11 and 13, exclusively in both conditions.


Derivative 2 was found to be highly cytotoxic under both aerobic and anaerobic conditions, meaning that it had no selectivity. The bioreductive assay showed that the bioreduction occurs in both conditions, probably explaining that the noselective cytotoxicity could be a consequence of the bioreductive events that happen. Bearing in mind the first cathodic potential of PDO 2, -0.92 V (Table 4), we could rationalize its normoxic toxicity in terms of an oxygen-dependent reversibility process yielding reactive oxygen species (initially O2•-) mediated by the CYP enzyme, according to recent descriptions (16), like aerobic cytotoxicity of QDO. According to our data, no differential enzymatic processes seem to participate in the biological response of this PDO in normoxia and hypoxia. Derivative 3 was not active in neither aerobic nor anaerobic conditions at the studied doses (Table 4, ref 31). Concomitantly, in this study, it was found to be neither bioreduced under aerobic nor anaerobic conditions. This could reinforce our hypothesis that PDO results in cytotoxicity for the reason of its bioreductive capability. In this case, the PDO 3 cathodic potential, -0.78 V (Table 4), may lie out of reductases range of effectiveness (44). As an attempt to get insight into the mechanism of action of PDO derivatives, we tried to identify some enzymes responsible for the bioreduction. Consequently, cytosolic-DTD was identified as an enzyme responsible for the hypoxic bioreduction of derivatives 1, 2, and 4 (Table 4). It was confirmed that in the case of derivatives 1, 2, and 4, cytosolic-DTD and, in the case of compound 4, microsomal-DTD, are responsible for transforming PDO into the corresponding phenazines in hypoxia. This information is very relevant taking into account that DTD levels are elevated in a number of tumor types, like nonsmall cell lung, colorectal, and breast carcinomas, when compared to the normal tissue (45). The detoxifying enzyme DTD can activate certain xenobiotics, such as antitumoral benzoquinones (46–48), allowing tumoral cytotoxicity without correspondingly high levels of toxicity to normal tissues. In addition, CYP was found to be responsible for generating the reduced end product, phenazine, in the case of the derivatives 2 and 4 in rat cytosol and for derivative 1 in rat microsomes. The enzymes XO and AO were found not to be involved in the bioreduction of any of our studied PDOs. In summary, according to our findings, the hypoxic metabolism of PDO by hepatic cytosol and microsomes revealed the formation of reduced metabolites, phenazine monoxides, and phenazines that concomitantly could produce cellular damage under low-oxygen concentrations. This bioreduction is catalyzed by two-electron reductases (Figure 5), DTD and CYP. Furthermore, according to Hecht et al.’s suggestions (49, 50), which explained the DNA damage of another phenazine N-oxide by HO• production, we could think that after DTD/CYP activation of the PDO this toxic reactive oxygen species could be released. Experiments that confirm this proposal are currently in progress. Moreover, the aerobic bioreduction of PDO by these hepatic systems only takes place in the aerobic cytotoxic derivative. The metabolism of the PDO by DTD/CYP reported here is analogous to XO-mediated conversion of quinoxaline N,N′dioxide to its mono-N-oxide, which occurs by direct two-electron reduction (51). The studies presented here provide the first evidence that the hypoxia-selective cytotoxin PDO 1 is able to selectively reduce under hypoxic conditions. The results afford a clear chemical basis for the medicinally interesting biological activities reported for this compound.


Acknowledgment. We acknowledge Marcelo Fernańdez for the purchase of the rats. This investigation was supported by Comisioń Honoraria de Lucha contra el Cańcer (Uruguay). M.L.L. and M.C. thank PEDECIBA/ANII and DINACYT for scholarships.

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TX800199V

Differential Enzymatic Reductions Governing the Differential Hypoxia

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