Peroxidase catalysed oxygen activation by arylamine carcinogens and phenol

185

Chem.-Biol. Zntemctions, 56 (1985) 185-199 Elsevier Scientific Publishers Ireland Ltd.

PEROXIDASE CAR(:INOGENS

VANGALA

CATALYSED OXYGEN AND PHENOL

V. SUBRAHMANYAM

Department of Biochemistry, Newfoundland (Canada)

ACTIVATION

RY ARYLAMINE

and PETER J. O’BRIEN Memorial

University

of

Newfoundland,

St.

John’s,

(Received June 6th. 1985) (Revision received August 26th. 1985) (Accepted September 4th. 1985)

SUMMARY

Peroxidase catalysed the formation of active oxygen in the presence of NADH or GSH and traces of HzOz and arylamine or phenolic substrates. Some oxygen activation occurred with some arylamines even in the absence of NADH or GSH. Oxygen consumption was proportional to the N,ADH oxidized or GSSG formed. Approximately 0.80 and 0.40 mol of oxygen were consumed per mole of NADH or GSH oxidized respectively. The requirement for trace amounts of hydrogen peroxide and arylamine or phenolic substrates suggest that redox cycling resulted in H,Oz formation. It is proposed that initially formed phenoxy radicals or arylamine cation radicals oxidize NADH or GSH to radicals which react with oxygen to form superoxide radicals and H’LO2

Key wom!s: Peroxidase - Arylamine Active oxygen - Glutathione - Phenol.

carcinogen

- Hydrogen

peroxide

-

INTRODUCTION

Reduced oxygen species, like the superoxide radical (0,) hydrogen peroxide (H&J and hydroxyl radical (OH’) are believed to be toxic to living organisms (reviewed in Ref. 1). A number of oxidases present in microsomes or mitochondria reduce molecular oxygen to Hz02 via 0,. In turn ferrous salts can reduce H202 to yield highly reactive OH’. Increased pOa has shown to result in increased H202 production in several bacteria and submil.ochondrial 0009-2797/85/$03.30 @ 1985 Elsevier Scientific Publishers Printed and Published in Ireland

Ireland Ltd

186 particles. Alternatively. cytotoxicity by oxygen intermediates may also be caused by oxidative stress as a result of intracellular species which can undergo cyclic one-electron oxidation-reduction reactions catalysed by reductases or peroxidases to radical species which can interact with oxygen to form activated oxygen species. Both species may attack macromolecules or lipids and change membrane permeability [2]. In the process other reactive radicals are generated. The metabolic activation of chemical carcinogens is widely believed to occur by two-electron oxidation to products which are subsequently converted to electrophiles. (:ovalent interaction of these electrophiles with a cellular macromolecule is believed to be the basis for the initiation of carcinogenicity [3-51. However, in recent years evidence has also accumulated for metabolic activation of chemical carcinogens by a oneelectron oxidation to free radicals which bind to macromolecules [6,7]. The peroxidase activity of prostaglandin(H)synthase activiates a wide variety of xenobiotics [6,8] and has been implicated in arylamine and nitrofuran derivatives induced bladder cancer [g-13] and in phenacetin induced kidney necrosis [14]. Horseradish peroxidase, a model peroxidase used in many studies, has also been shown to activate a wide variety of xenobiotics by a one-electron oxidation process [8,10 and references cited therein]. Rat mammary cell peroxidase, mouse uterine peroxidase and rat bone marrow peroxidase can also activate N-hydroxy-2-acetylaminofluorene [ 15,191, diethylstilbestrol [ 161 and phenol [ 171, respectively. Peroxidases have been implicated in diethylstilbestrol induced uterine toxicity [IS], benzidine induced Harderian gland tumors [6], trarzs-4aminostilbene induced Zymbal gland tumours [20] and benzene induced bone marrow toxicity and leukemia [17,18]. Recently it has been suggested that target organs for chemical carcinogenesis are the organs containing high levels of peroxidases [6,13,21]. The Zymbal gland, a sebaceous gland located in the external ear duct of rats is a common target tissue for the carcinogen trczns-4-aminostilbene [22], acetylaminofluorene [23], monomethylaminoazobenzene [24] and benzene [25]. The Harderian gland, a large gland located beneath the nictating membrane of eyes, is a target organ for benzidine derivatives in mice [26]. Lactoperoxidase is present in high levels in these glands [20,27], but they contain very low mixed-function oxidase levels [28]. The uterus, a target organ for diethylstilbestrol induced carcinogenesis, contains peroxidase [29] and prostaglandin synthase [30], but no mixed-function oxidase [29]. The salivary gland and thyroid gland are also target organs in human cancer and contain high concentrations of peroxidases [31] and little mixed-function oxidase [32]. We recently reported that peroxidases in intact cells can activate a variety of carcinogens to products that bind irreversibly to nuclear DNA [21] and that endogenous H202 is required for the activation. In this paper, we present evidence that peroxidase can mediate oxygen activation in the presence of chemical carcinogens.

187 MATERIALS

AND METHODS

2-Aminofluorene, 1-naphthylamine, 2-naphthylamine, 4-aminobiphenyl, N,N’-dimethyl4-aminoazobenzene N, N’-dimethyl-p-toluidine (DMPT), (DAB). N-methyl-4-aminoazobenzene (MAB), aminoazobenzene (AB). glutathione (GSH), NADPH, NADH and horseradish peroxidase (HRP) type VI, lactoperoxidase (LP), superoxide dismutase (SOD) type I, were obtained from Sigma Chemical Company (St. Louis, MO, U.S.A.). Phenol was obtained from Baker Chemical Company. Oxygen consumption by peroridase Oxygen consumption was measured by a Clarke type electrode at 20°C. The standard reaction mixtures contained in 2 ml of 0.1 M Tris-HCl. 1.0 mM EDTA buffer (pH 7.4) phenol (100 PM) or arylamine substrate (10 FM), HRP (10 pg), NADH (200 PM) or GSH (400 PM). Reactions were started by the addition of H,Oz (10 PM). Measurement of NADH oxidation The reaction mixtures, in 2 ml of 0.1 M Tris-HCl, 1.0 mM EDTA buffer (pH 7.4) contained phenol (100 PM) or arylamine substrate (10 PM), HRP (10 pg), NADH (200 PM). Reactions were started by the addition of Hz02 (10 PM) and the disappearance of NADH with time was followed at 340 nm using the Shimadzu UV-240 spectrophotometer until the NADH oxidation was complete (usually l-2 min). Measurement of mte constants Reaction mixtures (2 ml) of 0.1 M Tris-HCI, 1.0 mM EDTA buffer (pH 7.4) contained (unless otherwise stated) the following: NADH (100 FM), arylamine or phenolic substrate (10 PM), HRP (1 pg) or LP (10 pg). Reactions were started by the addition of H202 (10 PM) and the rate of NADH oxidation was followed at 340 nm. Measurement of GSSG formation The 2 ml reaction mixtures contained, in 0.1 M Tris-HW1.0 mM EDTA buffer (pH 7.4), phenol (100 PM) or arylamine substrate (10 PM), HRP (10 pg), GSH (400 PM). The reactions, were started by the addition of Hz02 (10 PM). After 10 min, NADPH (200 PM) was added to each reaction mixture and the amount of NADPH oxidized following the addition of glutathione reductase (1 unit) was determined [33]. Reduction of ferricytochrome C The 2 ml reaction mixtures of 0.1 M Tris-HCl buffer (pH 7.4) contained NADH (200 FM), HRP (1 pg), ferricytochrome C (20 vM) and phenol. Reactions were started by the addition of H,On (10 PM). Reductions of ferricytochrome C was monitored by the increase in absorbance at 550 nm 1341.

188 The concentrations of peroxidase, H202 and ferricytochrome determined by using the extinction coefficients reported previously

C were [35-371.

RESULTS

Table I shows that the oxidation of the arylamines 1-naphthylamine, 2-naphthylamine or DMPT by HRP and H202 resulted in rapid oxygen uptake. However, much more rapid oxygen uptake occurred when NADH or GSH are included in the reaction mixture. A similar oxygen uptake occurred if NADPH was used (results not shown). Little oxygen consumption was observed in the absence of one of HRP, H202 or arylamine. 2Aminofluorene, Caminobiphenyl or methylaminoazobenzene or phenol resulted in only little oxygen consumption in the absence of NADH or GSH under the conditions studied but resulted in extensive oxygen consumption in the presence of NADH or GSH. p,p’-Biphenol, hydroquinone, catechol, mesidine or aniline were not active even in the presence of NADH or GSH. Figure 1 shows the effect of varying HzOz concentration (Fig. 1A) or SOD (Fig. 1B) on the total oxygen consumption catalyzed by peroxidase in a

TABLE I PEROXIDASE PHENOL

MEDIATED

OXYGEN

ACTIVATION

BY ARYLAMINE

CARCINOGENS

AND

Incubation conditions: The reaction mixtures contained in 2 ml of 0.1 M Tris-HCI, 1.0 mM EDTA buffer (pH 7.4), phenol or arylamine substrate (concentrations as indicated), HRP (10 pg), NADH (200 PM) or GSH (400 FM). Reactions were started by the addition of H,O, (10 FM) and followed until complete (usually l-2 min). See Materials and Methods for the measurement of oxygen consumption. Mean + S.E.M. for 3 experiments are given. Substrate

None 2-Naphthylamine (0.01 mM) 1-Naphthylamine (0.01 mM) I-Aminobiphenyl(O.01 mM) MAB (0.01 mM) DMPT (0.01 mM) 2-Aminofluorene (0.01 mM) Mesidine (0.1 mM) Aniline (0.1 mM) Phenol (0.1 mM) o,o’-Biphenol (0.1mM) p,p'-Biphenol (0.1mM) Catechol (0.1 mM) Hydroquinone (0.1 mM)

Oxygen

consumed

(PM)

With NADH

With GSH

Minus NADH/GSH

0.5* 142.8 f 32.4zk 140.8 f 106.6 f 108.4 f 62.4+ 0.5* 0.5* 114.2 f 105.2 f 0.5* 0.5* 0.5*

0.5* 137.1 f 30.8zt 137.a* 76.2 f 86.5zt 69.8* 0.5* 0.5* 119.1 f 112.1 f 0.5* 0.5* 0.5+

0.5 f 0.5 16.5 f 3.5 28.8 f 4.4 11.5*4.4 8.5 k4.5 34.6 it 6.6 5.5 f 5.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5 f 0.5 0.5*0.5 0.5*0.5 0.5 * 0.5

0.5 14.6 4.8 16.4 10.2 10.6 8.2 0.5 0.5 14.4 15.4 0.5 0.5 0.5

0.5 15.7 5.2 14.1 12.2 13.4 5.2 0.5 0.5 13.2 14.1 0.5 0.5 0.5

189

io

;0

0

CONCN.

1 L

0

40

So

so

H,O,I)lMI

40

, 0.25

, 0.50

CONCN.

0

I

0.75

i! s ii

1.00

SoDoJM’

Fig. 1. Oxidation of NADH and GSH by HRP-H,O, in the presence of phenol and 2aminofluorene. The total oxygen consumed, NADH oxidized or GSSG formed (from 2 mol GSH) in the reaction mixture was measured as described in Materials and Methods. A: dependence on H,O, concentration. 2 ml reaction mixtures of 0.1 M Tris-HCI, 1.0 mM EDTA buffer (pH 7.4) contained phenol (200 PM) or 2-aminofluorene (20 PM), HRP (1 pg) and NADH (200 FM) or GSH (400 PM). Reactions were started by the addition of H,Oy. , in the presence of phenol; x x x, in the presence of 2-aminofluorene. B: dependence on SOD concentration. 2 ml reaction mixtures of 0.1 M Tris-HCI. 1.0 mM EDTA buffer (pH 7.4) contained aminofluorene (20 FM), HRP (1 pg) and NADH (200 PM) or GSH (400 *M), SOD concentrations as indicated in the figure.

190 reaction mixture containing phenol or 2-aminofluorene. NADH, H202 and HRP. Oxygen consumption was proportional to the NADH oxidized. Approximately 0.80 mol oxygen were consumed for the oxidation of 1 mol NADH. Similar results were obtained when GSH was used instead of NADH. GSSG was formed in stoichiometric amounts from the GSH and approx. 0.40mol oxygen were consumed for the oxidation of 1 mol GSH. The disappearance of NADH with time in the reaction mixture was followed at 340 nm. In the absence of arylamines or phenols, the oxidation of NADH by the H202-HRP reaction mixture was very slow at this pH. However, in the presence of trace amounts of arylamines or phenols, the oxidation of NADH was rapid. Figure 1B shows the effect of SOD on the amount of NADH oxidized or GSSG formed in relation to oxygen consumed. As the figure shows, addition of 1 FM SOD resulted in the complete oxidation of NADH and a 2-fold increase in the accompanying oxygen uptake. The addition of SOD also resulted in the complete oxidation of GSH and a 2-fold increase in the accompanying oxygen uptake. As shown in Fig. 2, phenol enhanced the initial reduction of ferricytochrome C in the H20,-HRP-NADH system. Increasing the concentration of phenol increased the initial rate of reduction of ferricytochrome C. Above 50 PM concentration of phenol, the rate was too fast to measure. No reduction of ferricytochrome C occurred with phenol alone in the absence of Hz02. The enhancement by phenol of the reduction rate was completely prevented by superoxide dismutase (1 PM) suggesting that superoxide was responsible for cytochrome C reduction. The extent of superoxide formation by the peroxidase-Hz02-thyroxine system was previously estimated from the inhibition of O2 uptake by ferricytochrome C and its prevention by superoxide dismutase [341. Figure 3

i,

/i

0.032

2 ul

3

0.024

d1:

0.016

0

10 CONCN.

20

20 PHENOL

i

40

a

(JJM)

Fig. 2. Reduction of ferricytochrome C during phenol mediated NADH oxidation See Materials and Methods for reaction conditions.

by HRP-H202.

191 I

A Pkn0l,NADH,Cyf

c

Phenol,NADH.Cyf

I

C. SOD

I

olmMCytc+OlpMSOD

L 0.02

mM

Cvf.C

05,uMS00

OlmMC”tc+

-

0 IBM

Cvt

c + l.O,uM SOD

Fig. 3. A: effect of ferricytochrome C and SOD on oxygen consumption by arylamine or phenol-NADH-HRP-H,O, system. 2 ml of 0.1 M Tris-HCI, l.OmM EDTA buffer (pH 7.4) contained NADH (200 PM), phenol (400 PM), HRP (10 pg) and ferricytochrome C (concentrations as indicated), Reactions were started by the addition of H,O, (100 FM). B: effect of SOD on inhibition by ferricytochrome C. Reaction conditions are as described in Fig. 3A.

shows the oxygen consumption by phenol in the HRP-HzOz-NADH system. A higher H202 concentration was also used so as to oxidize all the NADH in the system. Ferricytochrome C inhibited oxygen consumption indicating that superoxy radical formation was required for oxygen consumption [34]. Approximately 0.15mM ferricytochrome C was required to inhibit the oxygen consumption completely. However, addition of only 0.02 mM ferricytochrome C resulted in a 50% inhibition of oxygen consumption, probably because reduced cytochrome C can also be oxidized by superoxy radicals back to oxidized cytochrome C (ferricytochrome C) [34]. Super-oxide dismutase (1 PM) prevented the inhibition by ferricytochrome C. Similar results were obtained when phenol was replaced by aminofluorene or other arylamine substrates. Ferricytochrome C, however, did not affect the amount of NADH oxidized in this system, indicating that superoxy radicals do not oxidize NADH. Figure 4 shows that the rate of NADH oxidation is first order with respect to phenol concentration when fixed concentrations of other reactants were used. The rate can be calculated from -

d[NADH]

dt

J

= &[Ph] [NADH] = kIIPh]

0

50 CONCN.

150 100 PHENOL

200 t)Ihdj

Fig. 4. Apparent first order rate constants (k,) were plotted against concentrations of phenol. 2 ml reaction mixtures Of O.lM Tris-HCl, l.OmM EDTA buffer (pH 7.4) contained, phenol (concentrations as indicated in the figure), HRP (1 pg), NADH (200 PM). The reactions were started by the addition of H,O, (20 PM).

where &[NADH] = k, is the apparent first order rate constant (min-‘) in which [Ph] is the initial concentration of phenol. The rate constants, however, were independent of H202 concentration provided the HzOz concentration was above 30 PM. Below these concentrations, NADH oxidation was not complete (see Fig. 1A). However, the initial rates for NADH oxidation were not affected. NADH oxidation measured with other compounds were also apparently first order reactions. Table II shows the apparent first order rate constants for NADH oxidation for the compounds tested under the conditions used for following oxygen uptake. It can be seen that compounds such as aniline, mesidine, catechol and p,p’-biphenol which did not activate oxygen were highly effective at catalysing NADH oxidation. However, hydroquinone did not catalyse NADH oxidation indicating that the semi-quinone or quinone products did not oxidize NADH. 2-Naphthylamine was found to be the most active compound followed by 2-aminofluorene, 1-naphthylamine and 4-aminobiphenyl. At these peroxidase concentrations, the rate of NADH oxidation was dependent on peroxidase concentrations indicating that the rate of peroxidase-HzOz catalyzed oxidation of the arylamine or phenolic compounds was rate limiting. In the case of p,p’-biphenol, the rate of NADH oxidation was not affected over the range of 0.1-500 pg peroxidase concentrations. p,pBiphenol is the most effective peroxidase donor known [18] and therefore the rate of p,p’-biphenol oxidation was not rate limiting. Table II also shows the rate constants for NADH oxidation at high peroxidase concentrations. Under these conditions, the rate was independent of peroxidase concentrations and therefore reflects the ability of the various arylamine cation radicals in oxidizing NADH.

193 TABLE II FIRST-ORDER RATE CONSTANTS OF NADH BY HRP-HxO,

IN ARYLAMINE

AND PHENOL MEDIATED

OXIDATION

All the substrates used were of 10 pM concentrations except for p,p’-biphenol which was used at a concentration of 5 FM. Mean * S.E.M. for 3 experiments are given. N.M., too fast to measure. Substrate

None 2-Aminofluorene 1-Naphthylamine P-Naphthylamine 4-Aminobiphenyl DMPT MAB AB Mesidine Aniline Phenol o,o’-Biphenol p,p’-Biphenol Hydroquinone Catechol “Reaction mixtures (100 PM) substrate of H20n (10 *M). “Reaction mixtures were started by the

k,(apparent)

k, X min

X min

HRP”

Lp”

HRPh

0.00 0.32 kO.08 0.1 *0.02 0.8 50.2 0.14 zto.04 0.10 *to.03 0.06 kO.02 0.04 *to.01 0.08 50.02 0.04 *0.01 0.02 *0.01 0.005 * 0.001 0.03 *0.01 0.00 0.18*0.05

0.00 0.44*0.10 0.28 * 0.06 0.8 kO.2 0.12 f 0.03 0.12*0.03 0.04 * 0.01 0.03 f 0.01 0.16*0.03 0.10*0.02 0.42 f 9.08 0.08+0.02 0.06 f 0.02 0.00 0.95*0.10

0.00 N.M. N.M. N.M. N.M. 0.42 t 0.06 0.34*0.05 0.31 f 0.04 0.14*0.03 0.10 f 0.02 0.03 f 0.01 0.03 f 0.01 0.04 f 0.01 0.00 0.04 i 0.01

of 2 mIO.l M Tris-HCl, l.OmM EDTA buffer (pH 7.4) contained NADH (10 PM). HRP (1 pg), or LP (10 pg). Reactions were started by the addition contained NADH (50 PM), addition of H,O, (50 PM).

substrate

(0.5 PM),

HRP (100 pg). Reactions

DISCUSSION

Figure 1 showed that trace amounts of phenol or arylamines can catalyse the oxidation of NADH in a reaction mixture containing peroxidase and H202. Other investigators have shown that some phenols can act as catalysts in the oxidation of NADH by hydrogen peroxide and peroxidase (341. The mechanism is believed to be due to the initial oxidation of phenol to phenoxy radicals by peroxidase-H202 and the reaction of phenoxy radicals with NADH to form NAD radicals. The reaction of NAD radicals with oxygen forms 0; [34]. Our results showed that 0.80 mol oxygen were consumed per mole of NADH oxidized which is close to the value of 0.83 mol oxygen consumed per mole of NADH oxidation reported by Takayama and Nakano [34] where thyroxine was used as catalyst. They also reported that 54% of the total flux of electrons from NADH to oxygen resulted in superoxy radical production. The mechanism in the presence of GSH also appears to be similar except that 0.40 mol oxygen were consumed per mole of GSH oxidized.

194 Figure 1 showed that a small amount of H202 in the presence of peroxidase and a small amount of arylamine or phenolic donor catalyzed the oxidation of a large excess of NADH and GSH. Since superoxide radicals do not oxidize NADH [34], they presumably dismutate to H202 thus allowing the reaction to proceed. The addition of superoxide dismutate to the reaction mixture resulted in a twofold increase in the total NADH or GSH oxidized. This is probably the result of the increased H202 levels. Superoxy dismutase also prevents the inactivation of the peroxidase as a result of compound III formation by superoxy radicals [40]. A mechanism for the oxygen activation can be described by the following equation: 2ArNHz + H,O, -- 2ArNH + 2HzO

(1)

With some arylamines, the radical formed is further oxidized by oxygen and superoxide radicals are formed. Oxygen uptake was previously reported for the peroxidase catalysed oxidation of DMPT [41]. Superoxide radicals are also formed during the peroxidase catalysed oxidation of halogenated N, N’dimethylanilines [42]. In the presence of NADH ArNH + NADH -

ArNHz + NAD’

(2)

This redox cycling by the arylamine would explain why only catalytic amounts of arylamine were required for NADH oxidation. Wilson [43] has reported a second order rate constant of 1.9 x 10’ M-’ s-’ for the following reaction: NAD+002+H++NAD++O;

(3)

This would also explain the observed stoichiometry of 0.8 mol O2 consumed per mole of NADH. Furthermore, the requirement for only catalytic amounts of HzOz could be explained if the HzOz formed by dismutation of the superoxide radicals (reaction 4) participated in reaction 1. 20;~HzO,+Oz

(4)

A different mechanism for superoxy radical formation in the presence of GSH is likely as the thiyl radicals react with 0, to form peroxy sulfenyl radicals which react further to form higher oxidation states of GSH including GSSG [44]. ArNH + GS + ArNHz + GS

(5)

The thiyl radicals formed can react with GS to form autoxidisable disulfide radical anions [45,46]. Other investigators have found a cysteine oxidase

activity for peroxidase [47] and a similar mechanism for the Hz02 formation has been proposed [45]. Barton and Packer [48] have reported second order rate constants of 3 x 10gM~’ s1 and 4 x lo* M-‘s-l respectively for the following reactions. However Quintiliani [49] has reported 6.6 x 10” M-’ s-l and 1.6 x 10’ M-’ s-l respectively for these reactions.

GS’ + GS- s GSSG’ GSSG1

(6)

+ O2 -+ GSSG + 0;

(7)

This would also explain the observed stoichiometry of 0.4 mol O2 consumed per mole GSH as 2 mol GSH are required to form 1 mol of GSSG. Autoxidizing thiols have been shown to be mutagenic [50] and cytotoxic to isolated rat hepatocytes [46,51]. Hydroxyl radicals were formed [51] and the following reaction was suggested: RSSR’

+ H,O,+

OH. + RSSR + OH-

(8)

Hydroxyl radicals readily cause cytotoxicity [52], DNA single strand breaks [53], chromosome aberrations (541 and have been implicated in tumor promotion [55]. Stier et al. [56] have proposed that the redox cycling of stable nitroxide radicals formed from carcinogenic arylamines may be of importance in arylamine induced carcinogenesis. Nakayama et al. [57] recently reported that superoxy radicals were formed during the autoxidation of N-hydroxy and nitroxy radical metabolites of carcinogenic arylamines including l- and 2-naphthylamine. The results presented here show that superoxy radicals are also formed during the autoxidation of the cation radical of l- and 2naphthylamine (Table I). At low catalytic concentrations, the non-carcinogenic arylamines mesidine, aniline, catechol or p,p’-biphenol were ineffective in catalysing oxygen activation although they were effective in mediating the oxidation of NADH. With these compounds, the following very rapid reaction could prevent NAD’ accumulation and the above chain reaction but result in NADH oxidation by redox cycling: NAD’ + ArNH’ --, NAD’

+ ArNH,

(9)

However, the quinone products of catechol and p,p-biphenol rapidly oxidize During the p,p’-biphenol-peroxidase-NADH-HZOZ catalysed NADH. the p, p’-biphenol existed as p, p’-biphenoquinone. p, p’oxidation, Biphenoquinone also rapidly oxidized NADH and p,p’-biphenol was formed (results not shown). Furthermore quinones oxidize NADH by hydride transfer rather than electron transfer [58]. It is therefore unlikely that radicals are responsible for the NADH oxidation. The lack of oxygen activation by hydroquinone could be attributed to the inability of benzoquinone to oxidize NADH and its ability to conjugate GSH [611.

196 Recently oxygen uptake and thiyl radical formation has been reported with peroxidase, GSH and higher non-catalytic concentrations of Hz02 and p-phenetidine [59]. This oxygen uptake was ascribed to the following reactions: GS‘ + 0, + GSOi

(IO)

2GSO; + Hz0 -+ GSOzH + GSOBH

(11)

The lack of super-oxide radical and HzOz formation would then explain why little oxygen uptake occurs at catalytic concentrations of phenetidine and H202. The mechanism of benzene induced haematopoietic toxicity and leukemia is thought to involve initial hydroxylation to phenol by the liver cytochrome P-450 monooxygenase system [60]. Further hydroxylation of phenol either at the ortho or para position by the monooxygenase system results in the formation of catechol and hydroquinone respectively [61]. The semiquinone and/or benzoquinone oxidation products bind to protein and glutathione ]61,62]. However, phenol did not bind to DNA following activation by liver microsomes and NADPH [ 181. Sawahata and Neal reported that bone marrow peroxidase can activate phenol to form biphenols [17] and it was proposed that p,p’-biphenoquinone is a reactive species involved in benzene toxicity does not bind to DNA although [ 171. However, p, p’-biphenoquinone extensive phenol binding to DNA occurred following a peroxidase oxidation [ 181. Instead bone marrow peroxidase could exert a toxic effect by mediating a one-electron oxidation of phenol or o,o-biphenol to radicals which react with NADH or glutathione to form superoxide radicals and HzOz. Plasma membranes of a variety of cells e.g. liver, erythrocytes and HeLa cells and the plant cell wall contain superoxide forming NAD(P)H oxidases [63]. Neutrophils also possess a transmembrane NAD(P)H oxidase which is believed to form HzOz for the antibacterial function of this cell [21]. A thiol (GSH) oxidase is also associated with the plasma membrane of renal tubular epithelial cells and intestinal epithelial cells [64]. In view of the role superoxide radicals may play in cytotoxicity [52] or carcinogenesis [55]. activation of these oxidase systems by catalytic concentrations of carcinogenic arylamines or phenol could have biological consequences. ACKNOWLEDGEMENTS

This research was supported by the National Cancer Institute of Canada and the National Research Council of Canada. We are thankful to Dr. Anver Rahimtula for carefully reading the manuscript and for helpful suggestions. REFERENCES 1 J. DiGuiseppi and I. Fridovich, The toxicology of molecular oxygen, CRC, Crit. Rev. Toxicol., 12 (1984) 315-342. 2 P.J. O’Brien, Peroxide mediated metabolic activation of carcinogens in: K. Yagi (Ed.), Peroxides in Biology and Medicine, Academic Press, New York, 1982, pp. 317-338.

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