Redox cycling of aromatic hydrocarbon quinones catalysed by digestive gland microsomes of the common mussel (Mytilus edulis L.)

AIJUATIC

TllHICOLU6Y Aquatic Toxicology 38 (1997) 83-99

Redox cycling of aromatic hydrocarbon quinones catalysed by digestive gland microsomes of the common mussel (Mytilus edulis L.) Anders

M. Sjiilin”, David

R. Livingstoneb%*

“Kristineberg Marine Station, Pl.2130. S-450 34 Fiskebiickskil, Sweden hNERC Plymouth Marine Laboratory, Citadel Hill, Plymouth PLl ZPB, UK

Accepted 30 September 1996

Abstract The NAD(P)H-dependent redox cycling of six aromatic hydrocarbon quinones (1,4-benzoquinone, tetramethyl-1,4-benzoquinone (duroquinone), 1,2- and 1,4_naphthoquinones, 9, lophenanthrenequinone, anthraquinone) by digestive gland microsomes of M. edulis was assessed in terms of oxygen consumption (Clark electrode) and reactive oxygen species (ROS) production (conversion of superoxide anion radical, 02- and hydrogen peroxide, HZOZ to hydroxyl radical, -OH-the latter detected by oxidation of 2-keto-4-methiolbutyric acid (KMBA) to ethylene): additionally, oxygen consumption was determined for the known ROS-generating 2-methyl-1,4-naphthoquinone (menadione) and benzo[a]pyrene diones (1,6-, 3,6- and 6,12-). Stimulated oxygen consumption was detectable for duroquinone, naphthoquinones, menadione and phenanthrenequinone only, whereas stimulated ROS production was evident for all ten quinones, indicating that the latter is a more sensitive measure of redox cycling. ROS production was greatest for 1,2-naphthoquinone and least for anthraquinone. Stimulated ROS production and/or oxygen consumption for the six aromatic hydrocarbon quinones was greater for the NADH- than the NADPH-dependent reactions. MiChaelissMenten kinetics with respect to quinone concentration were evident for both stimulated oxygen consumption and ROS production, and correlations between the two measurements provide strong evidence for the process of redox cycling (i.e. conversion of 02 to Oz-) occurring in digestive gland microsomes. Differences were also seen between the profiles for the two measurements, particularly for 1,4-naphthoquinone, indicative of other processes of oxygen consumption and/or ROS production occurring. Comparison of different quinones indicates that enzyme specificity and quinone structure are factors in determining ROS production. Overall, the results indicate a wide pro-oxidant potential for quinones formed from aromatic hydrocarbons by biotransformation and photo-oxidation processes. *Corresponding author Abbreviations: AH, aromatic hydrocarbon; keto-4-methiolbutyric acid

BaP, benzo[a]pyrene; DMF, dimethylformamide;

0166-445X/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIISO166-445X(96)00836-3

KMBA, 2-

84

A.M. $jdin,

D. R. LivingstonelAquutic

Touicologq

Keywords: Myths edulis; Digestive gland microsomes; species; Aromatic hydrocarbon quinones

38 (1997) X3-99

Redox

cycling;

Reactive

oxygen

1. Introduction Aromatic hydrocarbons (AHs) are widespread in the aquatic environment and are readily bioaccumulated in the tissues, particularly the digestive gland, of mussels and other molluscs (Burns and Smith, 1981; Widdows and Donkin, 1992). Sources of input include oil terminals, oil spills (Sol& et al., 1996), boating activities and industry (Livingstone, 1991). Potential deleterious effects of AH exposure in Mytilus sp. have been observed at various levels of biological organisation, including putative induction of the detoxication-toxication cytochrome P450 monooxygenase system (Livingstone et al., 1985; Livingstone, 1988; Michel et al., 1993b; SolC et al., 1996), DNA-adduct formation (Marsh et al., 1992; Venier and Canova, 1996), lipid peroxidation (Livingstone et al., 1990; SolC et al., 1996), decreased lysosomal stability (Lowe et al., 1995), altered immunocompetence (Coles et al., 1994), impaired reproduction (Lowe and Pipe, 1986) and reduced scope for growth (Widdows and Donkin, 1992). Additionally, although previous analysis of field studies failed to discern a relationship between AH exposure and cancer in bivalves (Mix, 1986), more recently a chemical aetiology for tumour formation was indicated for the oyster Crassostrea virginica exposed to a mixture of chemicals, including polycyclic aromatic hydrocarbons (PAHs) (Gardner et al., 1992). Production of reactive oxygen species (ROS), such as the superoxide anion radical (OZ-), hydrogen peroxide (HaOZ) and the highly reactive hydroxyl radical (*OH), is indicated to be a mechanism of pollutant-mediated toxicity in aquatic organisms (Winston and Di Giulio, 1991; Lemaire and Livingstone, 1993), including molluscs (Livingstone et al., 1990). Possible mechanisms leading to increased ROS production include redox cycling of contaminants such as quinones (see below) and nitroaromatics (Garcia Martinez et al., 1995; Hetherington et al., 1996), other free radical interactions of organic xenobiotics, uncoupling of membranebound electron transfer systems, and induction and autoxidation of enzymes such as cytochrome P450. The redox cycling of AH-quinones involves their univalent reduction to the semiquinone radical, catalysed by NAD(P)H-dependent flavoprotein reductases (Reaction (l)), which then autoxidises (i.e. reacts spontaneously with molecular oxygen) to produce 0~~ and regenerate the parent AH-quinone (Reaction (2)) so completing the redox cycle (Reaction (3)). The net result is the consumption of molecular oxygen and reducing equivalents and the production of 0~~ (Reaction (3)) (Mason, 1990): Reaction

(I) (reduction)

: NAD(P)H

+ HS + Q -+ NAD(P)+

+ Q-

A.M. Sjdin, D. R. Livingstone IAquatic Toxicology 38 (1997) 83-99

85

: Q- + O2 -+ Q + 02-

Reaction

(2) (autoxidation)

Reaction

(3) (redox cycle) : NAD(P)H

+ H+ + 0s +NAD(P)+

+ 0~~

where Q and Q* are the parent AH-quinone and semiquinone radical, respectively. In turn, Os- can give rise to other ROS and pro-oxidant species, for instance, by dismutation to H202, resulting in possible oxidative damage to key molecules such as DNA, lipid and protein (Kehrer, 1993). The degree of oxidative damage and oxidative stress incurred will depend on the effectiveness of antioxidant defences to detoxify ROS, and repair systems to, for example, remove damaged proteins and repair DNA lesions (Winston and Di Giulio, 1991; Kehrer, 1993). Previous studies have demonstrated the stimulation of NAD(P)H-dependent ROS production of digestive gland microsomes of the common mussel, Myths edulis, by the model AH-quinone redox cycling compound menadione (2-methyl1,4_naphthoquinone) (Livingstone et al., 1989) and the quinone metabolites derived from the biotransformation of benzo[a]pyrene (BaP), namely, BaP-1,6-, BaP-3,6and BaP-6,12-diones (Garcia Martinez and Livingstone, 1995) (Fig. 1). This, combined with the observations in Mytilus sp. and other molluscs of the biotransformation of BaP to quinones in vitro (Lemaire et al., 1993; Michel et al., 1993a) and in vivo (Michel et al., 1995) the presence of photo- and other oxidation products of AHs in tissues (Burns, 1993) and the elevation of flavoprotein reductase activities with exposure to AHs (Livingstone et al., 1986; Livingstone, 1987 ; Livingstone, 1988), indicates AH-quinone-mediated ROS production as a potential mechanism of AH toxicity. The present study describes the NAD(P)H-dependent redox cycling and ROS production by digestive gland microsomes of A4. edulis by six monocyclic and polycylic aromatic hydrocarbon quinones, namely, 1,Cbenzoquinone, tetramethyl1,bbenzoquinone (duroquinone), 1,2_naphthoquinone, 1,6naphthoquinone, 9,l Ophenathrenequinone and anthraquinone (Fig. 1). Unlike previous studies on menadione (Livingstone et al., 1989) and BaP-quinones (Garcia Martinez and Livingstone, 1995) which measured ROS production only, redox cycling in the present study was assessed in terms of both oxygen consumption and ROS production; oxygen consumption was also determined for menadione and the BaP-quinones, providing together results for a range of one- to five-ring AHs. The objectives of the study were: (1) to examine the redox cycling potential of a range of quinones derived from aromatic hydrocarbons which are widespread in the aquatic environment, e.g. naphthalenes, anthracene and phenanthrene in marine sediments and biota (Varanasi, 1989) duroquinone in pulp mill effluent (Niemela, 1990); (2) to provide evidence for redox cycling by the simultaneous measurement of oxygen consumption and ROS production; (3) to examine the relationship between the structure of aromatic hydrocarbon quinones and their potential for redox cycling and ROS production. The studies were carried out on digestive gland microsomes because they are a major site of xenobiotic metabolism and the flavoprotein reductase activities involved in redox cycling (Livingstone, 1991).

86

2. Materials

A.M.

Sjdin,

D. R. Living.p.FtonrIAyuutk

Tosic~ok)g~s 38 (1997) K3 99

and methods

Menadione, P-nicotinamide adenine dinucleotide reduced form (NADH), p-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), 2-keto-4-methiolbutyric acid (KMBA), dimethylformamide (DMF), ethylenediaminetetraacetic acid, sodium azide and catalase (EC 1.11.1.6) were obtained from Sigma Chemical Company Ltd, Poole, UK. The BaP-1,6-, -3,6- and -6,12-diones were from the NC1 Chemical Carcinogen Repository (Kansas City, USA). All other AH-quinones were from the Aldrich Chemical Co. (Gillingham, UK). Standard laboratory chemicals were of AnalaR grade from Merck Ltd, Lutterworth, UK. 2.2. Anitnals and preparation

of’ tnicrosotnes

Mussels (4.-5 cm shell length) were collected at low tide from a local clean site at Whitsand Bay, Cornwall, UK, and kept for several days at ambient temperature in recirculating seawater without food to clear gut contents. Digestive glands were dissected out, damp-dried, frozen in liquid nitrogen and stored at -70°C. Microsomes were prepared at 4°C essentially as described by Livingstone and Farrar (1984). Frozen digestive glands from 3040 mussels were homogenised in 1:s tissue wet weight :buffer volume ratio in IO mM Tris-HCl (pH 7.6) containing 0.5 M sucrose and 0.15 M KC1 using an electrically driven glass-Teflon PotterElvehjem homogeniser. The microsomes (100 OOOg pellet) were obtained by successive centrifugations of the homogenate and resultant supernatants of 5OOgX 10 min. 10 OOOgx 45 min and 100 OOOgx90 min. The microsomes were suspended in homogenisation buffer to give a protein concentration of about 10 mg ml-’ and stored at -70°C until required. Protein was measured by the method of Lowry et al. (1951). 2.3. Oxygen

cunsut~zption tneusuremmts

Oxygen consumption was measured with a digital Rank Brothers, Cambridge, UK oxygen monitor equipped with a Clark-type electrode essentially as described by Trudgill (1985). incubations were carried out at least in triplicate. The standard assay conditions in a final volume of 2 ml were: 25°C 100 mM KH~POJ-KzHPOd pH 7.6, 2 mM NAD(P)H, 0.5 or 0.75 mg microsomal protein and varying concentrations of AH-quinones added in 20 ~1 DMF (controls received DMF alone). Reactions were started by the addition of reduced coenzyme. Other conditions included varying the amount of NAD(P)H (to determine the saturating concentration of reduced coenzyme) and microsomal sample, and additions of sodium azide or catalase (see Section 3 for details). Rates of oxygen consumption were linear over the time period of measurement. Control (basal) rates of oxygen consumption were subtracted from rates in the presence of AH-quinones to obtain the net stimulated rates.

A.M.

Sjdin,

D.R. LivingstonelAquatic

2.4. ROS production (iron-EDTA-mediated

Toxicology

38 (1997) 83-99

87

KM3A oxidation)

ROS production was quantified by the formation of -OH from Os- and Hz02 via the iron-ETDA-catalysed Haber-Weiss reaction (Os-+Hs02 +*OH+OH-+Os) (Winterbourn, 1987). The NAD(P)H-dependent generation of *OH was detected by the oxidation of the scavenging agent KMBA to ethylene as described by Garcia Martinez et al. (1995). Incubations were carried out in duplicate in sealed 25 ml conical flasks in a shaking water-bath at 25°C. Contained in a final volume of 1 ml were 100 mM KsHP04-KHsP04 pH 7.4, 10 mM MgCls, 10 mM KMBA, 1 mM sodium azide, 0.5 mM NAD(P)H, 75 PM FeCls in 150 pM neutralised EDTA, approximately 1 mg microsomal protein and varying concentrations of AH-quinones added in 20 pl DMF (controls received DMF alone). Reactions were started by the addition of reduced coenzyme. Air samples (1 ml) were taken directly from the headspace of the sealed flasks at various time intervals after initiation of the reaction and injected directly into a Varian (Palo Alto, CA, USA) Model 27 gas chromatograph equipped with a Porapack Q-column (So-100 mesh) (Phase Separations Ltd., Deeside, UK) and a flame ionisation detector. Operating conditions were: column, 50°C; detector set at 180°C; and nitrogen (carrier gas) flow. 30 ml min-I. Quantification was by reference to ethylene standards prepared under the same conditions of incubation. Control rates were subtracted from rates in the presence of AH-quinones to obtain the net stimulated rates of KMBA oxidation. Chemical rates of KMBA oxidation (i.e. incubations containing all additions except microsomes) were also determined and subtracted from the stimulated rates. 2.5. Kinetic measurements and statistics Rates of oxygen consumption and ROS production were determined over a range of four to seven concentrations of AH-quinone. The Michaelis-Menten parameters of maximal rate ( Vnlax) and apparent K, (app. K,) were determined by weighted Lineweaver-Burk plots using the program Enzpack (Biosoft, Cambridge, UK). Rates of oxygen consumption and ROS production in absence and presence of AH-quinones over time were compared by two-way analysis of variance, and P < 0.05 was accepted as significant.

3. Results 3.1. Rates of oxygen consumption Using a fixed saturating concentration of menadione of 0.5 mM for ROS production (Livingstone et al., 1989) the stimulated rates of NADH- and NADPHdependent oxygen consumption were linear with a ten-fold range of microsomal protein concentration (0.2-2.0 mg ml-‘), and increased with increasing reduced coenzyme concentration, levelling off at 0.5 mM NADH or 2.0 mM NADPH (data not shown). The studies of the dependence of stimulated oxygen consumption

88

A.M.

~jdin,

D. R. Livingslone IAyuutic

Toxicology

38 (1997) 83-99

0

0

0 I I

1,4-benzoqulnone

(I

ICH3

menadione

Q

0

9,10-phenanthrenequinone

duroquinone 0 0

0

anthraqulnone

I,?-naphthoquinone 0 0

1,6-benro(a)pyrene

1,4-naphthoquinone 0

quinone

0

Fig. I. Chemical structures of the range of l-5-ring aromatic hydrocarbon It should be noted that only 1,6-benzo[a]pyrene quinone (dione) is shown.

quinones

used in the study.

on concentration of AH-quinone were therefore carried out at a microsomal protein concentration of 0.5 mg ml-’ (NADH studies) or 0.75 mg ml-’ (NADPH studies), and a saturating reduced coenzyme concentration of 2 mM. The Michaelis-Menten parameters for the dependence of stimulated rates of oxygen consumption on AH-quinone concentration are given in Table 1, and the profiles for the dependence of oxygen consumption on AH-quinone concentration for selected AH-quinones shown in Fig. 2(A)Fig. 3(A)Fig. 4(A)Fig. 5(A). Stimulated rates of oxygen consumption were detected for five out of the ten AH-quinones tested, but not for 1,4-benzoquinone, anthraquinone or any of the BaPdiones up to an AH-quinone concentration of 2 mM (Table 1). Michaelis-Menten kinetics were seen with respect to AH-quinone concentration, with correlation coefficients for the determination of app. K,,, and V,,,;,, of 0.96-0.99. Depending on the reduced coenzyme, maximal rates of stimulated oxygen consumption decreased in the order I ,2-naphthoquinone > 1,4_naphthoquinone or 9,10_phenanthrenequinone > duroquinone or menadione (Table 1). NADH-dependent rates of stimulated oxygen consumption were greater than NADPH-dependent rates for all five AHquinones, the greatest difference being seen for 1,6naphthoquinone (Fig. 4(A)). Thus the ratio of VI,;,, for NADH and NADPH for stimulated oxygen consumption was 6.3 for 1,4_naphthoquinone and in the range 1.8-3.3 for duroquinone, 1,2naphthoquinone, menadione and 9, lo-phenanthrenequinone. The app. K,, values for AH-quinone for stimulated oxygen consumption were lower for the NADHthan the NADPH-dependent reaction for the 1,2- and 1,bnaphthoquinones, but the

89

A.M. Sjiilin, D. R. Livingstone IAquatic Toxicology 38 (1997) 83-99

Table I Michaelis-Menten parameters for the dependence of NADH- and NADPH-dependent quinone-stimulated rates of oxygen consumption on the concentration of aromatic hydrocarbon quinones in digestive gland microsomes of M. edulis Quinone

NADH

NADPH V,,, (nmol min-’ mg-’ protein)

App. K,

(W

(PM)

V,,, (nmol min-’ rng-’ protein)

ND 2390 319 543 181 ND 107 ND ND ND

ND 27.9 418 93.2 21.2 ND 66.1 ND ND ND

ND 309 374 1930 52.2 ND 22.1 ND ND ND

ND 8.4 237 14.8 9.0 ND 30.7 ND ND ND

App. G

1,4-Benzoquinone Duroquinone I ,2-Naphthoquinone I ,4-Naphthoquinone Menadione Anthraquinone 9,10-Phenanthrenequinone Benzo[a]pyrene-l,6-dione Benzo[a]pyrene-3,6-dione Benzo[a]pyrene-6,12-dione The correlation Not detectable.

coefficients

for determination

of the kinetic parameters

were in the range 0.96-0.99.

ND,

reverse was seen for duroquinone, menadione and 9, IO-phenanthrenequinone (Table 1). The lowest app. Km for AH-quinone was seen for 9,10_phenanthrenequinone, for both reduced coenzymes (Table 1). The range of variation of kinetic parameters for different AH-quinones was greater for NADPH than NADH (I’,,,---15fold (NADH) and 2%fold (NADPH); app. K,,--22-fold (NADH) and 87-fold (NADPH)).

+

NA!JH

-

:

12-

+

NADPH

14 ,

--f

NADPH

I

A

T

00 Tetm.meihyl-l+Ben.?cquoquinone (mM)

Fig. (A) tion are

NADH

05

1.0

1s

7.0

Tetmmethyl-l,4-Benznqu1none (mM)

2. Dependence of the NADHand NADPH-dependent rate of stimulated oxygen consumption and ROS production (iron_EDTA-mediated oxidation of KMBA to ethylene) (B) on concentraof tetramethyl-1,Cbenzoquinone (duroquinone) in digestive gland microsomes of M. edulis. Values mean + SEM or f range.

A. M. Sjiilin, D. R. Livingstone IAquatic

90 +

NADH

-

38 (I 997) 83.-99 +

NADPH

350,

0 00

Toxicology

NADH

-

NADPH

,

025

0 50

0 75

I 00

I 25

I,?-Naphthoqumone (mM)

0 00

025

0 50

0.75

I 00

I 25

I,‘&Naphthoqumone (mM)

Fig. 3. Dependence of the NADH- and NADPH-dependent (A) and ROS production (iron_EDTA-mediated oxidation tion of 1.2-naphthoquinone in digestive gland microsomes 2 range.

rate of stimulated oxygen consumption of KMBA to ethylene) (B) on concentraof M eduh. Values are mean k SEM or

Rates of NADH- and NADPH-dependent stimulated oxygen consumption in the presence of 0.5 mM menadione were the same in the absence and presence of 1 mM sodium azide (added to inhibit contaminating catalase activity) (data not shown). Following pre-incubation in the presence of saturating concentrations of AH-quinone (duroquinone, 1,2_naphthoquinone, 1,6naphthoquinone, menadione or 9,l Ophenanthrenequinone) until a significant proportion of the oxygen present had been consumed, and subsequent addition of 100 units of commercial catalase activity, increased oxygen levels were seen, indicating the presence of Hz02 (i.e. oxygen production occurred via the catalase reaction: 2Hz02 +2H20+02). The amount of oxygen produced as a percentage of that consumed before the addition of catalase varied from 0 to 12% for the NADH-dependent reactions and from 0 to 19% for the NADPH-dependent reactions. 3.2. Rates qf ROS production ethylene)

(iron-EDTA-mediated

oxidation

of KMBA

to

All six AH-quinones tested showed stimulated rates of NADH- and NADPHdependent ROS production. Most rates were linear up to 30 min, the exceptions being anthraquinone (NADPH-dependent reaction) and high concentrations of 1,4naphthoquinone and 9, lo-phenanthrenequinone (NADH-dependent reaction) and 1,2-naphthoquinone (NAD(P)H-dependent reactions) which levelled off after 15 min (data not shown). The Michaelis-Menten parameters for the dependence of stimulated rates of ROS production on AH-quinone concentration are given in Table 2, and the rates of ROS production vs. AH-quinone concentration for selected AH-quinones shown in Fig. 2(B)Fig. 3(B)Fig. 4(B)Fig. 5(B). Michaelis-Menten kinetics were seen with respect to AH-quinone concentration, with correlation coefficients for the determination of app. K,,, and V,,,,, of 0.97-0.99, except for 0.65 for NADH-dependent ROS production by anthraquinone. NADH-dependent rates

A.M. +

Sjtilin, D. R. Livingstone IAquatic

NADH

+

Toxicology

NADPH

91

38 (1997) 83-99 +

NADH

--f

NADPH

1500 ,

125

50

25

-

1.0

20

30

I+Naphthoqumone

40 l+Naphthoqumone

(mM)

Fig. 4. Dependence of the NADHand NADPH-dependent (A) and ROS production (ironEDTA-mediated oxidation tion of 1,4_naphthoquinone in digestive gland microsomes + range.

(mtvf)

rate of stimulated oxygen consumption of KMBA to ethylene) (B) on concentraof M. edulis. Values are mean*SEM or

of stimulated ROS production were greater than NADPH-dependent rates for all AH-quinones, decreasing in the order 1,2_naphthoquinone > 1,4_naphthoquinone and 9, lo-phenanthrenequinone > duroquinone > 1,4-benzoquinone > anthraquinone (Table 2). The ratio of V,,,, for stimulated ROS production for NADHI NADPH was 3.84.4 for 1,2_naphthoquinone and 1,4-benzoquinone, and 1.331.8 for duroquinone, 1,4_naphthoquinone and 9, lo-phenanthrenequinone. The app. K,,,

Table 2 MichaelissMenten parameters lated rates of ROS production tion of aromatic hydrocarbon Quinone

1,4_Benzoquinone Duroquinone 1.2-Naphthoquinone 1,4-Naphthoquinone Menadione” Anthraquinone 9.10-Phenanthrenequinone Benzo[a]pyrene- 1,6-dione” Benzo[a]pyrene-3.6-dione” Benzo[a]pyrene-6,12-dione”

for the dependence of NADH- and NADPH-dependent (ironEDTA-mediated KMBA oxidation to ethylene) quinones in digestive gland microsomes of M. edulis NADH

quinone-stimuon the concentra-

NADPH

APP. K,, (PM)

V,,,, (nmol mini’ mg-’ protein)

App. K,,,

341 402 128 124 386 248 58.1 13.0 14.6 0.6

0.179 0.268 10.30 1.33

103 278 50.3 253 88 ND 230 6.7 7.8 ND

0.021 1.09 0.204 0.340 0.029

The correlation coefficients for determination of the kinetic except for anthraquinone (0.65). -, No information available; “Data from Livingstone et al. (1989). “Data from Garcia Martinez and Livingstone (1995).

(PM)

V,,,, (nmol mm’ rng-’ protein) 0.041 0.151 2.71 0.945 ND 0.867 0.022 0.022 ND

parameters were in the range ND, not detectable.

0.97-0.99.

A. M. Sjdin, +

NADH

D. R. Livingstone IAquatic

-t-

Toxicology

NADPH

38 (1997) 83-N +

60-

NADH

+

NADPH

1250 T

A

:

SO-

B

1000

E :

$

750

z

000

0 25

0 50

0 75

9,10-Phenanthrenequmnone

I 00

8

500

3 0 z %

250

I 25

0 0 00

0 25

0 50

0.75

9,10-Phenanthrmequmone

(mM)

1.oo

I !5

(mM)

Fig. 5. Dependence of the NADH- and NADPH-dependent rate of stimulated oxygen consumption (A) and ROS production (iron-EDTA-mediated oxidation of KMBA to ethylene) (B) on concentration of 9,10-phenathrenequinone in digestive gland microsomes of M. dulis. Values are mean f SEM or k range.

values for AH-quinone for stimulated ROS production were lower for the NADHthan the NADPH-dependent reaction for 1,4_naphthoquinone and 9,10-phenanthrenequinone only, but the reverse for all the others, including menadione and the BaP-1,6- and -3,6-diones (Table 2). The lowest app. K,,, for AH-quinone was seen for the BaP-diones. The range of variation of kinetic parameters for different AH-quinones was greater for NADH than NADPH ( V,,,,X- X 481-fold (NADH) x 66-fold (NADPH); app. K,,,- X 670-fold (NADH) and X 42-fold and (NADPH)). 3.3. Compurison

of rates of oxygen

consumption

and ROS production

Overall general similarities were evident between the profiles for dependence of stimulated oxygen consumption and ROS production on AH-quinone concentration (Fig. 2, Figs. 3 and 5) but marked differences were also apparent, namely, for 1,4_naphthoquinone (compare Figs. 4(A) and 4(B)). For the four AH-quinones for which measurements of both oxygen consumption and ROS production were made on the same biological sample (i.e. duroquinone, 1,2- and 1,6naphthoquinones and 9,10-phenanthrenequinone), good correlation was seen between the two processes for V,,,,, for both NADH and NADPH (correlation coefficients of 0.99 and 0.96, respectively) and app. K,,, for NADH but not NADPH (correlation coefficients of 0.99 and 0.24, respectively). For the same four AH-quinones (duroquinone, 1,2- and 1,4_naphthoquinone and 9, IO-phenanthrenequinone), the values for V,n,, of oxygen consumption exceeded those of ROS production by 59- to 103-fold (NADH) or 15to 87-fold (NADPH) (Tables 1 and 2). Similarly, values of app. K,,, for AH-quinone for both NADH- and NADPH-dependent reactions were 0.6- to 6.4-fold higher for stimulated oxygen consumption than ROS production, the exceptions for NADPH being the reverse for 9,10-phenanthrenequinone and similar values for duroquinone (Tables 1 and 2).

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4. Discussion

A range of one- to five-ring structurally related AH-quinones derived from diverse possible pollutant sources, such as the petroleum industry, pulp mill effluents and combustion processes (see the Introduction), were examined for their ability to redox cycle in digestive gland microsomes of a common bivalve mollusc, A4. edulis. The net result of redox cycling is the consumption of reducing equivalents and 02 and the production of 02-. Previous studies on digestive gland microsomes of M. edulis have demonstrated both xenobiotic-stimulated oxygen consumption and Osproduction for the nitroaromatics nitrofurantoin (N-(5nitro-2-furfurylidene)-l-aminohydantoin) (Garcia Martinez et al., 1995) and 4-nitroquinoline N-oxide (Garcia Martinez et al., 1992), but not for any AH-quinone. NADH- and NADPH-dependent oxidase activities have also been shown to be present in digestive gland microsomes of M. edulis (Livingstone et al., 1989). Overall the measurement of both stimulated oxygen consumption and ROS production for five AH-quinones (duroquinone, 1,2- and 1,Cnaphthoquinones, menadione and 9, lo-phenanthrenequinone) provides strong evidence for the NAD(P)Hdependent redox cycling of these compounds by digestive gland microsomes of A4. edulis. The fact that stimulated ROS production, but not oxygen consumption, is detectable for all ten AH-quinones examined so far (i.e. the above plus l,Cbenzoquinone, anthraquinone and BaP-diones) indicates that the former is probably a more sensitive assay of redox cycling than the latter. The production of Hz02 from 02 was demonstrated by the release of oxygen following the addition of catalase to the incubations, as has also been observed for redox cycling of nitrofurantoin (Garcia Martinez et al., 1995). However, unlike nitrofurantoin, no detectable inhibition of rates of stimulated oxygen consumption was seen in the absence of sodium azide (inhibitor of contaminating endogenous catalase activity), which, coupled with the indicated low presence of up to only 19% of consumed 0s as Hz02 (seen from addition of catalase), provides indirect evidence for a large part of the reduced 0s being present as Os-. The detected HsOs could presumably arise directly from autoxidation of cytochrome P450 (Premereur et al., 1986) or more likely from dismutation of Os- (20s-+2H+ + HsOs+Os). The absolute requirement of HsOs for significant iron-ETDA-mediated microsomal production of *OH via the Haber-Weiss reaction (HsOs+02- +*OH+OH-+Os) is well established for M. edulis (Livingstone et al., 1989; Winston et al., 1990; Garcia Martinez et al., 1992, Garcia Martinez et al., 1995, Garcia Martinez and Livingstone, 1995) and other marine invertebrates (Jewel1 and Winston, 1988, Jewel1 and Winston, 1989; Gamble et al., 1995; Hetherington et al., 1996). The role of 02~ in the generation of -OH via the iron-EDTA-mediated Haber-Weiss reaction is dismutation to produce the necessary HsOs and/or reduction of added Fe3+ to produce Fe*+ (i .e . 02-+Fe3+ +02+Fe2+), which is required to yield *OH from Hz02 via the Fenton reaction (i.e. HsOs+Fe*+ + *OH+Fe3+). The possibility exists that Fe3+ could be reduced directly to Fe *+ by flavoprotein reductases, as is observed for rat cytochrome P450 reductase (Winston et al., 1984) and the crayfish Procambrus clurkii (Jewel1 and Winston, 1989), so negating a role for 0~~. However, superoxide

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dismutase inhibition studies on menadioneand BaP- 1,6-stimulated production of *OH by digestive gland microsomes of M. edulis have demonstrated the involvement of 0~~ (Livingstone et al., 1989; Garcia Martinez and Livingstone, 1995). Rates of both AH-quinone stimulated oxygen consumption and ROS production were generally greater for NADH than NADPH, consistent with previous studies on digestive gland microsomes of M. edulis (Livingstone et al., 1989; Garcia Martinez and Livingstone, 1995) and hepatic microsomes of the flounder Platichthys flesus (Lemaire et al., 1994, but the reverse of that seen for hepatic microsomes of perch PercaJEuviatilis (Lemaire et al., 1994) and rat (Smith et al., 1985; Dicker and Cederbaum, 1991). The indicated higher rates of NADH- than NADPH-dependent redox cycling of AH-quinones are also consistent with the higher NADH- than NADPH-dependent flavoprotein reductase (Livingstone and Farrar, 1984) and lower NADH- than NADPH-dependent DT-diaphorase (Livingstone et al., 1989) activities in digestive gland microsomes of M. edulis. Although similarities were clearly evident between the profiles for the dependence of oxygen consumption and ROS production on AH-quinone concentration (as shown by the correlation coefficients that they are between the two measurements for V,,,, and app. I&), indicative reflecting the same process (i.e. redox cycling), quantitative and qualitative differences were also seen. The 16- to 104-fold greater rates of oxygen consumption than ROS production are presumably mainly due to the fact that the potent oxidant -OH will react with other molecules in the biological sample in addition to KMBA. Other factors are the stoichiometry of the reactions, which require three molecules of OZ consumed for every *OH produced (see Livingstone et al., 1989), and the possibility of oxygen consumption processes which do not result in the production of ROS. The qualitative differences were most apparent for 1,4_naphthoquinone, indicative of a discrepancy between oxygen consumption and ROS production in the process(es) being measured. Nothing is known of the metabolic role of 1,4naphthoquinone, except that it is related to menadione (2-methyl-1,4-naphthoquinone) or vitamin K:$, which has been observed to stimulate spawning in M. edulis (Livingstone et al., 1990). Overall, the mainly lower values for app. K,,, for AHquinone, and the wider range of values for different AH-quinones for both V,, and app. K,,, for ROS production compared with oxygen consumption possibly indicate that the former is a more specific measure of redox cycling than the latter. Assuming ROS production is a measure of redox cycling, the different activities and patterns observed for different AH-quinones may be determined by a number of factors, including: hpophilicity of the substrate; the topography, complement and substrate specificity of the microsomal flavoprotein reductases; the structure and redox potential of the AH-quinone. Little can be said about enzyme topography, except that interactions could take place both in the lipid membrane and on its surface because, for example, mammalian cytochrome P450 reductase protrudes from the endoplasmic reticulum into the aqueous environment (Guenthner et al., 1980) and the same is indicated for liver microsomes of P. flesus (Lemaire and Livingstone, 1994). Uptake of substrate into the microsomal membrane may be a factor, but no correlation was observed between rates of ROS production and AHquinone lipophilicity, e.g. rates were lower for three-ring anthraquinone than one-

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ring 1,4-benzoquinone, and indicated to be similar for one-ring 1,4-benzoquinone and five-ring BaP-diones (see Garcia Martinez and Livingstone, 1995). Nothing is known of the substrate specificity of individual microsomal flavoprotein reductases of A4. edulis digestive gland, but cytochrome P450 reductase and cytochrome b:, reductase are indicated to be present (Livingstone and Farrar, 1984), and the involvement of hepatic cytochrome P450 reductase in NADPH-dependent redox cycling of xenobiotics has been demonstrated in P. Jesus (Lemaire and Livingstone, 1994). The greater range of values of V,, and app. K, for different AH-quinones for NADH compared with NADPH is consistent with more pathways of electrontransport indicated for NADH than NADPH in digestive gland microsomes of M. edulis, namely, both NADH-cytochrome c and NADH-ferricyanide, but only NADPH-cytochrome c, reductase enzymic activities were detected (Livingstone and Farrar, 1984). The different ratios between NADH- and NADPH-dependent reactions for different AH-quinones for V,, or app. K, are presumably determined by differences in enzyme complement and/or substrate specificity. The redox potential and chemical structure of the AH-quinone substrate are both indicated to influence redox cycling and ROS production. The lower V,,, rate of ROS production for 1,Cbenzoquinone compared with duroquinone, 1,2-naphthoquinone, 1,4_naphthoquinone and 9, lo-phenanthrenquinone is consistent with its lower reduction potential (E) for Q/Q’ (Wardman, 1989) of +78 compared with -89 to -260 mV for the other AH-quinones (the more electronegative is the reduction potential for Q/Q, the more likely is redox cycling to occur). Also, recent mammalian studies have shown that compared with compounds with more electronegative reduction potentials, 1,4-benzoquinone is more likely to undergo two-electron than one-electron reduction by cytochrome P450 reductase (Butler and Hoey, 1993). In contrast, the importance of substrate structure and enzyme substrate specificity is illustrated by the about ten-fold greater rates of ROS production of 1,2-naphthoquinone (E -89 mV) compared with 1,Cnaphthoquinone (-140 mV), 9,10-phenanthrenequinone (- 124 mV) and duroquinone (-260 mV). Similarly, the only slightly higher rates of NADH-dependent ROS production of tetramethyl- 1,Cbenzoquinone (duroquinone) compared with 1,6benzoquinone (E -260 and +78 mV, respectively) may be due to the presence of methyl-groups. The present and previous studies (Livingstone et al., 1989; Garcia Martinez and Livingstone, 1995) on the redox cycling of AH-quinones clearly indicate a widespread potential for ROS production by this group of xenobiotics in digestive gland of M. edulis and other molluscs (Livingstone et al., 1990). The compounds include industrial quinones, e.g. duroquinone in pulp mill effluents (Niemela, 1990) and those derived by environmental photo-oxidation (Burns, 1993) or biotransformation (Michel et al., 1995) of parent AHs from such wide sources as the petroleum industry and combustion. Biotransformation could be particularly important in bivalves because of their apparent feature, compared with higher invertebrates (crustaceans, echinoderms) and vertebrates, of metabolising BaP (and therefore possibly other AHs) to predominantly diones (Livingstone, 1991; Den Besten et al., 1992, Den Besten et al., 1993 ; Lemaire and Livingstone, 1993 ; Michel et al., 1993a, Michel et al., 1995, Porte et al., 1995). The parent AHs of the AH-quinones

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examined show a wide range of water solubilities, e.g. naphthalene compared with BaP (log (octanol/water) coefficients of 3.3 and 6.42, respectively) (Hawker and Connell, 1986) but are all readily bioaccumulated by bivalves from the watercolumn, sediment or biota (Livingstone, 1991; Widdows and Donkin, 1992). Oxidised lipid (Livingstone et al., 1990), DNA (Marsh et al., 1993) and protein (Kirchin et al., 1992) have been detected in the digestive gland of M. edulis. However, the extent to which enhanced ROS production by AH-quinones could increase oxidative damage will depend upon the effectiveness of antioxidant defences, which are indicated to be very efficient in digestive gland of M. edzdis; for example, no or minimal increases were seen in levels of %hydroxydeoxyguanosine and lipid peroxides (malonaldehyde equivalents) following exposure of M. edulis to 100 ppb menadione for 4 days (Marsh et al., 1993) or to either 50 ppb BaP or 1 ppm menadione for 6 days (Livingstone et al., 1990). However, equally well, the widespread nature of oxidised products of AHs, including quinones, produced by photooxidation (Burns, 1993) and possibly also by biotransformation (Michel et al., 1995) in molluscs, is only just becoming appreciated and could be of potential toxic significance. Thus, ROS production could play a role in chemical carcinogenesis in bivalves, such as that caused by a chemical mixture including the PAHs BaP and 1,2_benzanthracene in C. virginica.

Acknowledgements

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