Chem -Bwl Interactwns, 85 (1992) 187-197

187

Elsewer Scientific Publishers Ireland Ltd.

REDUCTION PROPERTIES OF NITRATED NAPHTHALENES: RELATIONSHIP BETWEEN ELECTROCHEMICAL REDUCTION POTENTIAL AND THE ENZYMATIC REDUCTION BY MICROSOMES OR CYTOSOL FROM RAT LIVER

NOBUHISA IWATAa, KIYOSHI FUKUHARAb, KAZUHIRO SUZUKIa, NAOKI MIYATAb and ATSUSHI TAKAHASHI a

aDwiswn of Xenobwtw Metabolucm and D~spos~twn and bDw~swnof Organw Chemistry, Natwnal Institute of Hyg~enw Sciences, Tokyo 158 (Japan) (Recmved June 19th, 1992) (Revision received August 31st, 1992) (Accepted September 14th, 1992)

SUMMARY

The nitroreductase activities of rat liver microsomes and cytosol towards various nitrated naphthalenes (1-, 2-mononitro-, 1,3-, 1,5-, 1,7-, 1,8-dinitro-, 1,3,5- and 1,3,8-trinitronaphthalenes) were characterized as follows. (1) The rates of reduction of nitrated naphthalenes in either macrosomal or cytosolic incubation were found to increase in the order of trinitro- > dinitro- > mono-nitronaphthalene, although, in the case of microsomal nitroreduction, trinitronaphthalenes were reduced more rapidly than in cytosol. (2) The effective cofactors, electron donors, in the nitroreduction of nitrated naphthalenes in cytosol were NADH and hypoxanthine, but not NADPH. (3) The nitrated naphthalenes with a nitro group at a ~-position appear to be more easily reduced among the various isomers. The cytosolic nitroreductase actiwties towards the nitrated naphthalenes were closely related to the single-electron reduction potentials measured by cyclic voltammetry and hence, there was a good relationship between the logarithm of nitroreductase activities and the electrochemical reduction potentials. In microsomes, nitroreductase activities were rather less well related to electrochemical reduction potentials.

Key words: Nltronaphthalene -- Nitroreductase -- Reduction potential -NADPH-cytochrome c reductase -- DT-diaphorase -- Xanthine oxidase Correspondence to N Iwata, I:hwsion of Xenoblotlc Metabolism and Dmposltion, National Institute of Hygmmc Scmnces, 18-1 Kamlyoga, 1-chome, Setagaya-ku, Tokyo 158, Japan 0009-2797/92/$05 00 © 1992 Elsewer Scientific Publishers Ireland Ltd Printed and Pubhshed in Ireland

188

INTRODUCTION

Nitroarenes are notorious environmental pollutants formed by the nitration of polycyclic aromatic hydrocarbons. They have been detected in ambient air and numerous combustion emissions [1-4]. Their mutagenicity, genotoxmity and carcinogenicity are well established [1 - 7]. The number of fused rings and the substituted position and multiplicity of nitro groups on the parent compound greatly influence in their toxicity [1-7]. Even among the simplest nitrated polycyclic aromatic hydrocarbons, i.e. nitrated naphthalenes, the toxic potency varies markedly. For example, while 2-nitronaphthalene is a potent carcinogen in monkeys, the 1-nitro isomer has no carcinogenicity [1,3,4,7] Dimtronaphthalenes are much more mutagenic than mononitronaphthalenes [1,3,5,7]. The reduction of the nitro group requires nitroreductase, which is present in bacterial and mammalian cells [8-18]. Several enzymes have been shown to possess significant nitroreductase activity towards various kinds of mtro compounds. They are NADPH-cytochrome c reductase, cytochrome P-450, DTdiaphorase and xanthine oxidase. The active forms of a series of nitroarenes are implicated in the metabolic reduction of nitro groups, i.e., the primary basis of biological action of nitroarenes appears to be a reduction of the nitro group to give the corresponding N-hydroxylamines via the nitroso intermediates [8,9]. Indeed, the mutagenicity of the nitroarenes in Salmonella typhzmumum strata TA98NR, which lacks one of the nitroreductases, is decreased [1,3,7,19,20]. N-Hydroxy-1- and N-hydroxy-2-naphthylamine, which are N-hydroxy metabolites of 1- and 2-nitronaphthalene, respectively, are potent tumorigens [21]. The tumorigenicity of N-hydroxy-l-naphthylamine is more potent than that of Nhydroxy-2-naphthylamine [21]. However, in the case of parent compounds, this is quite the reverse [1,3,4,7]. Therefore, the facility of the reductive metabolism of nitroarenes to hydroxylamines is also one of the factors determining the toxicity of these nitroarenes, as well as the ability of their N-hydroxy metabolites to undergo O-acetylation. Nevertheless, little has so far been revealed regarding the structure-activity relationships for the enzymatm reduction of nitroarenes in the liver. The present report describes the rates of reduction of several nitrated naphthalenes, including mono-, di- and trinitrated naphthalenes, by rat liver microsomes and cytosol in relation to their electrochemmal redox properties. MATERIALS AND METHODS

Materials Cytochrome c and superoxide dismutase were purchased from Sigma Chemical Co., St. Louis, MO. Pyridine nucleotides were purchased from Oriental Yeast Co. Ltd., Osaka, Japan. 1-Nitropyrene, 1-, 2-nitronaphthalenes, 1,5- and 1,8-dinitronaphthalenes were purchased from Tokyo Kasei Industries Co., Tokyo, Japan. 1,3-Dinitronaphthalene was purchased from Aldrich Chemical Co., Milwaukee, WI. 1,7-Dinitronaphthalene and 1,3,5- and 1,3,8-trinitronaphthalene were synthesized by nitration of 2-nitronaphthalene as described previously [22] and these structures were confirmed by the analysis of mass spectrography and high-

189

resolution 400 MHz proton NMR: 1,7-dimtronaphthalene, EI-MS m/z (rel. intensity) 218 (100, M÷), 172 (33, M+-N02), 126 (90, M÷-2NO2), 114 (79); ~H-NMR (CDC13) ~7.81 (t, H3), 8.15 (d, Hs, J = 8.9 Hz), 8.27 (d, H4, J = 8.4 Hz), 8.42 (d, H6, J -- 8.9 Hz), 8.46 (d, H2, J -- 7.6 Hz), 9.62 (s, Hs). 1,3,5-trinitronaphthalene, ELMS m/z (rel. intensity) 263 (83, M÷), 171 (14, M+-2NO2), 125 (66, M÷-3N02), 75 (100); 1H-NMR (CDC13) ~8.06 (t, H7), 8.54 (d, H6, J = 8.0 Hz), 8.86 (d, Hs, J -- 8.8 Hz), 9.06 (s, H2), 9.81 (s, H4). 1,3,8-trinitronaphthalene, EIMS m/z (rel. intensity) 263 (10, M+), 217 (81, M÷-NO2), 171 (22, M+-2N02), 113(100); 1H-NMR (CDC13) 57.96 (t, H6), 8.46 (d, H5, J = 8.4 Hz), 8.50 (d, H7, J - - 7 . 6 Hz), 9.03 (s, H5), 9.15 (s, H2). Other nitrated naphthalenes were reagent-grade, confirmed to be chromatographmally >99% pure and used without further purification. Partially succmoylated cytochrome c (succinoylated Cyt. c) was prepared according to the method of Kuthan et al. [23]. Other chemicals of reagent grade were obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan.

Enzyme preparatwn Rat liver cytosol and microsomes were prepared from male F344 rats (> 200 g, obtained from Japan SLC, Shizuoka, Japan). Rats were killed by decapitation and the livers were quickly removed and perfused with 1.15% KCI. Liver cytosol was prepared from a homogenate in four volumes (w/v) of 10 mM Tins - HC1 (pH 7.4) containing 1.15% KCI, 0.1 mM EDTA, 1.0 mM dlthlothreitol and 0.01 mM phenylmethylsulfonyl fluoride, by centrifugation of the 9000 × g supernatant at 105 000 x g for 1 h. Microsomes (105 000 × g pellet) were washed once with 1.15% KC1 and resuspended in the original homogemzation buffer. All preparations were stored at - 80°C. Enzyme assay Aerobic cytosolic or microsomal nitroreductase activltms were assayed essentially as described by Djurid et al. [13]. In this assay system, the facihty of firstelectron transfer to nitro compounds was measured. Further, the rate of succinoylated Cyt. c reduction is indicative of aerobm nitroreductlon to intermediates that could not be detected by HPLC. The incubation mixture contained 20 #M or 40 ~M nitrated naphthalene (naphthalenes were dissolved in dimethylsulfoxide, final concentration 1%, v/v), 50 ~M succmoylated Cyt. c, 0.1 mM pyridine nucleotides (or 50 tLM hypoxanthine) and 0.1-0.6 mg/ml cytosolic or microsomal protein in 100 mM potassium phosphate buffer (pH 7.4) to a final volume of 1.0 ml. The reaction was initiated by the addition of either substrate or enzyme. Reduction of the succinoylated Cyt c was monitored spectrophotometrically at 37°C by following the increase in absorbance at 550 nm against a reference, which contained all the reactants but no substrate. An extinction coefficient of Ess0 21 mM- 1 cm- 1 was used for quantification [24]. The non-enzymatic reductions were little m the case of nitrated naphthalenes. Each enzymatm reaction was at linear ranges with both time and enzyme protein concentration. Protein concentration was determmed using the mmro BCA kit (Pierce Chemical Co., IL) with bovine serum albumin as a standard. nm

=

190

Electrochemwal reduction by cychc voltammetry Measurement of electrochemical reduction potentials of various nitro compounds was performed using an electrochemmal analyzer, BAS 100B (Bioanalytical Systems, Inc., West Lafayette, IN) as descmbed previously [19,25]. Dimethylformamide containing 0.1 M tetraethylammonium perchlorate was used as a solvent. After transfer of the solution containing the test chemical into the cell, the solution was purged of oxygen by bubbhng N2 gas for 10 min. Cyclic voltammograms were recorded at a scan rate of 100 mV/s while mamtaining the test solution under a steady stream of N2 gas.

Molecular orb,tal calculatwns Energies of the LUMOs were obtained from semiempirical MNDO (moderately neglected differential overlap) methods [26] by using standard parameters in 'Pasocon MOPAC/386', which is based on the MOPAC (ver. 5.0 QCPE No. 455) of Toray Center. RESULTS

Chemical characteristws of reduction of n~trated naphthalenes Table I lists the LUMO energy levels and the polarographm reduction potentials, together with the chemical structures, of nitrated naphthalenes used in this study. Nitrated naphthalenes showed a reversible redox wave owing to the redox cycling between nitro group and nitro anion radical and the reduction potentials (E1/2) in DMF were between -500 and -1100 mV versus a standard calomel

TABLE I LUMO E N E R G Y L E V E L S C A L C U L A T E D BY MNDO A N D P O L A R O G R A P H I C R E D U C T I O N P O T E N T I A L S OF N I T R A T E D N A P H T H A L E N E S 8

1

1,4,5 and 8 a re a-positions 2,3,6 and 7 a re 3-positions 5

4

Compound

P o l a r o g r a p h i c red uc t i on p o t e n t i a l (mV)

LUMO e n e r g y level (eV) m ground state

No

1-Nltronaphthalene 2 -Nltro naphthalene 1,3-Dmltronaphthalene 1,5-Dmltronaphthalene 1,7-Dmltronaphthalene 1,8-Dmltronaphthalene 1,3,5-Trmltronaphthalene 1,3,8-Tmmtronaphthalene

- 1031 - 1049 - 710 - 827 - 775 - 927 - 519 - 578

-

1 2 3 4 5 6 7 8

MNDO Mo&fied n e g l e c t of & a t o m m overlap

1 181 1 398 2 075 1 896 1 867 1 860 2 580 2 587

191

electrode. The reduction potentials of trinitrated naphthalenes were more than 500 mV higher than those of mononitrated naphthalenes and the order of ease of electrochemical reduction was 1,3,5- >-_ 1,3,8- > 1,3- > 1,5- > 1,7- > 1,8- > 1- ~- 2-nitrated naphthalene. The LUMO energy levels of these nitrated naphthalenes calculated with the MNDO method were between -1.1 and - 2 . 6 eV. The order of ease of one-electron reduction was 1,3,5- = 1,3,8- > 1,3- > 1,5-- 1,7- -- 1,8- > 2- > 1-nitrated naphthalene. Reduction properties determined by the electrochemical measurement and the semi-empirical molecular orbital calculation are rather well correlated, as shown in Fig. 1. Among the dinitrated naphthalenes, 1,3-dinitronaphthalene, whmh has two nitro substltuents on one benzene ring was more easily reduced than the other dinitronaphthalenes (1,5-, 1,7- and 1,8-), whmh have one nitro substltuent on each benzene ring. 1,8Dinitronaphthalene, that has two nitro substituents at the adjoining peripositions, was reduced easily compared to 1,3-, 1,5- and 1,7-dmitronaphthalenes. A similar situation was found for 1,3,8- and 1,3,5-trinitronaphthalenes. These results show that the reduction properties of nitrated naphthalenes can be roughly predicted by using chemical calculation.

M~crosomal n~troreductase aztw~ties Nitrated naphthalene-mediated reduction of succinoylated Cyt. c by rat liver microsomes is shown in Table II. The microsomal nitroreductase activities were highest towards 20 ~M trinitronaphthalenes (95.6 and 29.9 nmol/mg protein/rain) and moderate activities were detected towards 20 #M dinitronaphthalenes (0.47-4.83 nmol/mg protein/min) and, to a lesser extent, towards 20 ~M mononitronaphthalenes (0.16 and 0.36 nmol/mg protein/ram). The extent of

"2501

==

-750

o

e6

o=

1

r:0.961

"o Q rr

~

-125• -3.0

, -2.5

i -2.0

, -1.5

- .0

LUMO Energy level (ev) Fig 1 Plot of LUMOenergy levelvs reductionpotential for mtronaphthalenes Numberson the hne are the compoundnumbers of varmus mtrated naphthalenes shown m Table I

192 TABLE II NITROREDUCTASE ACTIVITIES OF RAT LIVER MICROSOMES TOWARDS NITRATED NAPHTHALENES Reduction of succmoylated Cyt c was momtored at 550 nm m I ml cuvettes and was performed with 20 or 40 ~M nitrated naphthalene, 50 #M succlnoylated Cyt. c, 0 1 mM NADPH, 0 6 mg/ml mmrosomal protein and, where indicated, 0 1 mg/ml superomde dmmutase as described m Materials and Methods Each value m the mean of duplicated assays Values m parentheses indicate mdiwdual determinants in duphcate Rate of succlnoylated cytochrome c reduction (nmol/mg protein/ram) - SOD Compound (conc)

20 ~M

1-Nitropyrene 0 96 (0.97, 1-Nltronaphthalene 0 16 (0 18, 2-Nltronaphthalene 0.36 (0.28, 1,3-I:hnitronaphthalene 3.36 (3 69, 1,5-I:hnitronaphthalene 0.47 (0.38, 1,7-Dimtronaphthalene 4 83 (4 65, 1,8-Dlmtronaphthalene 0.95 (1.02, 1,3,5-Trimtronaphthalene 95.61 (102 6, 1,3,8-Trimtronaphthalene 29 90 (28.2,

+ SOD 40/~M 0.96) 1.19 (1 02, 1 37) 0 15) 0 16 (0 20, 0.12) 0 44) 0.41 (0 41, 0 41) 3 02) 4 01 (3 05, 4 97) 0.55) 0 35 (0 41, 0.29) 5 00) 6 21 (5.70, 6.72) 0 87) 1 08 (0.90, 1.25) 88.6) 127 01 (107 0, 147 0) 31 6) 47 75 (48 9, 46 6)

20 #M 0 87 0 20 0 10 1 41 0 21 2 27 0 38 65.92 21 06

(0 84, (0 21, (0 12, (1 40, (0 17, (1 98, (0 37, (62 1, (22 8,

0 89) 0 19) 0 08) 1 42) 0 25) 2.56) 0.40) 69 7) 19 3)

nitroreduction may be associated with the positions of nitro groups on naphthalene. For example, dinitronaphthalenes with a nitro group at a ~-position (1,3- and 1,7-dinitronaphthalene) were more easily reduced than a-substituted compounds (1,5- and 1,8-dinitronaphthalene). Nitroreductase activities towards mononitronaphthalenes and dinitronaphthalenes were not increased when higher concentrations were used.

Cytosolic nitroreductase activities Measurement of cytosolic nitroreductase activity was performed in the presence of NAD(P)H, cofactors of DT-diaphorase (Table IIIa), and hypoxanthine, a cofactor of xanthine oxidase (Table IIIb). Substrate specificity observed in rat liver cytosolic incubation with each cofactor was essentially the same as that in microsomal incubation. However, when each cofactor was used, cytosolic nitroreductase activities towards trinitronaphthalenes were somewhat lower than microsomal nitroreductase activities. When pyridine nucleotide-dependent nitroreductase activities were measured, greater activities towards all nitrated naphthalenes tested were observed in the presence of NADH, in comparison to those with NADPH. The hypoxanthine-dependent activities showed comparable rates to the NADH-dependent activities.

193

TABLE IIIa NITROREDUCTASE ACTIVITIES OF RAT LIVER CYTOSOL WITH NAD(P)H TOWARDS NITRATED NAPHTHALENES Incubation was performed as described In Table II with 0 1 mg/ml cytosohc protein and 0 1 mM NADPH or NADH Each value is the mean of duphcated assays Values in parentheses lndmate ln&wdual determinants in duphcate Rate of succmoylated cytochrome c reduction (nmol/mg proteln/mm) Compound (conc) 1-Nitropyrene 1-Nitronaphthalene 2-Nitronaphthalene 1,3-Dmltronaphthalene 1,5-Ihmtronaphthalene 1,7-I:hmtronaphthalene 1,8-Ihmtronaphthalene 1,3,5-Tnmtronaphthalene 1,3,8-Tnmtronaphthalene

NADPH NADH NADPH NADH NADPH NADH NADPH NADH NADPH NADH NADPH NADH NADPH NADH NADPH NADH NADPH NADH

20 ~M

40 ~M

0 40 (0 40, 0 40) 0 73 (0 51, 0 96) 0 15 (0 18, 0 11) 0 40 (0 37, 0.44) 0 23 (0 22, 0.24) 1 03 (1 03, 1 03) 0 61 (0 92, 0.30) 3 43 (3 49, 3 38) 0 51 (0 37, 0 66) 2 15 (2 02, 2 28) 0 35 (0 55, 0 15) 3 67 (3 49, 3 86) 0 27 (0.28, 0 26) 1.10 (1 29, 0 92) 3 15 (3 03, 3.30) 16.12 (15 4, 16 8) 1 88 (2 30, 1 47) 6 33 (5 51, 7 16)

0 50 (0 50, 0.50) 1 34 (1.51, 1 18) 0 18 (0 22, 0 15) 0 50 (0.44, 0 55) 0 29 (0.40, 0.18) 1 07 (1 10, 1 03) 0 99 (0 83, 1.16) 4 17 (4 22, 4 11) 0 95 (0 64, 1 25) 2 77 (2 64, 2 90) 0 54 (0 51, 0 57) 4 50 (4 22, 4.77) 0 35 (0 37, 0.33) 1 16 (1.29, 1 03) 2.87 (2 66, 3.08) 16 58 (15.3, 17.8) 1 42 (1 38, 1 47) 7 95 (7 16, 8 74)

Superoxide-mediated reduction of n~tronaphthalene Since it is well known that NADPH-supplemented liver microsomes [23,27,28] and the xanthine oxidase/xanthine system [23,29,30] generate superoxide, we investigated whether or not superoxide generation under these conditions may be involved in nitrated naphthalene reduction (Tables II and IIIb). Superoxide dismutase resulted in a decrease in the reduction of succinoylated Cyt. c by microsomes in the order of mononitro- (70%) > dinitro- (60%) > trinitronaphthalene (30%), but had no influence in the case of 1nitronaphthalene. In contrast, the rates of succinoylated Cyt. c reduction in the cytosolic incubation with hypoxanthine were not appreciably inhibited by superoxide dismutase. Relationship between the electrochemical reduction potentials and the nitroreductase activities As shown in Fig. 2, the enzymatic nitroreductase activities towards various nitrated naphthalenes in cytosol were closely related to the single-electron reduc-

194 TABLE IIIb NITROREDUCTASE ACTIVITIES OF RAT LIVER AWARDS NITRATED NAP~HALENES

CYTOSOL WITH

HYPOXANTHINE

Incubation was performed as described m Table II with 0 1 mglml cytosohc protem and 50 pM hypoxanthme Each value IS the mean of duphcated assays Values m parentheses mdicate mdirndual determmants m duplicate Rate of succmoylated cytochrome c reduction (nmobmg protem!min) -SOD

+ SOD

Compound (cone )

20 pM

1-Nltropyrene 1-Nltronaph~~ene 2-Nltronaphthalene 1,3-Dmitronaphthalene 1,5-~nlt~naphth~ene 1,7Dmitronaphthalene 1,8-~nit~naph~~ene 1,3,5-Trimtronaphthalene 1,3,8-T~nltronaph~~ene

0 26 0 45 0 96 4 34 2 43 3 58 131 14 70 8 03

(0 28, (0 41, (0 99, (4 71, (2 51, (3 55, (1.32, (14 5, (7.47,

40 aM 0 25) 0 50) 0 93) 3 97) 2 35) 3 60) 129) 15 0) 8 59)

0 96 (1 19, 0 58 (0 58, 109 (102, 4 17 (4 46, 197 (1 57, 4 05 (3 97, 165 (157, 15 74 (15 9, 10 10 (9 71,

20 pM 0 73) 0 58) 3 16) 3 88) 2 37) 4 13) 173) 15 6) 10 49)

0 28 (0.12, 0 45 (0 50, 0 ‘79(0 99, 3 35 (3 47, 3 30 (4 21, 3 92 (4 71, 120 (107, 13 34 (12 2, 7 19 (7 35,

0 45) 0 41) 0 58) 3 22) 2 40) 3 14) 132) 14 5) 7.02)

8

20

1.0

-1100

-1000

-900

-800

-700

-600

-500

Polarographic reduction potential (mV) Fig 2 The relationship between the poiarographic reduction potentials and the mlcrosomal or cytosohe mtroreductase actnnhes The values along the abscissa are the polarographlc reduction potentials (- mV) and the values along the ordmate are the loganthmlc values of the mlcrosomal or cytosol~c mtroreductase activities All mtrated naphthalenes were used at 20 pM The microsomal mtroreductase actnnhes m the presence of superoxlde dismutase (0, T = 0 939), eytosohc mtroreductase actnnties usmg either NADPH (*, r = 0 951), NADH (0, r = 0 954) or hypoxanthme (m, T = 0 966) m the presence of superoxlde dzsmutase are shown The number of the mtrated naphthalenes and their angle-electron reduction potentials are shown m Table I

195

tion potentials measured by the electrochemical assays and hence there was a good relationship between the logarithm of nitroreductase activities and electrochemical reduction potentials. In microsomes, nitroreductase activities were also quite closely related to electrochemical reduction potentials. DISCUSSION

The present report has demonstrated that nitrated naphthalenes are reduced in both microsomes and cytosol. NADPH-cytochrome c reductase, DTdlaphorase and xanthine oxidase may be involved in the nitroreduction. Each showed very similar substrate specificities, with minor exceptions. The results obtained here agree well with those reported for 1-nitropyrene by Djurid et al. [13]. In general, the rate of reduction of nitrated naphthalenes was found to increase in the order of trinitro- > dinitro- > mononitro-naphthalene. In the case of microsomal nitroreduction, trinitronaphthalenes were reduced more readily than in cytosol. This may be due to not only differences in enzyme specificity but also the hydrophobic environment in microsomal membranes. Nitrated naphthalenes with a nitro group at a 3-position appear to be rather easily reduced. This selectivity may result from the differences in the electron densities of the respective nitro groups caused by the differences of orientation between a and 3-nitro substituents with respect to the naphthalene ring. Djuric et al. [13] indicated that reduced nitro- and nitrosopyrene intermediates formed by rat hver microsomes and cytosol could directly reduce succinoylated Cyt. c. Since the rates of succinoylated Cyt. c reduction in microsomal or cytosolic incubations with the nitrated pyrenes were not appreciably inhibited by superoxide dismutase, the reduced mtrated pyrenes directly reduced succinoylated Cyt. c [13]. However, as shown in Table II, the rates of succinoylated Cyt. c reduction in microsomal incubations with the mtrated naphthalenes were decreased by the addition of superoxlde dismutase. Therefore, in liver microsomes, succinoylated Cyt. c reduction may involve both direct reduction by reduced nitronaphthalene intermediates and the indirect reduction mediated by superoxide. Since the rates of succinoylated Cyt. c reduction in the cytosohc incubation with hypoxanthine were not appreciably inhibited by superoxide dismutase, in this system, the reduced nitronaphthalene intermediates were directly reducing the succmoylated Cyt. c. In general, DT-diaphorase activities towards menadlone or dichlorophenol indophenol using NADPH as a cofactor in normal rats were greater than with NADH [13], or similar [15]. However, greater nitroreductase activities towards the nitrated naphthalenes were observed m cytosot when NADH was used as a cofactor, in comparison to NADPH. The cofactor requirements of the cytosolic nitroreductase activitms towards dinitropyrenes are also similar to our results in this respect [13,15]. Hajos and Winston [15] have observed that, m the case of dinitropyrenes, the purified DT-diaphorase showed greater nitroreductase activities when NADH was used as a cofactor, m comparison to NADPH. Thus, the nitroreductase activity of DT-diaphorase appears to require NADH preferentially, differing from the case of quinone reduction.

196

Both NADPH-cytochrome c reductase and DT-diaphorase catalyze the reduction of not only nitro groups but also quinone to hydroquinone [31 - 33]. The initial rates of quinone enzymatic reduction by these enzymes were not related to the reduction potentials of varmus quinones including naphthoquinones [31,32], although nitroreduction activities towards the nitrated naphthalenes were closely related to the reduction potentials (Fig. 2). The extent of autoxidation of semiquinones and hydroquinones or their alkyl derivatives is controlled by the reduction potentmls of the hydroquinone/semiquinone and semiquinone/quinone couples, the nature of the substituents, solvent cage and solvation energy, pH and temperature [32]. Based on their autoxidation and structural features, the stabilities of nitro radicals may be different from those of semiquinone radicals. The toxic potencies of nitroarenes are known to be extremely dependent on the chemical structures. The results of our study clearly showed that, even among a series of closely related nitronaphthalenes, the reduction properties were drastically different depending on the structures and the reduction rates of these nitroarenes were also dependent on the enzyme-preparations and the cofactors. For nitroarenes, the metabolic reduction to nitrosoarenes is one of the most decisive steps in their biological activation, so the reduction properties described herein should be helpful in predicting the potential hazard arising from the huge quantities of nitroarenes detected as environmental pollutants from their structures. REFERENCES 1 H S. Rosenkranz and R. Mermelstem, Mutagemclty and genotoxmlty of mtroarenes All mtrocontmnlng chemmals were not created equal, Mutat. Res, 114 (1983) 217-267 2 F.A Beland, R H Heflmh, P C Howard and P P Fu, The ~n wtro metabohc activation of mtro polycychc aromatm hydrocarbons, in R G Harvey (Ed), Polycychc Hydrocarbons and Carclnogenems, American Chemmal Society, Washington, DC, 1985, pp 371-396 3 H ToMwa and Y Ohmshl, Mutagemclty and carcmogemclty of nltroarenes and their sources m the environment, CRC Cnt Rev. Toxmol, 17 (1986) 23-60 4 H ToMwa, R Nakagawa, K Honkawa and A Ohkubo, The nature of the mutagemclty and carclnogemclty of mtrated, aromatm compounds m the enwronment Enwron, Health Perspec, 73 (1987) 191-199 5 H ToMwa, R. Nakagawa and Y Ohmshl, Mutagemc assay of aromatm mtro compounds with Salmonella typh~mur~um, Mutat Res, 91 (1981) 321-325 6 W -Z Whong and G S Edwards, Genotoxlc actlwty of mtroaromatlc explomves and related compounds m Salmonella typh~mumum, Mutat Res, 136 (1984) 209-215 7 E C McCoy, E J Rosenkranz, L A Petrullo, H S Rosenkranz and R Mermelstem, Structural

8 9 10

11

basra of the mutagemclty m bacteria of mtrated naphthalene and derivatives, Enwron Mutagen, 3 (1981) 499-511 D E Rmkert, Metabohsm of mtroaromatm compounds, Drug Metab Rev, 18 (1987) 23-53 P P Fu, Metabohsm of mtro-polycychc aromatm hydrocarbons, Drug Metab Rev, 22 (1990) 209 - 268 R W Hart, P P Fu and A Turturro, Nltro-polycychc aromatm hydrocarbons structural features, genotomclty and risk evaluatmn, m P Pohtzer and F J Martm, Jr (Eds), Chemmal Carcinogens, Elsewer, Amsterdam, 1988, pp 264-290 Z Djtmc, Metabohsm and DNA binding of mtropyrenes, Rev. Bmchem Tomcol, 10 (1989) 1-40

197 12 13 14

15

16 17 18 19

20 21

22 23 24 25

26 27 28

29

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R Kato, A. Takahashi and T Oshlma, Characteristics of mtro reduction of the carcmogemc agent, 4mtroqumolone N-oxide, Blochem Pharmacol , 19 (1970) 45 - 55 Z Dmnc, D W Potter, R.H Hefhch and F A. Beland, Aerobm and anaerobic reduction of nitrated pyrenes m vitro, Chem -Biol Interact., 59 (1986) 309-324 Z. D~unc, E K Flfer, Y Yamszoe and F A Beland, DNA bmdmg by 1-mtropyrene and 1, 6dmitropyrene zn vctro and tn tnwo effects of mtroreductase mduchon, Carcmogenesis, 9 (1988) 357-364 A K D HaJos and G.W Wmston, Dmitropyrene mtroreductase activity of punfied NAD(P)Hqumone oxidoreductase role m rat hver cytosol and mduchon by Aroclor-1254 pretreatment, Carcmogenesm, 12 (1991) 697 - 702 N Harada and T Omura, Participation of cytochrome P-450 m the reduction of mtro compounds by rat liver mlcrosomes, J Blochem , 87 (1980) 1539 - 1554 K Tatsuml, S Krtamura and N Narsu, Reductive metabohsm of aromatic mtro compounds mcludmg carcmogens by rabbit hver preparations, Cancer Res , 46 (1986) 1089- 1093 S L Bauer and P C Howard, The kmetlcs of 1-mtropyrene and 3mtrofluoranthene metabohsm using bovme hver xanthme oxldase, Cancer Lett , 54 (1990) 37-42 K Fukuhara, N Mlyata, M Matsm, K Matsm, M Ishidate, Jr and S Kamiya, Synthesis, chemical properties and mutagemclty of 1,6- and 3,6-dmitrobenzo[a]pyrenes, Chem Pharm Bull , 38 (1990) 3158-3161 N Sera, M Kai, K Honkawa, K Fukuhara, N Mlyata and H Tohwa, Detection of 3,6dmitrobenzo[a]pyrene m airborne particulates, Mutat Res , 263 (1991) 27-32 K.L Dooley, F A Beland, T J Bucel and F F Kadlubar, Local carcmogematy, rates of ahsorptlon, extent and persistence of macromolecular bmdmg, and acute histopathologmal effects of N-hydroxy-1-naphthylamme and N-hydroxyl-2naphthylamme, Cancer Res , 44 (1984) 1172- 1177 E R Ward and C.D Johnson, Polymtronaphthalenes Part III A quantitative study of the further nitration of the ten dmltronaphthalenes, J Chem Sot , (1961) 4814-4321 H Kuthan, V Ullnch and R W Estabrook, A quantitative test for superoxide radicals produced m biologmai systems, Blochem J , 203 (1982) 551- 558 B F Van Gelder and E C Slater, The extmction coefficient of cytochrome c, Blochim Blophys Acta, 58 (1962) 593 - 595 K Fukuhara, A Hakura, N Sera, H Tokiwa and N Mlyata, l- and 3-Nltro-6-azabenzo[o]pyrenes and their N-oxides highly mutagenic mtrated azarenes, Chem Res Toxic01 , 5 (1992) 149 - 153. M J.S. Dewar and W Thlel, Ground states of molecules 38 The MNDO method Approxlmations and Parameter, J Am Chem Sot , 99 (1977) 4899 - 4907 A Bovens, N Oshmo and B Chance, The cellular production of hydrogen peroxide, Blochem J , 128 (1972) 617- 630 S.D. Aust, D.L Roeng and T.C Pederson, Evidence for superoxlde generation by NADPHcytochrome c reductase of rat hver mmrosomes, Bioehem Biophys Res Acta, 47 (1972) 1133- 1137 J M. McCord and I Fndovmh, Superoxide dismutase An enzymatic function for erythrocuprem (hemocuprem), J Blol Chem , 244 (1969) 6049-6055 I Fndovmh, Superoxlde dismutases, Ann Rev Blochem , 45 (1975) 147- 159 G. Powis and P.L. Appel, RelatIonshIp of the single-electron reduction potential of qumones to their reduction by flavoprotems, Blochem Pharmacol , 29 (1980) 2567- 2572 G D Bufflnton, K C)lhnger, A Brunmark and E Cadenas, DT-dlaphorase-catalysed reductron of 1,4-naphthoqumone derivatives and glutathlonyl-qumone conJugates Effect of substltuents on a&oxidation rates. Blochem J , 257 (1989) 561- 571 K Glhnger, G.D. Buffinton, L Ernster and E. Cadenas, Effect of superomde dismutase on the autoxldation of substituted hydro- and semi-naphthoqumones, Chem -Biol Interact, 73 (1990) 53-76