Pharmac. Ther, Vol. 53, pp. 261-273, 1992

0163-7258/92 $15.00 © 1992 Pergamon Press Ltd

Printed in Great Britain. All rights reserved

Specialist Subject Editor: J. L. HOLTZMAN

XENOBIOTIC METABOLISM BY PROSTAGLANDIN H SYNTHASE T. E. ELING* and J. F. CURTIS Eicosanoid Biochemistry Section, Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, National Institutes of Health, P.O. Box 12233, Research Triangle Park, NC 27709, U.S.A. Abstract--During the metabolism of arachidonic acid by prostaglandin H synthase many chemicals including carcinogens are metabolized. These chemicals are metabolized by either the peroxidase activity of prostaglandin H synthase, the peroxyl radicals generated during arachidonic acid oxygenation, or a combination of these two mechanisms. In many cases, the chemical metabolism results in the formation of reactive metabolites that have mutagenic activity and potential carcinogenic activity. In other cases, the chemicals are detoxified. Chemical metabolism that occurs during arachidonic acid oxygenation may be an important determinate of chemical toxicity in extra-hepatic tissues. CONTENTS 1. 2. 3. 4.

Introduction Biochemistry of Prostaglandin H Synthase Mechanism of Oxidation Biological Implications of Metabolism During Arachidonic Acid Oxidation 4.1. Mutagenic activation 4.2. Carcinogenicity 5. Detoxifications Catalyzed by Prostaglandin H Synthase 6. Summary Acknowledgements References

261 262 263 266 266 268 269 270 270 270

1. I N T R O D U C T I O N

Exposure to chemicals is recognized as an important cause of carcinogenesis. The chemicals are, in themselves, not carcinogenic but are metabolized by the enzyme systems present in the body to carcinogenic metabolites. This process of metabolic bioactivation usually results in the formation of an electrophilic metabolite that reacts with cellular DNA. The resultant altered nucleic acids are the eventual molecular lesions that ultimately lead to the development of the cancer. The oxidation of chemicals can cause not only an enhancement of the toxicological activity of the chemical, but also lead to a loss of activity or results in detoxification of the chemical. For example, the introduction of an oxygen atom can serve as a means of attaching polar moieties such as glutathione and glucuronic acid. As a result, the chemical has increased water solubility which results in its increased excretion. The metabolism of a chemical is thus an important determinant in chemical toxicity. The cytochrome P-450 family of enzymes is recognized as the most established enzyme system for converting chemicals to reactive electrophiles that are carcinogenic. Other enzyme systems *Corresponding author. Abbreviations--2-AF, 2-aminofluorene; PHS, prostaglandin H synthase; PAH, polycyclic aromatic hydrocarbons; BP, benzo(a)pyrene; BP-7,8-diol, BP-7,8-dihydroxy-7,8-dihydro; GS', glutathionyl free radical; GSH, glutathione; 2-NA, 2-naphthylamine; PGG2, prostaglandin G2; PGH2, prostaglandin H2; IQ, 2-amino-3methylimidazo[4,5-f]-quinoline. 261

262

T.E. ELINGand J. F. CURTIS

however may also bioactivate chemicals. The peroxidative oxidation of chemicals that occurs during arachidonic acid oxidation by prostaglandin H synthase (PHS) also results in the formation of electrophiles and bioactivates chemical carcinogens. This enzyme system is involved in the bioactivation of chemicals in extrahepatic tissues where the monooxygenase system has low activity. We will review and discuss the oxidation of chemicals by PHS with particular emphasis placed on the bioactivation of chemicals to carcinogenic metabolites. Other reviews on this subject are recently published (Smith et al., 1991; Eling et al., 1990) and should also be consulted for a more complete understanding of this system.

2. BIOCHEMISTRY OF PROSTAGLANDIN H SYNTHASE Arachidonic acid is metabolized by the prostaglandin H synthase dependent pathway into prostaglandins, prostacyclin and thromboxane and by lipoxygenases to leukotrienes and hydroxyfatty acids (Pace-Asciak and Smith, 1983). Prostaglandin H synthase is the initial enzyme which commits arachidonic acid to prostaglandin formation (Fig. 1). The activity of this enzyme is of primary importance in the control of the formation of prostaglandins. Two catalytic activities co-purify with PHS: cyclooxygenase and peroxidase (Miyamoto et al., 1976). The cyclooxygenase catalyzes the formation of the cyclic endoperoxide hydroperoxide, prostaglandin G2 (PGGz). The peroxidase then reduces the hydroperoxide to the corresponding alcohol, prostaglandin Hz (PGH2). PGH2 is then converted into thromboxanes, prostacyclin and prostaglandins E2 and F2 by other enzymes. PHS activity is found in almost every mammalian tissue that has been investigated. The highest concentrations of PHS are found in ram seminal vesicles and thus they are the primary source of PHS for investigative studies. Relatively high levels of PHS are found in kidney medulla, platelets, vascular endothelial cells, the alimentary tract, brain, lung and bladder (Christ and Van Dorp, 1972; Yoshimoto et al., 1986; Zenzer and Davis, 1988; Pace-Asciak and Rangaraj, 1977; DeWitt et al., 1983). Prostaglandin H synthase is present in both the endoplasmic reticulum and nuclear membrane (Rollins and Smith, 1980; Smith and Bell, 1978; Huslig et al., 1979). PHS isolated and purified from bull and ram seminal vesicles (Miyamoto et al., 1976; Hemler et al., 1976; Van Der Ouderaa et al., 1977; Mevkh et al., 1985) has a MW of about 70,000 Da and is a glycoprotein (Van Der Ouderaa et al., 1977). The PHS is a heme containing enzyme but the heme is often removed during purification to form the apoenzyme. Other hemoproteins, such as oxyhemoglobin, methemoglobin and metmyoglobin, will reconstitute the apoenzyme to PHS activity (Ogino et al., 1978). Interestingly, manganese protoporphyrin IX reconstituted enzyme has cyclooxygenase activity but no peroxidase activity. Full length cDNAs (2.8 kilobase pairs) coding for PHS from ram seminal vesicles (DeWitt and Smith, 1988; Merlie et al., 1988; Yokoyama et al., 1988) have been isolated and sequenced. The nucleotide and amino acid sequence of PHS are known. The peptide consists of 600 amino acids which includes a 24 residue signal peptide. There are four potential asparagine-linked glycosylation sites as well as several hydrophobic sites which may represent points of interaction with the membrane. Human PHS was recently cloned and sequenced and is similar to ram PHS (Hla et al., 1986; Yokoyama and Tanabe, 1989; Funk et al., 1991). The cyclooxygenase and peroxidase activities of PHS are intimately related which may account for several unusual kinetic observations. First a short lag period is observed after the addition of arachidonic acid before full catalytic activity is seen. The addition of a variety of lipid hydroperoxides to the enzyme shortens the lag time (Hemler et al., 1979). Low concentrations of peroxides (10 -8 M) are apparently required to initiate cyclooxygenase activity (Kulmacz and Lands, 1983). Another unusual kinetic feature of PHS is the self-inactivation that occurs during catalysis. PHS is irreversibly inactivated within 1 to 2 min after the addition of arachidonic acid. Both the cyclooxygenase and the peroxidase are sensitive to inactivation. Preincubation of PHS with micromolar concentrations of arachidonic acid or peroxides inactivates PHS. Inactivation of PHS requires peroxidase turnover and is prevented by the presence of a peroxidase reducing cosubstrate. For example, 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) inactivates PHS with an IC50 of 4 laM in the absence of phenol compared with an IC50 of 92/~M in

Hydroperoxide-dependent oxidation

~

263

Arschldonlc Acid

PGG2

ms

Hydroperoxy-Fatty Acids

Prostaglandin H Synthase (Pero,,-'~)

PGH2

TXB2

Hydroxy-Fstty Acids

Leukotdenes

PGE2 PGF2~ PGI2

FIG. 1. The metabolism of arachidonic acid by prostaglandin H synthase and lipoxygenase pathways. the presence of phenol (Markey et al., 1987). PHS apoenzyme is not sensitive to peroxide inactivation, indicating the importance of the heme prosthetic group in the inactivation process (Markey et al., 1987). The cyclooxygenase activity of PHS catalyzes the insertion of 2 mol of oxygen into 1 mol of arachidonic acid. The initial reaction is a hydrogen abstraction at the aUylic C-13 of arachidonic acid forming a carbon-centered radical, which rearranges to C-11. 'lne C-11 radical, which was detected with electron spin resonance using spin traps (Mason et al., 1980; Schreiber et al., 1986), reacts with molecular oxygen leading to the formation of a cyclic 9,1 l-endoperoxide and a carbon-centered radical at C-15. This C-15 radical reacts with a second mol of oxygen forming a peroxyl radical which then abstracts hydrogen to form PGG2. More detailed descriptions of this mechanism are available in other reviews (Eling et al., 1990; Smith and Marnett, 1991; Marnett and Eling, 1983). PHS cyclooxygenase is inhibited by nonsteroidal anti-inflammatory drugs. Indomethacin, ibuprofen and numerous other drugs inhibit the cyclooxygenase activity of PHS. Aspirin irreversibly inhibits the cyclooxygenase by acetylation of a serine residue (Smith and Marnett, 1991). Other anti-inflammatory drugs are reversible inhibitors. In contrast to PHS cyclooxygenase, there are no known inhibitors which are specific for PHS peroxidase. Compounds which inhibit PHS peroxidase-dependent cooxidation reactions, such as methimazole, probably do so by directly reducing cosubstrate free radicals rather than by inhibiting the enzyme (Petry and Eling, 1987).

3. MECHANISM OF OXIDATION PHS-dependent bioactivation of xenobiotics occurs by three principle mechanisms. First, the peroxidase directly oxidizes the carcinogen or chemical. Secondly, peroxyl radicals generated during prostaglandin biosynthesis are potent oxidizing agents which react with chemicals resulting in their oxygenation. A third mechanism of bioactivation involves the formation of secondary oxidant species which is derived from a metabolite catalyzed by the peroxidase. As stated previously, PHS processes two activities, a cyclooxygenase which generates the peroxide PGG2 and a peroxidase that reduces PGG2 to PGH2. Chemicals capable of supplying electrons to the peroxidase undergo a one electron oxidation during the peroxide reduction. PHS is unique in that it supplies the peroxides necessary for the peroxidase to oxidize chemicals. A large number of compounds can serve as reducing cosubstrates for PHS peroxidase (Markey et al., 1987). Functional groups which are easily oxidized by the peroxidase include phenols and aromatic amines. These functional groups are common structural features of xenobiotics which

264

T.E. ELINGand J. F. CURTIS ROOH

ROH

PP(IX) - Fe .3

PP'(IX) - Fe+4=O (Compound I)

(RestingEnzyme)

+ X H -. ~

X H P(IX) - Fe+4=O (Compound II)

FIG. 2. Proposed mechanism for the prostaglandin peroxidase.

produce toxic and carcinogenic responses. Upon reaction with peroxide (Fig. 2), ferric protoporphyrin IX, in the Fe(III) state present in the resting peroxidase, undergoes a two electron oxidation. An intermediate (compound I) is formed in which the protoporphyrin is in the Fe(V) state and contains a porphyrin (n) cation radical with the structure (protoporphyrin IX'+FeWO). Compound I is reduced to compound II at the expense of an electron donor (cosubstrate) which undergoes a one electron oxidation. Compound II has the structure ((protoporphyrin IX) FeWOH-) and is reduced to the resting enzyme (Fe(III)) at the expense of a second electron donor molecule. In some cases, compound I, particularly with sulfur containing chemicals (P16 and Marnett, 1989), directly transfers the oxygen atom to an acceptor molecule and is reduced to the resting enzyme. Thus, electron or oxygen transfer can occur with PHS peroxidase. Since the electron donor transfers a single electron to the peroxidase, a free radical metabolite of the electron donor is generated (Fig. 3). Compounds that undergo peroxidase-catalyzed one-electron oxidation (Boyd and Eling, 1984), are typically converted to highly reactive products. Direct oxidation of cosubstrates by the peroxidase represents a potentially important mechanism of PHS-catalyzed bioactivation. Unlike a Peroxidase mediated reaction, the second mechanism of PHS-dependent bioactivation is indirectly linked to the cyclooxygenase activity of the enzyme (Fig. 3). During the cyclooxygenase-catalyzed conversion of arachidonic acid to PGG2 (Fig. 1), peroxyl radicals are formed as reaction intermediates. These lipid peroxyl radicals represent a source of activated oxygen and can in turn directly bioactivate procarcinogens or promutagens. Peroxyl radicals are also formed from hyperperoxides that are formed from unsaturated fatty acids by lipoxygenases. For example, 15-1ipoxygenase oxidizes arachidonic acid to 15-hydroperoxy-eicosatetraenoic acid (15-HPETE) which is subsequently degraded by the lipoxygenase to intermediates which contain peroxyl radicals. This mechanism of PHS-dependent oxidation is important in the epoxidation of several carcinogenic bay region polycyclic aromatic hydrocarbon (PAH) diols and is discussed in more detail later in the review. Additional information on peroxyl radical-dependent bioactivations is available in recent review articles (Smith et al., 1991; Eling et al., 1990; Reed, 1987). The third mechanism of PHS-dependent bioactivation is based on the fact that the peroxidase catalyzes one-electron oxidation of chemicals to often highly reactive free radicals (Fig. 3). These radicals under the right circumstances, can react with a second chemical that results in bioactivation of this secondary compound. For example, the nonsteroidal anti-inflammatory drug phenylbutazone is oxidized by PHS peroxidase to a carbon-centered free radical. The carbon-centered free radical traps molecular oxygen to form a peroxyl radical (Reed et al., 1984). This peroxyl radical

Hydroperoxide-dependcnt oxidation

265

I. Peroxidase-medlated pathway: ROOH 2AH

ROH

"~,.~ _...~ peroxidase

Ex=n~:

©-

2A°

ROH

OH

© O°

peroxidase

il. P e r o x y l r a d i c a l - m e d i a t e d p a t h w a y : ArachidonicAcid

cyclooxygenase :

= PGG2

[ROO']

×0

Example: Roo" ~

~ . . . , Roll

HO

HO OH

ROH

OH

IlL Cosubstrate-derlved oxidant: ROOH 1) 2AH

ROH

~'~ J f =, 2A" peroxidase

2) 2A" + 202 AO0"

~ 2AOO° AOH

3) XH ~

XO

Example: SO~

02 peroxidase

= o3soo" ~

OH

o3so.

HO OH

FIG. 3. Reaction mechanisms for chemical oxidations that occur during arachidonic acid metabolism.

266

T.E. ELINGand J. F. CURTIS TABLE 1. Chemicals Converted to Mutagens During Arachidonic Acid Metabolism in Ames Salmonella Tester Strains Chemicals Benzidine and analogs Benzidine N-Acetylbenzidine 2,4-Diaminoanisole 2,5-Diaminoanisole Aromatic amines 2-Aminofluorene 2-Naphthylamine Heterocyclic aromatic amines 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) 2-Amino-3,4-dimethylimidazo[4,5-f]quinoline (MelQ) Polycyclic aromatic hydrocarbons 7,8-Dihydroxy-7,8-dihydrobenzo(a)pyrene (BP-7,8-diol) ! ,2-Dihydro- 1,2-dihydroxychrysene (Chrysene- 1,2-diol) 3,4-Dihydro-3,4-dihydroxybenzo(a)anthracene (BA-3,4-diol) Cyclopentenyl[c,d]pyrene (CPP)

can bioactivate other compounds by the second mechanism discussed above. Another example of this mechanism of indirect bioactivation is the potentiation of benzo(a)pyrene-anti-diolepoxide formation by bisulfite (Reed et al., 1986). Bisulfite is oxidized by the peroxidase to a bisulfite anion free radical which in turn reacts with molecular oxygen to form a peroxyl radical. This peroxyl radical can react with BP-7,8-dihydroxy-7,8-dihydro (BP-7,8-diol) to yield the anti-diolepoxide. We have proposed that this reaction may in part be responsible for the observed enhancement in BP-induced lung tumors by co-exposure to bisulfite (Reed et al., 1986).

4. BIOLOGICAL IMPLICATIONS OF METABOLISM D U R I N G ARACHIDONIC

ACID OXIDATION 4.1. MUTAGENICACTIVATION Mutagenicity assays are used to demonstrate the formation ofmutagenic metabolites which arise during PHS-dependent oxidation. Salmonella bacterial tester strains, a source of PHS activity (either ram seminal vesicle microsomes or purified PHS) and test chemicals are incubated and the formation of the mutagen is detected by the standard assay. These studies indicate several polycyclic aromatic hydrocarbons and aromatic amines were activated to mutagenic products (Table 1) by PHS. Polycyclic aromatic hydrocarbons are carcinogenic environmental contaminants. Most polycyclic aromatic hydrocarbons require several enzymatic reactions to form their ultimate mutagenic and carcinogenic metabolites. The first reaction is an epoxidation catalyzed by cytochrome P-450. This epoxide is hydrolyzed by epoxide hydrolase to a PAH dihydrodiol. The final step is epoxidation of the dihydrodiol to a highly reactive diolepoxide. The epoxidations are catalyzed by cytochromes P-450, but peroxyl radicals formed during arachidonic acid metabolism can also catalyze the epoxidation of some PAH-diols to the PAH diolepoxides. Benzo(a)pyrene metabolism has served as a model for these studies. The bay region 7,8-diol of benzo(a)pyrene (BP) was activated to mutagenic products during arachidonic acid oxidation by PHS (Marnett et al., 1978). The epoxidation and mutagenicity of BP-7,8-diol was dramatically reduced by inhibiting the formation of peroxyl radicals. Neither BP nor the non-bay region BP-diols were activated to mutagenic metabolites by PHS. The bay region diols of benzo(a)anthracene and chrysene were also activated to mutagenic products by PHS (Guthrie et al., 1982). Epoxidation of the PAH cyclopenteno(c,d)pyrene (CPP) is a unique example of PAH epoxidation by peroxyl radical. It was directly epoxidized to a diol (Reed and Ryan, 1990;

Hydroperoxide-dependent oxidation

267

TABLE2. Formation of Aromatic Amine Mutagens by PHS Relative mutagenicity Chemical TA-98 pYG- 121* 2-Aminofluorene ++ ++++ Benzidine + ++ Acetylbenzidine +++ +++++ 2-Naphthylamine ++ NT 2,4-Diaminoanisole + NT 2,5-Diaminoanisole ++ NT Aniline NT Acetylaminofluorene Trp-l-P Glu-P-1 + IQ ++ ++++ MelQ ++ ++++ o-anisidine + p-anisidine + +++ *High acetylase activity.

Reed et al., 1988) by the peroxyl radicals without prior conversion by cytochrome P-450 and epoxide hydrolase. These studies indicate that the production of peroxyl radicals during the metabolism of arachidonic acid by PHS is an important mechanism of activation of PAH metabolites to mutagens. The PHS will also activate aromatic amine carcinogens to mutagens. Benzidine, 2-aminofluorene (2-AF), 2-naphthylamine (2-NA) and 2,5-diaminoanisole were metabolized by PHS to products which were mutagenic to standard Salmonella tester strains (Robertson et al., 1983). In contrast to PAH, the formation of aromatic amine mutagens is dependent upon the peroxidase activity of PHS. The PHS-dependent mutagenic metabolite(s) of most aromatic amines are unknown. The mutagenic metabolite of 2-AF aromatic amines catalyzed by PHS may be a free radical but definite data supporting this conclusion are lacking (Boyd and Eling, 1984; Boyd et al., 1983, 1985; Krauss and Eling, 1985). Benzidine was metabolized by PHS to free radical intermediates capable of forming adducts with glutathione, N-acetylcysteine and DNA (Josephy et al., 1983; Wise et al., 1985; Josephy and Iwaniw, 1985; Yamazoe et al., 1988). However, horseradish peroxidase also catalyzes the formation of the same free radical metabolites of benzidine as PHS yet does not activate benzidine to mutagenic products. Furthermore, benzidine diimine, the two-electron oxidation product of benzidine formed during the peroxidative metabolism of benzidine also lacks mutagenicity (Josephy and Subden, 1984). The activation of benzidine and other aromatic amines (Table 2) by PHS was also studied using Salmonella tester strains with varying amounts of acetyl CoA-dependent arylamine N-acetyl transferase/aryl hydroxylamine O-acetyltransferase activity (Watanabe et al., 1987). Salmonella tester strains with high acetyltransferase (Josephy et al., 1989) activity are extremely sensitive to PHS-dependent benzidine mutagenicity (Petry et al., 1988). While mutagenicity was not observed in a Salmonella strain devoid of acetyltransferase activity (TAI538/I,8-DNP) (Petry et al., 1988). In some Salmonella strains, PHS activates N-acetylbenzidine to a more potent mutagen than benzidine. These studies suggest that the formation of N-acetylbenzidine occurs prior to mutagenic activation by PHS. Other data suggests that additional but undefined acetylation events are also important (unpublished observations). Heterocyclic aromatic amine carcinogens formed from the pyrolysis of amino acids or proteins are also activated to mutagenic metabolites by PHS. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and several methylated IQ derivatives are converted by PHS peroxidase to extremely potent mutagens (Wild and Degen, 1987; Petry et al., 1988). For example, the specific mutagenic activity (revertants/nmol) catalyzed by PHS of IQ is approximately 50-100 fold greater than the aromatic amine bladder carcinogens. Like benzidine, PHS-mediated IQ mutagenicity was enhanced or diminished in Salmonella strains with high or low acetyltransferase activity, respectively (Petry et al., 1988).

268

T.E. ELINGand J. F. CURTIS 4.2. CARCINOGENICITY

Many of the chemicals metabolized to mutagens during arachidonic acid oxidation induce carcinogenesis in experimental animals and man. Tumors are frequently observed in tissues that have relatively high PHS activity. Many aromatic amines that are oxidized by the peroxidase activity of PHS to mutagens are established human bladder carcinogens while the PAHs epoxidized by peroxyl radicals generated during arachidonic acid oxidation induce skin and lung neoplasia. Determining the relative contribution of PHS and peroxyl radicals in the bioactivation of aromatic amines and polycyclic aromatic hydrocarbons is fraught with the same problems and difficulties that have plagued investigations of the bioactivation of enzyme systems. One approach to determine the biological importance of peroxidase- and peroxyl radical-mediated bioactivation is to either enhance or inhibit these metabolic processes. A second and more successful approach is to measure unique metabolites or DNA adducts, for the peroxidase- and peroxyl radical-dependent bioactivation, in tissues that were exposed to the carcinogen. Non-steroidal anti-inflammatory drugs such as indomethacin or aspirin inhibit the cyclooxygenase activity of PHS and thus inhibit bioactivation of carcinogens dependent on PGG2 generated by the cyclooxygenase and the peroxidase activity of PHS. The cyclooxygenase can also trigger events that lead to the formation of peroxyl radicals that convert PAH to the ultimate carcinogenic metabolites, the diolepoxides. However, this approach is flawed since other enzyme systems not inhibited by the anti-inflammatory drug produce peroxides and peroxyl radicals. Thus, even if the cyclooxygenase activity of PHS is blocked, other sources can supply the peroxide or the peroxyl radicals necessary for their bioactivation. These studies require the use of a peroxidase inhibitor or a specific inhibitor of peroxyl radical-mediated metabolism, which are not available. The most successful approach to determining the biological importance of peroxidase- and peroxyl radical-mediated bioactivation relies upon the formation and measurement of unique markers for these reactions. Peroxidase-dependent metabolisms are one-electron oxidations compared to the two-electron oxidations catalyzed by the cytochrome P-450 monooxygenase. Thus, the potential exists for different metabolites to be formed by the peroxidase and monooxygenases. Moreover, in many cases the electrophilic metabolite formed by the peroxidase is different from that catalyzed by monooxygenases and thus different adducts with cellular macromolecules can be formed. For PAH, the peroxyl radicals form the same metabolites as monooxygenases but the stereochemistry of the metabolites is different (Pruess-Schwartz et al., 1989). The mechanistic difference between the peroxidase/peroxyl radical-dependent systems and monooxygenases serves as a rational basis for the development of unique markers that can be used in vivo to test for the biological importance of bioactivation that occur during arachidonic acid oxidation (Eling et al., 1986a; Pruess-Schwartz et al., 1989). Characterization of the DNA adducts of aromatic amines catalyzed by PHS provide unique biochemical markers to measure peroxidase bioactivation in vivo of some aromatic amine carcinogens. The peroxidase-catalyzed DNA adducts are different from monooxygenase-dependent DNA adducts. Kadlubar and coworkers characterized several peroxidase-catalyzed DNA adducts of benzidine (Yamazoe et al., 1988, 1986) and 2-NA (Yamazoe et al., 1985) and we have partially characterized the peroxidase-catalyzed DNA adducts of 2-AF (Krauss and Eling, 1985). Our data indicate the formation of two uncharacterized adducts of the peroxidase which are different than N-(deoxyguanosin-8-yl)-2-AF (dG-2-AF) which is formed by the reaction of N-hydroxy-2-AF with DNA. Krauss et al. reported the detection of peroxidase-catalyzed N- (deoxyguanosin-8-yl)-2-AF (2-AF-DNA) adducts in the kidney and bladder epithelia of dogs fed radiolabeled 2-AF (Krauss et al., 1989). The 2-AF-DNA adduct (dG-2-AF) detected in the liver was the one catalyzed by the monooxygenase system. Kadlubar observed peroxidase-catalyzed benzidine and 2-NA DNA adducts in the bladder urothelium but not in the liver of dogs after treatment of the dogs with radiolabeled carcinogens (Yamazoe et al., 1985). Both 2-AF and benzidine induce urinary bladder cancer but not liver cancer in dogs. The presence of the peroxidase-derived DNA adducts of 2-AF, benzidine and 2-NA suggests that they may be of biological importance in aromatic amine-induced canine bladder cancer. Since the dog is the model for human bladder cancer and human bladder epithelial cells contain relatively high amounts of PHS, it is tempting to speculate that PHS bioactivation is also important in the human disease.

Hydroperoxide-dependent oxidation

269

The stereoselective difference between the epoxidation of BP-7,8-diol by peroxyl radicals and the monooxygenase enzyme is an excellent marker for determining the contributions of these two enzyme systems. The (+)BP-7,8-diol is converted by peroxyl radicals to the anti-diolepoxide while the monooxygenase system forms the syn-diolepoxide (Panthananickal et al., 1983). Measurement of the ratio of anti to syn diolepoxide by HPLC analysis of their stable hydrolysis products, the tetrols, gives an excellent assessment of the contribution of these two bioactivation systems. This stereochemical approach was used for investigations with mouse keratinocytes in vitro (Eling et al., 1986a) and mouse skin in vivo (Pruess-Schwartz et al., 1989). The data from these investigations established that peroxyl radicals are responsible for the epoxidation in intact skin or isolated keratinocytes from control animals. However, treatment of the mice with fl-napthoflavone, an inducer of certain cytochromes P-450, enhanced the total epoxidation and shifted the epoxidation to a monooxygenase-dependent pathway. The use of this stereochemical approach needs to be extended to other tissues where polycyclic aromatic hydrocarbons induce tumors to establish the contribution of the peroxyl radicals and the monooxygenase systems in the initiation of the neoplasia.

5. DETOXIFICATIONS CATALYZED BY PROSTAGLANDIN H SYNTHASE Activation of chemicals has been the major focus of peroxidase and peroxyl radical mediated metabolism. However, other studies indicate a role in detoxification of chemicals including several carcinogens. Glutathione is a cofactor for the detoxifying enzymes glutathione transferases which act to form water soluble conjugates of potentially carcinogenic epoxides (Ross, 1988). Glutathione also can reduce free radicals generated by PHS and peroxyl radicals. As a consequence of this reduction, glutathione is oxidized to a glutathionyl free radical (GS') as described below (Eling et al., 1986b). The formation of GS" eventually can lead to a reaction that detoxifies potential carcinogens and toxicants. Two mechanisms are established by which PHS peroxidase forms GS'. Glutathione serves as a cosubstrate for PHS peroxidase and is oxidized to GS" (Eling et al., 1986b). Using either ram seminal vesicle microsomes or purified PHS, we detected the formation of GS" by electron spin resonance which was dependent upon a functioning peroxidase, glutathione (GSH) and a peroxide source (Eling et aL, 1986b). In the second mechanism, the peroxidase first oxidizes a chemical that is a more efficient reducing cosubstrate than GSH (e.g. phenols, aromatic amines) (Markey et al., 1987). The cosubstrate-derived radical produced by this oxidation is then reduced by GSH forming both GS" and the parent reducing cosubstrate. With efficient reducing cosubstrates such as aminopyrine (Eling et al., 1985), phenol (Stock et al., 1986) and acetaminophen (Eling et al., 1985) this process leads to markedly enhanced levels of GS'. GS" can add directly to compounds, thereby forming GSH conjugates. This new route for formation of GSH conjugates, mediated by peroxidases through the production of GS" (Stock et al., 1989) is compared to the glutathione (s) transferase (GST)/monooxygenase pathway in Fig. 4. In the presence of GSH, PHS and arachidonic acid, styrene was directly converted to two isomeric glutathione conjugates in contrast to the four isomeric GSH conjugates formed by GST-catalyzed addition of GSH to styrene oxide. The mechanism for the formation of glutathione conjugates via the peroxidase-mediated pathway is illustrated in Fig. 5. GS" adds to a double bond proximal to a highly conjugated system such as an aromatic ring. This results in the formation of a carbon-centered radical which reacts with molecular oxygen to yield a peroxyl radical. The peroxyl radical is then reduced to the alcohol yielding the glutathione conjugate. Thus glutathione conjugation occurs in the absence of epoxide formation and catalysis by glutathione transfers. Other studies (Foureman and Eling, 1989) showed that an exocyclic double bond, as found in styrene, is required for a compound to undergo GS" addition. For example the precocenes, anti-juvenile hormones, possess this structural arrangement. Several PAH dihydrodiols, which possess structural similarity to styrene, were tested for their ability to undergo GS" addition catalyzed by PHS-peroxidase (Foureman and Eling, 1989). For example, BP-9,10-diol, BP-7,8-diol, BA-3,4-diol and BA-1,2-diol are all extensively converted to GSH conjugates by this mechanism.

270

T.E. ELINGand J. F. CURTIS

" OC";C o O ~?H--

8

. (R,S)

[~

CH2--OH

(R,S)

?0H-CH2~sG H (R,S)

FIG. 4. Reaction mechanism for peroxidase-catalyzed glutathione conjugate formation. Peroxidase-catalyzed formation of GSH conjugates is therefore a general mechanism and may represent an alternative route to detoxication of a wide variety of compounds.

6. S U M M A R Y The cooxidation reactions that occur during arachidonic acid metabolism are dependent upon the peroxidase activity of PHS. For chemicals that are not substrates for the peroxidase, the epoxidation reactions that occur are dependent upon the subsequent formation of peroxyl radicals. A large and diverse number of chemicals are metabolized by an equally large and diverse number of chemical reactions. The unifying theme is the free radical nature of these oxidations. The subsequent reactions that these chemicals undergo are dictated by the nature of the free radical and the environment in which the radical is generated. Ample evidence now exists that these free radical-mediated reactions can contribute to the formation of toxic metabolites, but in some cases can also participate in the detoxification of chemicals. The overriding factor for this type of metabolism to occur is the relative concentrations of PHS and peroxyl radicals with respect to other activating systems, particularly the monooxygenase system, in the specific tissue. In vivo investigations support the importance of the peroxidase and peroxyl radical systems in both activation and detoxification of chemicals in extrahepatic tissues. Ackowledgements--The authors wish to acknowledge Mrs Kathy Robinson for her editorial assistance in

preparing this manuscript.

REFERENCES

BOYD,J. A. and ELING,T. E. (1984) Evidence for a one-electron mechanism of 2-aminofluorene oxidation by prostaglandin H synthase and horseradish peroxidase. J. biol. Chem. 259: 13885-13896. BoYo, J. A., HARVAN, D. J. and ELING, T. E. (1983) The oxidation of 2-aminofluorene by prostaglandin endoperoxide synthetase. Comparison with other peroxidases. J. biol. Chem. 258: 8246-8254.

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BOYD, J. A., ZEIGER, E. and EL1NG,T. E. (1985) The prostaglandin H synthase-dependent activation of 2-aminofluorene to products mutagenic to S. typhimurium strains TA98 and TA98NR. Mutat. Res. 143: 187-190. CHRIST,E. J. and VAN DORP. D. A. (1972) Comparative aspects of prostaglandin biosynthesis in animal tissues. Biochim. biophys. Acta 270: 537-545. DEWITT, D. L. and SMITH,W. L. (1988) Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence. Proc. natn. Acad. Sci. U.S.A. 115: 1412-1416. DEWITT, D. L., DAY, J. S., SONNENBURG,W. K. and SMITH,W. L. (1983) Concentrations of prostaglandin endoperoxide synthase and prostaglandin I 2 synthase in the endothelium and smooth muscle of bovine aorta. J. clin. Invest. 72: 1882-1888. ELING, T. E., MASON, R. P. and SIVARAJAH,K. (1985) The formation of aminopyrine cation radical by the peroxidase activity of prostaglandin H synthase and subsequent reactions of the radical. J. biol. Chem. 260: 1601-1607. ELING, T. E., CURTIS, J., BATTISTA,J. and MARNETT, L. J. (1986a) Oxidation of (+)-7,8-dihydroxy-7,8dihydrobenzo(a)pyrene by mouse keratinocytes: Evidence of peroxyl radical- and monooxygenasedependent metabolism. Carcinogenesis 4: 1957--1963. ELING,T. E., CURTIS,J. F., HARMAN,L. S. and MASON,R. P. (1986b) Oxidation of glutathione to its thiol free radical metabolite by prostaglandin H synthase. A potential endogenous substrate for the hydroperoxidase. J. biol. Chem. 261: 5023-5028. ELING,T. E., THOMPSON,D. C,, FOUREMAN,G. L., CURTIS,J. F. and HUGHES,M. F. (1990) Prostaglandin H synthase and xenobiotic oxidation. A. Rev. Pharmac. Toxicol. 30: 1-45. FOUREMAN, G. L. and ELING, T. E. (1989) Peroxidase-mediated formation of glutathione conjugates from polycyclic aromatic dihydrodiols and insecticides. Arch. Biochem. Biophys. 269: 55-68. FUNK, C. D., FUNK, L. B., KENNEDY, M. T., PONG, A. S. and FITZGERALD,G. A. (1991) Human platelet/ erythroleukemia cell prostaglandin G/H synthase: cDNA cloning, expression and gene chromosomal assignment. FASEB J. 5: 2304-2312. Gu~Rm, J., ROBERTSON,I. G. C., ZEIGER,E., BOYD,J. A. and ELING,T. E. (1982) Selective activation of some dihydrodiols of several polycyclic aromatic hydrocarbons to mutagenic products by prostaglandin synthetase. Cancer Res. 42: 1620-1623. HEMLER, M., LANDS, W. E. M. and SMITH, W. L. (1976) Purification of the cyclooxygenase that forms prostaglandins. Demonstration of two forms of iron in the holoenzyme. J. biol. Chem. 251: 5575-5579. HEMLER,M. E., COOK,H. W. and LANDS,W. E. M. (1979) Prostaglandin biosynthesis can be triggered by lipid peroxides. Arch. Biochem. Biophys. 193: 340-345. HLA, T., FARRELL,M., KUMAR,A. and BAILEY,J. M. (1986) Isolation of the CDNA for human prostaglandin H synthase. Prostaglandins 32: 829-845. HUSLIG, R. L., FOGWELL,R. I. and SMIT,, W. L. (1979) The prostaglandin forming cyclooxygenase of ovine uterus: Relationship to luteal function. Biol. Reprod. 21: 589-600. JOSEPHY,P. D. and IWANIW,D. C. (1985) Identification of the N-acetylcysteine conjugate of benzidine formed in the peroxidase activation system. Carcinogenesis 6: 155-158. JOSEPHY,P. D. and SUBDEN,R. E. (1984) Hydrogen peroxide-dependent activation of benzidine to mutagenic species. Mutat. Res. 141: 23-38. JOSEPHY,P. D., ELING,T. E. and MASON,R. P. (1983) Cooxidation of benzidine by prostaglandin synthase and comparison with the action of horseradish peroxidase. J. biol. Chem. 258: 5561-5569. JOSEPHY, P. D., Cmu, A. L. H. and ELING, T. E. (1989) Prostaglandin H synthase-dependent mutagenic activation of benzidine in a Salmonella typhimurium Ames tester strain possessing elevated N-acetyltransferase levels. Cancer Res. 49: 853-856. KRAtJSS, R. S. and ELING,T. E. (1985) Formation of unique arylamine: DNA adducts from 2-aminofluorene activated by prostaglandin H synthase. Cancer Res. 45: 1680-1686. KRAUSS,R. S., ANGERMAN-STEWART,J., DOOLEY,K. L., KADLUBAR,F. F. and ELING,T. E. (1989) The formation of 2-aminofluorene-DNA in vivo: Evidence for peroxidase-mediated activation. Biochem. Toxicol. 4: lll-ll7. KULMACZ, R. J. and LANDS, W. E. M. (1983) Requirements for hydroperoxide by the cyclooxygenase and peroxidase activities of prostaglandin H synthase. Prostaglandins 25: 531-540. M ARKEY,C. M., ALWARD,A., WELLER,P. E. and MARNETT,L. J. (1987) Quantitative studies of hydroperoxide reduction by prostaglandin H synthase. Reducing substrate specificity and the relationship of peroxidase to cyclooxygenase activities. J. biol. Chem. 262: 6266--6279. MARNETT, L. J. and ELING,T. E. (1983) Cooxidation during prostaglandin biosynthesis: A pathway for the metabolic oxidation of xenobiotics. Rev. Biochem. Toxicol. 5: 153-172. MARNETT, L. J., REED, G. A. and DENNISON,D. J. (1978) Prostaglandin synthetase-dependent activation of 7,8-dihydro-7,8-dihydrobenzo(a)pyrene to mutagenic species. Bioehem. biophys. Res. Commun. 82: 210-216. MASON, R. P., KALYANARAMAN,B., TAINER, B. E. and ELING, T. E. (1980). A carbon-centered free radical intermediate in the prostaglandin synthetase oxidation of arachidonic acid. Spin trapping and oxygen uptake studies. J. biol. Chem. 255: 5019-5022. MERLIE, J. P., FAGAN, n., MUDD, J. and NEEDLEMAN,P. (1988) Isolation and characterization of the complementary DNA for sheep seminal vesicle prostaglandin endoperoxide synthase (cyclooxygenase). J. biol. Chem. 263: 3550-3553.

272

T. E. ELINGand J. F. CURTIS

MEVKH,A. T., SUD'INA,G. F., GOLUB,N. B. and VARFOLOMEEV,S. D. (1985) Purification of prostaglandin H synthetase and a fluorometric assay for its activity. Anal. Biochem. 150: 91-96. MIYAMOTO,T., OGINO,N., YAMAMOTO,S. and HAYAISHI,O. 0976) Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. biol. Chem. 251: 2629-2636. OGINO, N., OHKI, S., YAMAMOTO,S. and HAYAISHI,O. (1978) Prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. Inactivation and activation by heme and other metalloporphyrins. J. biol. Chem. 253: 5061-5068. PACE-ASCIAK,C. R. and RANGARAJ,G. (1977) Distribution of prostaglandin biosynthetic pathways in several rat tissues. Formation of 6-keto-prostaglandin F~. Biochim. biophys. Acta 486: 579-582. PACE-ASCIAK,C. R. and SMITH,W. L. (1983) Enzymes in the biosynthesis and catabolism of the eicosanoids: Prostaglandins, thromboxanes, leukotrienes and hydroxy fatty acids. Enzymes 16: 543-603. PANTHANANICKAE,A., WELEER,P. and MARNETT,L. J. (1983) Stereoselectivity of the epoxidation of 7,8dihydrobenzo(a)pyrene by prostaglandin H synthase and cytochrome P-450 determined by the identification of polyguanylic acid adducts. J. biol. Chem. 258:4411-4418. PETRY,T. W. and ELING,T. E. (1987) The mechanism for the inhibition of prostaglandin H synthase-catalyzed xenobiotic oxidation by methimazole. Reaction with free radical oxidation products. J. biol. Chem. 262: 14112-14118. PETRV,T. W., ELING,T. E., CHIU,A. L. H. and JOSEPHY,P. D. (1988) Ram seminal vesicle microsome-catalyzed activation of benzidine and related compounds: Dissociation of mutagenesis from peroxidase-catalyzed formation of DNA-reactive material. Carcinogenesis 9: 51-57. PLL P. and MARNETTE,L. J. (1989) Alkylaryl sulfides as peroxidase reducing substrates for prostaglandin H synthase: Probes for the reactivity and environment of the ferryl-oxo complex. J. biol. Chem. 264: 13983-13993. PRUESS-SCHWARTZ,D., NIMESHEIM,A. and MARNETT, L. J. (1989) Peroxyl radical and cytochrome P-450 metabolic activation of (+)-7,8-dihydroxy-7,8-dihydrobenzo(a)-pyrene in mouse skin in vitro and in vivo. Cancer Res. 49:1732 1737 REED, G. A. (1987) Cooxidation of xenobiotics: Lipid peroxyl radicals as mediators of metabolism. Chem. Phys. Lipids 44: 127-148. REED, G. A. and RYAN, M. J. (1990) Peroxyl radical-dependent epoxidation of cyclopenteno[c,d]pyrene. Carcinogenesis 11:1825 1829. REED, G. A., BROOKS,E. A. and ELING,T. E. (1984) Phenylbutazone-dependent epoxidation of 7,8-dihydroxy7,8-dihydrobenzo(a)pyrene. A new mechanism for prostaglandin H synthase-catalyzed oxidations. J. biol. Chem. 259:5591-5595 REED, G. A., CURTIS,J. F., MOTTLEY,C., EUNG, T. E. and MASON,R. P. (1986) Epoxidation of (_+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene during (bi)sulfite autoxidation: Activation of a procarcinogen by a cocarcinogen. Proc. natn. Acad. Sci. U. S. A. 83: 7499-7502. REED,G. A., LAVTON,M. E. and RYAN,M. J. (1988) Metabolic activation of cyclopenteno[c,d]pyrene by peroxyl radicals. Carcinogenesis 9: 2291-2295. ROBERTSON,I. G. C., SWARAJAH,K., ELING,T. E. and ZEmER, E. (1983) Activation of some aromatic amines to mutagenic products by prostaglandin endoperoxide synthetase. Cancer Res. 43: 476-480. ROLLINS,T. E. and SMITH, W. L. (1980) Subcellular localization of prostaglandin-forming cyclooxygenase in Swiss mouse 3T3 fibroblasts by electron microscopic immunocytochemistry. J. biol. Chem. 255: 4972-4975. Ross, D. (1988) Glutathione, free radicals and chemotherapeutic agents. Mechanisms of free radical-induced toxicity and glutathione-dependent protection. Pharmac. Ther. 37: 231-249. SCHREmER,J., ELING,T. E. and MASON,R. P. (1986) The oxidation of arachidonic acid by the cyclooxygenase activity of purified prostaglandin H synthase: Spin trapping of a carbon-centered free radical intermediate. Arch. Biochem. Biophys. 249: 126-136. SMITH, B. J., CURTIS,J. F. and ELING,T. E. (1991) Bioactivation of xenobiotics by prostaglandin H synthase. Chem. biol. Interact. 79: 245-264. SMITH, W. L. and BELL,T. G. (1978) Immunohistochemical localization of the prostaglandin-forming cyclooxygenase in renal cortex. Am. J. Physiol. 235: F451-457. SMITH, W. L. and MARNETT, L. J. (1991) Prostaglandin endoperoxide synthase: structure and catalysis. Biochem. biophys. Acta 1083: 1-17. STOCK, B. H., BEND,J. R. and EL1NG,T. E. (1986) The formation of styrene glutathione adducts catalyzed by prostaglandin H synthase. A possible new mechanism for the formation of glutathione conjugates. J. biol. Chem. 261: 5959-5964. VAN DER OUDERAA,F. J., BUYTENHEK,M., NUGTEREN,D. H. and VAN DORP, D. A. (1977) Purification and characterization of prostaglandin endoperoxide synthetase from sheep vesicular glands. Biochim. biophys. Acta 487: 315-331. WATANABE,M., NOHMI,T. and ISHIDATE,JR, M. (1987) New tester strains of Salmonella typhimurium highly sensitive to mutagenic nitroarenes. Biochem. biophys. Res. Commun. 147: 974-979. WILD,D. and DEGEN,G. H. (1987) Prostaglandin H synthase-dependent mutagenic activation of heterocyclic aromatic amines of the IQ type. Carcinogenesis 8: 541-545. WISE, R. W., ZENSER,T. V. and DAVIS, B. B. (1985) Prostaglandin H synthase oxidation of benzidine and o-dianisidine: Reduction and conjugation of activated amines by thiols. Carcinogenesis 6: 579-583.

Hydroperoxide-dependent oxidation

273

YAMAZOE,Y., MILLER,D. W., WEISS,C. C., DOOLEY,K. L., ZENSER,T. V., BELAND,F. A. and KADLUBAR,F. F. (1985) DNA adducts formed by ring-oxidation of the carcinogen 2-naphthylamine with prostaglandin H synthase in vitro and in the dog urothelium in vivo. Carcinogenesis 6: 1379-1387. YAMAZOE,Y., ROTH, R. W. and KADLUBAR,F. F. (1986) Reactivity of benzidine diimine with DNA to form N-deoxyguanosin-8-yl-benzidine. Carcinogenesis 7:179-182. YAMAZOE, Y., ZENSER, T. V., MILLER, D. W. and KADLUBAR,F. F. (1988) Mechanism of formation and structural characterization of DNA adducts derived from peroxidative activation of benzidine. Carcinogenesis 9: 1635-1641. YOKOYAMA,C and TANABE,T. (1989) Cloning of human gene encoding prostaglandin endoperoxide synthase and primary structure of the enzyme. Biochem. biophys. Res. Commun. 165: 888-894. YOKOYAMA,C., TAKAI,T. and TANABE,T. (1988) Primary structure for sheep prostaglandin endoper~xide synthase deduced from cDNA sequence. FEBS Lett. 231: 347-351. YOSHIMOTO,T., MAGATA, K., EHARA, H., MIZUNO, K. and YAMAMOTO,S. (1986) Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies. Biochim. biophys. Acta 877: 141-150. ZENZER,T. V. and DAVIS,B. B. (1988) Arachidonic acid metabolism by human urothelial cells: Implication in aromatic amine-induced bladder cancer. Prostaglandins Leuk. Essent. Fatty Acids: Rev. 31: 199-207.