Oxygen and Xenobiotic Reductase Activities of Cytochrome P450

Critical Reviews in Toxicology, 25(1):25-65 (1 995) Oxygen and Xenobiotic Reductase Activities of Cytochrome P450 Arnold R. Goeptar, Heleen Scheerens, and Nico P. E. Vermeulen* LeidewAmsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands * Adress all correspondence to: Nico P. E. Venneulen, LeiWAmsterdam Center for Drug Research, Division of Molecular Toxicology, Vrije Universiteif De Bcelelaan 1083, 1081 HV Amsterdam, 7hc Netherlands. ABSTRACT: The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (OF.) most likely by autooxidation of a P450 ferric-dioxyanion complex. The formation of reactive oxygen species (Of‘, hydrogen peroxide, and, notably, hydroxyl free radicals) presents a potential toxication pathway, particularly when effective means of detoxication are lacking. Under anaerobic conditions, P450 may also be involved in the reduction of xenobiotics. During the xenobiotic reductase activity of P450, xenobiotics are reduced by the ferrous xenobiotic complex. After xenobiotic reduction by P450, xenobiotic free radicals are formed that are often capable of reacting directly with tissue macromolecules. Unfomnately, the compounds that are reductively activated by P450 have little structural similarity. The precise molecular mechanism underlying the xenobiotic reductase activity of P450 is, therefore, not yet fully understood. Moreover, description of the molecular mechanisms of xenobiotic and oxygen reduction reactions by P450 is limited by the lack of knowledge of the three-dimensional (3D) structure of the mammalian P450 proteins. KEY WORDS: cytochrome P450, reductive activity, uncoupling, free radicals, oxygen reductase, xenobiotic reductase. 1. INTRODUCTION Cytochrome P450 (P450) refers to a group of iron-containing hemoproteins whose reduced car- bon monoxide complexes exhibit W-visible spec- tra with Soret maxima around 450 nm.’ P450 enzymes were discovered in the 1950s and are known to play a major role in the metabolism of drugs, carcinogens, steroids, pesticides, hydro- carbons, and natural products. Endogenous com- pounds metabolized by P450 enzymes include steroids , prostanoids , eicosanoids , fat-soluble vi- tamins, fatty acids, and mammalian alkaloids.* The oxidative metabolism of substrates by P450 is generally recognized. However, P450 also is involved in the reduction of molecular oxygen and certain substrates. In this review, the molecu- lar mechanisms determining oxygen and substrate reduction by P450 are discussed. An attempt is made to provide new insights into the diverse mechanisms of electron transfer processes during these P450-mediated reduction reactions. A. Evolution of Cytochrome P450 It is believed that early in the evolution, Earth had an anaerobic reducing atmosphere. At that time, so-called “abiogenic” chemicals were form- ing associations to amino acids. The apoprotein of P450 could have been derived from abiogenic amino acids3 some 2 to 3 billion years ago (Fig- ure 1). Due to its low reduction and oxidation potential (around -350 mV), P450 could have functioned in reductive reactions exclusively at the time the atmosphere was mainly composed of hydrogen, nitrogen, methane, ammonia, carbon monoxide, and carbon dioxide. Chemical or pho- 1040-8444/95/$.50 0 1995 by CRC Press, Inc. 25 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. (P450 evolutiond 4L I 3 billion 2 billion Y- Reductive activity n 1 billion 400 million Year Y- 154 P450 genes 1993 I60 60 40 ao 1111 m E ? FIGURE 1. Schematic representation of the evolutionary development of cytochrome P450. The evolution of cytochrome P450 is believed to have begun 3 billion years ago. At that time, the atmosphere was mainly composed of hydrogen, nitrogen, methane, ammonia, carbon monoxide, and CO,. In this anaerobic environ- ment, the early activity of cytochrome P450 was limited to reductive reactions only. tochemical reduction could have provided elec- trons to early P450 in the absence of flavoproteins and reducing cofactors. The absence of 0, in the atmosphere would have made oxidative activity of P450 unlikely. The early activity of P450s might have been limited to reductive reactions under anaerobic conditions. The appearance of 0, in the atmosphere about 2 billion years ago (Figure 1)4 must have had a profound effect on the organisms that were used to living under anaerobic conditions. The appear- ance of 0, was presumably accompanied by the appearance of ozone (0,) in the upper atmosphere, and the absorption of damaging solar ultraviolet radiation by 0, and ozone probably permitted the evolution of oxygen-evolving photosynthetic or- ganisms? The diatomic oxygen molecule (0-0) is a diradical due to the presence of two unpaired 26 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. electrons in different n*2p antibonding orbital^.^ 0, is a good oxidizing agent; however, due to spin restrictions according to Pauli’s principle, diatomic oxygen can only be reduced by accepting elec- trons sequentially. The addition of the first elec- tron to molecular dioxygen produces the superox- ide anion radical (O;.). A second electron yields a peroxide anion (OF) that has no unpaired elec- trons. Protonation of 05 yields hydrogen perox- ide (H,O,). Little is known about three- and four- electron reduced 0, intermediate^.^ In aqueous solutions, 0 5 . dismutates enzymatically (by su- peroxide dismutase) or nonenzymatically to H,O, and 0,. In the presence of Cu+ or Fez+, extremely reactive and potentially cytotoxic hydroxyl radi- cals (OH) are formed in the so-called Fenton reaction: Fez+ + H,O, + Fe3+ + ‘OH + -OH The damaging effects of oxygen on anaerobic life forms may have been due to oxygen free radical-mediated oxidation of essential cellular membranes and macromolecules.5 Anaerobic re- actions proceed in reducing environments mainly by electron donation reactions in the presence of suitable electron acceptors. The gradual appear- ance of 0, under reductive conditions could have resulted in gradual formation of OF., H,O, and .OH. It has been suggested that initially P450 may have participated in the removal of unwanted 0, entering the cell.3 However, the reductive activity of early P450 forms most likely would have in- creased rather than decreased oxygen toxicity as a result of direct 0, reduction to reactive oxygen species. It seems, therefore, more likely that other enzyme(s) may have controlled 0, toxicity in ancient living organisms. The remarkable ubiq- uity of superoxide dismutase (SOD) activity as well as the fact that it was highly conserved evolutionarly has led researchers to suggest that the initial physiological function of this enzyme was to detoxify byproducts of the aerobic “lifestyle”.6 B. Emergence of the Cytochrome P450 Superfamily The emergence of new genes encoding P450 began approximately 1 billion years ago when animals and plants emerged (Figure 1): At that time, animals began to eat plants and the latter responded by producing phytoalexins. Subse- quently, the animals produced new P450 genes to detoxify these phytoalexins. In fact, a rapid ap- pearance of new P450 genes encoding new forms of P450 can be seen during the past 400 million years.8 The f i s t experimental evidence for the exist- ence of P450 proteins was obtained in the late 1950s (Figure 1). In 1958, it was found that pig and rat liver microsomes treated with dithionite and purged with carbon monoxide showed a unique Soret maximum at 450 nm.9J0 The “pig- ment” responsible for this unusual absorption was called P (for pigment) 450. It was named “cyto- chrome P450” and further characterized as a P450 hemoprotein in the 1960s., In 1967, two major forms of P450 were characterized, namely, phenobarbital- and 3-methylcholanthrene-induc- ible forms.ll In 1987, 31 different genes were described, and an updated list presented only 2 years later contained 71 genes.’, In 1990, the “P450 superfamily” consisted of 154 different genes described in a total of 23 eukaryotes and 7 prokaryotes (Figures 1 and 2).13 C. Membrane Topology and Structure of Cytochrome P450 Most of the P450 isoenzymes in eukaryotes are found in membranes, mainly in the endoplas- mic reticulum and mitochondria. Liver microso- mal P450 constitutes 20% of the microsomal pro- tein (2 to 3% of the hepatic protein ~ontent).~ The current concepts of membrane topology for mi- crosomal P450 monooxygenases as proposed by Brown and Black are shown in Figure 3.14 By using trypsinized barbiturate-induced rabbit liver microsomes, they proposed two alternative mod- els (Figure 3). Model “A” is a bitopic N,-C,, topology , and model “B” is a polytopic, two- anchor hairpin model. In both models, strong contacts between the catalytic heme domain and the membrane are indicated (Figure 3), possibly mediated by peptide segment 3 16-330.14 In con- trast to microsomal P450s, the membrane topol- ogy of mitochondria1 P450 is less well character- ized. Certain features, such as the lack of a hydrophobic NH,-terminal anchor, are clearly 27 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. FIGURE 2. Cytochrome P450 gene family as described by Nebert et al., DNA Cell. Biol., 10, 1, 1991. The numbers between brackets indicate the designed numbering for the individual cytochrome P450s within a subfamily. different from the microsomal cytochromes P450.Is P450 is a hemoprotein consisting of an iron protoporphyrin IX heme moiety and a single polypeptide chain or apoprotein of 45,000 to 55,000 Da.I6 The iron of the heme prosthetic group is located at the center of the protoporphyrin ring. Four ligands of the heme iron can be attributed to the porphyrin ring. The fifth (axial) ligand is a thiolate anion from a cysteinyl residue of the apoprotein. The sixth (axial) ligand is probably a hydroxyl group from an amino acid residue, or a moiety with a similar field strength such as a water molecule. The iron of P450 exists in high-spin (Fe3+- P450,,) and low-spin (Fe3+-P450,) forms that are usually in equilibrium and possess different absorbance wavelengths (390 and 419 nm, re- spectively) (Figure 4).17 In Fe3+-P450,, the five 3d electrons of the iron are maximally paired to give a net spin of 1/2, whereas in the Fe3+-P450,, the five 3d electrons are maximally unpaired to give a net spin of 5/2 (Figure 4). Substrate inter- action with P450 can induce spin equilibrium shifts that can be followed spectrophotometri- cally.I7 Such spectral shifts reflect changes in the coordination environment of the ferric ion with the Fe3+-P450,, to Fe3+-P450, shift indicative of a strong field ligand axially coordinated to the central iron. A Fe3+-P450, to Fe3+-P450,, shift is indicative of an interaction between a hydropho- bic substrate and the apoprotein. The Fe3+-P450,, 28 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. A B FIGURE 3. Membrane topology of cytochrome P450 on the endoplasmic reticu- lum. The topological alternatives given (A) and (B) are modeled using the primary structure of hepatic rnicrosomal rabbit isoenzyme as described by Brown, C. A. and Black, S. D., J. Biol. Chem., 264, 4442, 1989. Model "A" is a bitopic N,,-C, topology and model "B" is a polytopic, two-anchored hairpin model. to Fe3+-P450Hs shift has been categorized as a type 1 interaction and a Fe3+-P450Hs to Fe3+-P450, shift is called a type 11 interaction (Figure 4).17 A reverse type I interaction between a hydrophobic substrate and P450 has also been described;18 however, the exact nature of this substrate-in- duced Fe3+-P450Hs to Fe3+-P450, conversion is not clear. In its resting state, the ferric iron is predomi- nantly Fe3+-P450,, probably with one water molecule acting as the sixth ligand trans to the thiolate (Figure 4). Because most P450 substrates are relatively hydroph~bic,'~ their binding prob- ably displaces water from the sixth ligand posi- tion such that the ferric iron preferentially lies out of the plane of the porphyrin ring (Figure 4).19 This involves a redistribution of the five 3d or- bital occupancy on the ferric iron to favor the Fe3+-P450Hs form, which cannot remain in the plane of the porphyrin ring due to an increase in ionic size.m The proximal thiolate ligand remains attached to the ferric iron in both Fe3+-P450Ls and Fe3+-P45OHS complexes.2l Much of our understanding of the structure and mechanism of the P450 system has been derived from work done with bacterial enzymes. For instance, P450CI (P450,, or P450 101) is a well-characterized, soluble bacterial P450 en- zyme that allows certain strains of Pseudornonas putida to utilize camphor as a carbon source.22 It is the only P450 enzyme for which a crystal structure is a~ailable.2~ Figure 5 shows the over- all topography of P450CI derived from highly refined X-ray structure. The protein of P450CI consists of a helix-rich domain (right side, Fig- ure 5) and a helix-poor domain (left side, Figure 5). The helix-rich domain of P450 comprises about half of the amino acid residues which are divided among 13 different helical (A to L) seg- ments. The helix-poor domain consists of anti- parallel beta (p) structures (p, to p,). The crystal structure of P450CI shows that the heme is em- bedded in the apoprotein between helices I and C and strand p3.23 Helix L lies below the heme and includes the cysteine (Cys357) thiolate ligand to the ferric iron. Interestingly, a comparison of the crystal struc- ture of P450CI and a large number of eukaryotic P450 sequences has demonstrated a significant amino acid homology.23 The most highly con- served regions in P450 are the heme-binding site and the central region of helix I, the oxygen bind- ing site, which presumably constitutes part of the substrate-binding site. 29 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. binding 1 + S\/. R-NH, A T binding low-spin high-spin FIGURE 4. Schematic representation of the spin state changes of the heme iron of cytochrome P450. Cytochrome P450 is present in a low-spin form (ferric iron is in the plane of the potphyrin ring) and in a high-spin form (ferric iron is out of the plane of the porphyrin ring). Binding of a type I substrate to the catalytic site of cytochrome P450 induces a low spin-to-high spin conversion, whereas a type I I substrate induces a high spin to low-spin conversion. In the low-spin state, the five 3d electrons are maximally paired and in the high-spin state these five 3d electrons are maximally unpaired. Experiments designed to probe stereo- selectivity and regioselectivity for mammalian P- 450 isoenzymes have provided some insight into the dimension of the P450 active site, albeit largely on the two-dimensional plane parallel to the por- phyrin ring.14 Such an approach has been used to predict the active site topology of highly purified P450IIB 1 and P450IA 1. These P450 isoenzymes are strongly induced by phenobarbital- and 3-methylcholanthrene, causing a 15- to 40-fold increase in the expression of the respective isoen- zymes (Table 1).=J6 The stereoselective oxygen- ation of the polycyclic aromatic hydrocarbons benzo [a] p yrene ,27,28 benzo [a] an thracene ,29 chry- sene30 and phenanthrene,3O by P450IA1 suggests that the catalytic site of P450IA accommodates 30 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. A 3746 B 67-77 B 89-96 C 106-126 D 127-145 E 149-169 F 173-185 G 192-214 H 218-225 I 234-267 J 267-276 K 280-292 L 359-378 FIGURE 5. Schematic representation of the cytochrome P45OCI structure. Helices are indicated by bars and fbstructure by ribbons. The camphor is hidden from view beneath helix F. The loop connecting helices F and G, the loop connecting the fbstrands of p5, and the helix p’ define the entrance of the substrate pocket. (From Poulos, T. L., Phamaceut. Res., 5, 67, 1988. With permission.) planar molecules with relatively large area-to- depth values (Table 1).I9J1 Ortiz de Montellano has clarified the active site topology of rat liver P450IIB 1 by investigating the regiochemistry of heme alkylation by terminal olefins and acety- l e n e ~ . ~ ~ The catalytic site of P450IIB1 seems to accommodate globular molecules, with relatively low area-to-depth values (Table l ) . 1 9 7 3 2 More re- cently, molecular modeling techniques were used to derive a predictive model for the active site of P45011D6.33 For this purpose, substrates with ba- sic nitrogen atoms were fitted into one model by postulating an interaction of the basic nitrogen atom with a negatively charged carboxyl group on the P450IID6 protein (Figure 6). The carboxy- late group was assumed to serve as an anchoring site on the P450IID6 protein. Moreover, molecu- lar modeling techniques were used to construct a 3D model for P450IID6 on the basis of the crystal structure of P450CI.33 The validity of this 3D model was established by fitting debrisoquine into a proposed planar pocket near the heme region formed by residues Va1370, Pro371, Leu372, Trp316, and part of the oxygen-binding site of P45011D6.34 D. Interaction between Cytochrome P450 and NADPH-Cytochrome P450 Reductase Metabolic reactions catalyzed by the microso- mal P450 require the transfer of electrons from NADPH to P450 by NADPH-cytochrome P450 reductase (RED).35 RED is a flavoprotein of ap- proximately 78,000 Da36 known to contain 1 mol of flavin adenine dinucleotide (FAD) and 1 mol of flavin mononucleotide (FMN) per mole of enzyme.37 The FAD moiety of purified RED has been recognized as the site of electron entry into the enzyme, whereas FMN is the electron-donat- ing site to P450 (Figure 7).38 RED contains a polar catalytic domain and a hydrophobic mem- brane-binding domain at the amino terminal.39 The membrane-binding domain is within the first 55 to 60 N-terminal amino acids of RED.40 When 31 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. TABLE 1 Hepatic Forms of Cytochrome P450 Cytochrome P450 Inducers Substrates IA IA l 8-Naphthoflavone, 3-methylcholanthrene, 7-EthoxyresorufinI aroclor, 2,3,7,8-tetrachlorodibenzo-pdioxin benzo[a]pyrene, IA2 IlB 1181 1182 1184 1106 llD IIE llEl IllA IVA IIIAl Isosafrole, 3-rnethylcholanthrene, B-naphthoflavone, aroclor, 2,3,7,8-tetra- chlorobenzo-pdioxin Phenobarbital, aroclor Phenobarbital Phenobarbital Noninducible Ethanol, ether, acetone, dirnethylsulfoxide Pregnenolone 1 6a-carbonitrile Clofibrate benzo[a]anthracene, chrysene, phenanthrene 2-Amino-3-methylimi- dazo[4,5-flquinoline, 2-Amino-3’,5-dirnethyI- irnidazo[4,5-flquinoline 7-Penthoxyresorufin, olefins, acetylenes, 7,12-Dirnethylbenzo[a]an- thracene 7,12-Dimethylbenzo[a]an- t h racene Benzphetarnine Debrisoquine, propranolol, sparteine, timolol, rnetroprolol KNitrosodirnethylarnine, ethanol, chlorzoxazone Testosterone Prostaglandins From Murray, M. and Reidy, G. F., Pharmacol. Rev., 42, 85, 1990. Wth permission. a 6-kDa hydrophobic domain4* is removed by protease treatment, the solubilized reductase can no longer reduce P450,4* although it retains its ability to reduce cytochrome c and femcyanide. Thus, this 6-kDa membrane-binding domain may play a crucial role in the interaction between RED and P450. In addition to hydrophobic forces, RED may also interact with P450 by electrostatic interac- tions, i.e., by pairing of oppositely charged amino acid residues. However, evidence in support of this hypothesis is rather contradictory. Previ- ously, the amino acids of RED involved in the interaction with P450 were identified with a dif- ferential labeling technique using the water- soluble carbodiimide l-ethyl-3-[3-(dimethyl- amino)propyl]-carbodiimide and methylamine to modify carboxyl residues on RED.43 More re- cently, it was shown that charges on RED and P450 may decrease the stability of the electron transfer complex, most likely due to charge re- pulsion.44 E. Mechanism of Cytochrome P450 Reduction by NADPH-Cytochrome P450 Reductase Purification of P450 isoenzymes and RED and subsequent experiments with these enzymes reconstituted in an artificial membrane such as dilauroylphosphatidylcholine have shown maxi- mal turnover when both enzymes were in a 1:l molar However, the 20: 1 stoichiometry of P450 to RED in liver microsomes4 raised ques- tions about the mechanism of electron transfer 32 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. FIGURE 6. Active site model postulated for cytochrome P45011D6 when substrates are fitted onto the template molecules debrisoquine and dextrometorphan. The oxidation sites (3) of all substrates are superimposed. The basic nitrogen atoms are either fitted onto the basic nitrogen atom of debrisoquine (2) or onto that of dextrometorphan (1) and interact with one of the carboxylic oxygen atoms (0, and O,, respectively) of a protein carboxylate group. (From Koymans L., Vermeulen, N. P. E., van Acker, S. A. B. E., te Koppele, J. M., Heykants, J. J. P., Lavrijsen, K., Meuldermans, W., and Donne-Op den Kelder, G. M., Chem. Res. Toxicol., 5,211, 1992. With permission.) from NADPH via RED to P450 and about the functional interaction of the proteins. The spin equilibrium of P450 has been sug- gested as a controlling factor for the biphasic reduction kinetics of the heme protein.4749 Ac- cording to this model, Fe3+-P450H, is more rap- idly reduced than Fe3+-P450,. The midpoint po- tential in Fe3+-P450, is about -350 mV, thus, electron transfer to Fe3+-P45& from NADPH, which has a redox potential of -365 mV, is ther- modynamically un fav~rab le .~~ Fe3+-P450H, is known to have a redox potential of -175 mV, so that it is thermodynamically favorable to reduce Fe3+-P45oH,.J1 Thus, substrates shifting the equi- librium toward the HS configuration might en- hance the initial rate and extent of the electron flow to P450 and drive the enzyme system to a more rapid The spin equilibrium be- tween Fe3+-P450, and Fe3+-P450Hs is described by K,= k,/k2. The rate of Fe3+-P450, to Fe3+- P45oHs conversion controls the rate of reduction of P450, indicating that the equilibrium rate con- stants k, and k2 are smaller than the forward re- duction rate constant k3 in the presence of type I substrates of P450. kl p- Fe2+-~450,s Fe3’-p45q, 4- Fe3’-p4s& k2 -350 mV -175 mV Based on the reaction kinetics and the extent of P450 reduction in microsomes, a rigid arrange- ment of RED and a cluster of P450s has been proposed.s2 Moreover, an interaction between P450 and RED by random collisions has been suggested.53 In addition, a modified “cluster model” where a number of P450 molecules were grouped around a central RED molecule has also 33 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. FIGURE 7. Schematic model of the interaction be- tween cytochrome P450 and NADPH-cytochrome P450 reductase on the endoplasmic reticulum. The flavin adenine dinucleotide (FAD) moiety is recognized as the site of electron entry in the enzyme, whereas the flavin mononucleotide (FMN) moiety is the electron- donating site to cytochrome P450. (From Strobel, H. W., Fang, W.-F., Takazawa, R. S., Stralka, D. J., Newaz, S. N., Kurzban, G. P., Nelson, D. R., and Beyer, R. S., Basic Life Sci., 39, 61, 1986. With permission.) been postulated.” The rapid reduction phase was thought to reflect the reduction of clustered P450s, whereas the slow reduction phase was thought to result from the reduction of unaggregated satellite P450s after lateral diffusion and collision (Fig- ure 8). Measurements of the kinetics of P450 re- duction and substrate hydroxylation in reconsti- tuted systems have shown that random collision of P450 and RED is responsible for their func- tional intera~tion.~~ Inasmuch as P450 was also shown to be rotationally mobile when reconsti- tuted with equimolar amounts of purified RED, the formation of a monomolecular complex be- tween P450 and RED was An attractive model to explain the reduction kinetics of P450 by RED has been proposed by Backes and Eyer (Figure 9).57 According to this model, P450 (P450IIB4) may exist in two confor- mations with respect to RED. In a “functionally competent complex” electrons are readily trans- ferred from RED to P450. In the “nonfunctionally competent complex,” P450 binds to RED but does not allow a rapid electron transfer. Moreover, substrate binding to P450 can enhance both the rate of association and the affinity of the RED- P450 complex and consequently the efficiency of electron flow from RED to P450.57 This view was conf i ied recently for several other P450 isoen- zymes, including P450IIB1 and I E L 5 * II. MONOOXYGENASE AND PEROXIDASE ACTIVITY OF CYTOCHROME P450 A. Monooxygenase Activity of Cytochrome P450 Much of our understanding of the steps in- volved in monooxygenation reactions catalyzed by P450 is based on the model of P450CI devel- oped by Gunsalus and associates. P450CI is in- volved in the 5-exo hydroxylation of camphor Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. ~~ ~ lustered cytochromes P45 I t FIGURE 8. Model illustrating the clustered cytochrome P450s and unaggregated satellite cytochrome P450s. The rapid phase of cytochrome P450 reduction is thought to reflect clustered cytochromes P450, whereas the slow phase of reduction results from reduction of unaggregated “satellite” cytochromes P450 upon lateral diffusion and collision. and related t e r p e n e ~ . ~ ~ An iron-sulfur protein (putidaredoxin) serves as an electron donor to P450CI and, therefore, resembles the mitochon- drial P450 enzymes more closely than P450s lo- cated on the endoplasmic reticulum, where a fla- voprotein (RED) acts as electron donor. The P450 isoenzymes catalyze a wide vari- ety of monooxygenase reactions, including hy- droxylations, epoxidations, N-demethylations, O-dealkylations, S-oxidations, deaminations, sulfoxidations, desulfurations, and oxidative dehalogenations.@’ The various stages in the monooxygenase catalytic cycle of P450 are shown in Figure 10. An important feature of the monooxygenation cycle is the binding of a sub- strate to the catalytic site of P450, which results in a conformational change in the apoprotein triggering electron transfer from RED to P450. Subsequently, 0, binds to the [Fez+-P450-sub- strate] complex to form [Fez+-P450-substrate] [O,] or the equivalent [Fe3+-P450-substrate][0,-] com- plex, which is then reduced by a second electron from RED (or perhaps, in some cases, from re- duced nicotinamide adenine dinucleotide [NADH] via cytochrome b, and NADH-cytochrome b, re- ductase).16 Capture of the second electron is thought to generate a [Fe3+-P450-substrate][0,2-l complex (Figure 10). Protonation of the peroxo- oxygen followed by 0-0 bond cleavage:’ results in the formation of a P450-substrate complex with 35 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. cytochrome P450 I I // -- \\ \ \ a a I N ADPH-c y toc hrome P450 reductase - n I functional competent I complex FIGURE 9. Proposed model for the equilibrium between *functional" and "nonfunctional" interactions of cytochrome P450 and NADPH-cytochrome P450 reductase in membranous systems. In the functional compe- tent complex, electron transfer from NADPH-cytochrome P450 reductase to cytochrome P450 occurs rapidly, whereas the nonfunctional complex does not allow a rapid electron transfer. 0, cytochrome P450; 0, NADPH- cytochrome P450 reductase. a single oxygen bound to the ferric iron, that is, the "oxenoid" intermediate [Fe3+O] (Figure 10). Subsequent insertion of one atom of oxygen into the substrate restores Fe3+-P450 (Figure 10, pathway I):, It is generally appreciated that P450 enzymes catalyze monooxygenase reactions by inserting oxygen into substrates (Figure 10, pathway I). Information now available suggests that radical intermediates are formed during oxygenation and oxidation reactions catalyzed by P450.63 For in- stance, the menthofuran obtained from cis-(Z)- CD, pulegon derives primarily (273%) from oXi- dation of the truns-CH, group by an initial proton abstraction, followed by spin delocalization and 'OH-recombination (Figure 11). The fact that menthofuran formation is only possible with a cis- rather than trans-hydroxymethyl group as well as the observation that there is no isomeriza- tion of the cis- and trans-methyl groups of pulegone or its hydroxymethyl derivatives strongly indicate that the cis- and truns-methyl groups must rotate prior to menthofuran formation. P450 sometimes also catalyzes dehydrogena- tion rather than monooxygenation reactions, that is, from paracetamol (acetaminophen) to its reac- tive metabolite N-acetyl-p-benzoquinoneimine.@ The mechanism of paracetamol oxidation indi- cates that initial hydrogen abstraction from the phenolic hydroxyl group is favored over initial hydrogen abstraction from the acetylamino nitro- gen group.@ B. Peroxidase Activity of Cytochrome P450 P450 has also been shown to utilize peroxides such as cumene hydroperoxide and t-butylhydro- peroxide and peracids to support N - and O-deallcylations, aliphatic hydroxylations, and ole- fin e p o x i d a t i ~ n s . ~ ~ ~ This bypass of the usual sequential 0, reduction is usually referred to as 36 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. Anaerobic condition (s.I I e- FIGURE 10. Reaction cycle of cytochrome P450. Cytochrome P450 is conveniently indicated as P450 Fe” and the substrate as SH. After binding of a substrate to the catalytic site of P450, the P450substrate complex is reduced in a one-electron reaction by NADPH-cytochrome P450-reductase. Subsequent binding of molecular oxygen followed by a second electron transfer results in substrate monooxygenation (pathway I). Uncoupling reactions compete with substrate oxygenation (pathways II and 111). The efficiency of the P450-mediated oxygenation refers to the percentage of reducing equivalents utilized toward substrate oxidation as opposed to molecular oxygen reduction. Substrates also can be reduced directly by ferrous P450 under anaerobic conditions (pathway IV). the “peroxide shunt”.61 180-labeling studies have established that the oxygen atom incorporated into the substrates is indeed derived from a perox- ide.68 The catalytic push required to cleave the 0-0 bond resides with the ability of the Cys heme ligand to donate electron density to the peroxo- ferric system.23 The oxenoid intermediate is also able to cata- lyze N-demethylation reactions by an initial one- electron oxidation of an amine nitrogen to an aminium cation radical.67 Subsequent depro- tonation of the aminium cation radical followed by recombination with the nascent heme iron- bound hydroxyl radical yields the unstable carbinolamine, which decomposes to formalde- hyde and a secondary a m i r ~ e . ~ ~ 111. OXYGEN REDUCTASE ACTIVITY OF CYTOCHROME P450 A. Molecular Oxygen Reduction of Cytochrome P45OCI For monoxygenation reactions catalyzed by P450,0, must be activated before insertion of an oxygen atom into a substrate can occur (Figure 10). However, the P450 reaction cycle can be short-circuited in such a way that 0, is reduced to 0,‘ and/or H202 instead of being utilized for sub- strate oxygenation (Figure 10, pathways I1 and III).21 This compromising side reaction is of- ten referred to as or the “oxidase activity” of P450.69 The event that commits P450 37 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. FeOHI3' - CHp H3C Cis-(Z)-trideuteriomethyl pulegon Menthofuran FIGURE 11. Proposed mechanism for the oxidation of pulegon to menthofuran. Cis-(Z)-trideuteriomethyl pulegon is oxidized by an initial proton abstraction followed by spin delocalization and .OH recombination. [Fe0I3+ represents the activated fenyl oxygen of cytochrome P450. (From Ortiz de Montellano, P. R., TIPS, 10, 354, 1989. With permission.) to reduce 0, is the starting point for this discus- sion. In P450C1, the redox potential of the ferric iron changes upon substrate binding to the cata- lytic site.7o The heme spin equilibrium shift from -300 mV (Fe3+-P450,) in the absence of sub- strate to -173 mV in the presence of camphor (Fe3+-P450,s)70 suggests that the ferric iron is rapidly reduced when camphor is bound to the catalytic site of P450CI. This has significant mechanistic implications, as the source of elec- trons required in the catalytic cycle is putida- redoxin, which exhibits a redox potential near -196 mV.70 In the absence of camphor, the substrate pocket of P450CI is occupied by tetrahedrically ordered water molecules with one water molecule coordinated to the ferric iron.21 In fact, the coordinated water ligand may explain why the substrate-free enzyme is mainly Fe3+-P450,s.70 With camphor as substrate, W-P45q, is almost completely converted to FG+-P45b (Figure 12), whereas norcamphor and camphane permit the enzyme to retain a higher fraction (about 50%) of Fe3+-P450,.21-70 The reason is that substrates smaller than camphor cannot form a hydrogen bond with Tyr96 (Figure 13) or are more free to move within the active site of P450CI.21 Thus, substrates that loosely fit in the active site of P45OCI are potential uncouplers of the P450-me- diated monooxygenation cycle.21 With camphor as substrate, 100% of the electrons derived from NADH can be accounted for as oxygenated prod- uct, whereas with norcamphor only 12% of the electrons can be accounted for by product hy- droxylation." In the latter case, uncoupling of the P450 reaction cycle results in the reduction 0, to 0;. and H,O,. The crystallographic structure of P450CI sug- gests that Thr252 is a key residue of the 0,- binding site (Figure 14).72 The hydrogen bond between the hydroxyl group of Thr252 interacts with the carbonyl oxygen of Gly248 in the inter- nal solvent ~hannel.7~ The solvent channel stabi- 38 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. Substrate Camphor Norcamphor Camp hane Structure formule High spin 95% 46% 46% FIGURE 12. Interaction between cytochrome P45OCI and camphor, norcamphor, and camphane. With camphor as substrate, the low-spin state of cytochrome P45OCI is almost completely converted to the high- spin state. With norcamphor and camphane as substrates, about 50% of the low-spin state of cytochrome P45OCI is retained. (From Poulos, T. L. and Raag, R., FASEB J., 6, 674, 1992. With permission.) lizes the distal helix I in a somewhat distorted conformation limiting the size of the 0,- binding groove (Figure 14) and thus preventing water from entering the catalytic site of P450CI. The crystal structure of the P450CI mutant Thr252Ala (in which Thr252 has been replaced by Ala) clearly shows that water is responsible for increased uncoupling of the enzyme during cam- phor hydr~xylation.~~ Uncoupling of the P450 cycle is probably due to steric interactions be- tween solvent and 0,. Moreover, the higher di- electric environment with water present will sta- bilize Fe3+-P450, relative to Fe2+-P45OHS. It is well known from model heme complexes that a protic environment destabilizes the heme-oxygen complex during formation of O;.74 B. Oxygen Reductase Activity of Membrane-Bound Cytochrome P450 Although microsomal membrane-bound P450s monooxygenate substrates, as does I P45OC1, the mechanism of the oxidase or un- coupling reaction of membrane-bound P450s may differ from that of P450CI in some re- spects. For example, electrons necessary for 0, reduction originate from a flavoprotein RED in the case of microsomal P 4 5 0 ~ ~ ~ and from the nonheme protein putidaredoxin in the case of P450CI.70 The autooxidation rate of [Fe2+- P450][0,] in microsomal P450s is fast,75 while that in P450CI is Microsomal P450s have a conserved “threonine cluster” at the putative distal while this cluster is absent from P450CI.78 The conserved “threonine” of microsomal P450s is composed of Thr319, Thr321, and Thr322 of P450IA. An ionic acid at 318 is also conserved as Glu or Asp and perhaps Val320 for most P450s. It has been shown by site-directed mutagenesis that the Glu3 18Ala and Va1320Ser mutants use 0, more efficiently to produce H,O, than to hydroxylate 7-ethoxycoumarin when compared with the wild type P450IA enzymes.78 Obviously, Glu3 18 39 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. f FIGURE 13. Edge-on view of the heme region of camphor-bound cytochrome P45OCI. The camphor molecule is anchored via a unique hydrogen bound to Tyr96 (/////). In addition, hydrophobic contacts with neighboring aliphatic and aromatic side chains keep camphor properly fixed. (From Poulos, T. L., Finzel, B. C., and Howard, A. J., 5hchemistry, 25, 5314, 1986. With permission.) plays an important role in the catalytic function of substrate hydroxylation by P450IA enzymes.78 Mutations in this region of P450 appear to un- couple the P450 reaction cycle under concomitant formation of 0,’ and H,O,. However, it remains unclear how Val320 contributes to the uncou- pling or oxidase activity of this enzyme. Phenobarbital-inducible P450s (P450IIB) and ethanol-inducible P450s (P450IIEl) exhibit high rates of oxygen Ethanol pretreat- ment of rats results in a three-fold increase in the rate of microsomal NADPH ~xidation,’~ which corresponds to the increase in P450IE1 seen in rat liver microsomes.82 Especially, liver microso- ma1 P450IIE1 exhibits a high rate of oxygen re- duction even in the absence of substrate^.^^ It has been shown that “uncoupling” of P450IIE1 may play an important role in the lipid peroxidation process in liver microsomes from ethanol- or ac- etone-pretreated rats.83 The mechanism underlying uncoupling of the P450 monooxygenase cycle is not clear and is subject to discussions. The shift from Fe3+- P450L, to Fe3+-P45OHS of microsomal P450s favors reduction of Fe3+-P450 to Fe2+-P450, and subsequent binding of 0, to Fe2+-P450 occurs.75 The fact that 0, in its ground state is a triplet system (3Cg-0,) with two unpaired electrons suggests that Fe2+-P450 has to be in its HS state before 3Cg-0, binding can occur. In addition, 3Cg-0, binding and spin pairing causes Fe2+- P450,, to return to its Fe2+-P450, state, while 0, becomes a singlet species (‘Ag).l9 Electron transfer from Fe2+-P45OLS to ‘Ago, gives rise to Fe3+-P450H, and O;..I9 In fact, a one-electron transfer from electron donors to ‘Ago2 readily produces 0, Moreover, ferrous porphyrin dioxygen complexes readily autooxidize to the ferric state via 0;. g e n e r a t i ~ n . ~ ~ Considering the respective redox potentials, electron trans- 40 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. ‘ Cyr.357 FIGURE 14. Regions of the distal helix I in the immedi- ate vicinity of the oxygen-binding site (shaded area) of cytochrome P45OCI. The formation of a hydrogen bond between Gly248 and Thr252 is indicated (/////). The inter- action between Gly248 and Thr252 is thought to prevent water from entering the catalytic site of cytochrome P45OCI. (From Poulos, T. L., Finzel, B. C., and Howard, A. J., J. Mol. Biol., 195, 687, 1987). fer from Fez+-P450,, (+50 mV)86 to form 0,‘ is more favored if 0, is in the IAg state (+ 650 mV) than in the 3Xg-0, state (-160 mV).*9987 In addition, ‘Ago, is a good electron acceptor due to its low E(LUM0) value (-0.2 eV). It is ob- vious that as a result of “uncoupling” of the P450 reaction cycle, 0, is reduced in a direct one-electron reduction to OF.. A more appro- priate name for this mechanism of 0, reduction by P450 could therefore be “oxygen reductase activity” of P450. 0, ‘ liberated from the [Fe2+-P4501 [O;] com- plex, in combination with iron, can yield ‘OH, which is a potent initiator of lipid peroxidation.88 The role of P450 in the lipid peroxidation process is supported by the following arguments: (1) boiled P450s are not capable of inducing lipid peroxi- dation, (2) the rate of lipid peroxidation is strongly associated with increased production of 0;. and H,O, upon addition of increasing amounts of P450 in reconstituted systems of purified enzymes, and (3) lipid peroxidation is inhibited by carbon mon- oxide.88 Experimental data have shown that oxy- gen free-radical intermediates may contribute substantially to liver injury mediated by the oxy- gen reductase activity of P450IIEl.@ C. Xenobiotic-Induced Oxygen Reductase Activity of Cytochrome P450 Compounds known to induce the oxygen reductase activity of P450 include (1) “pseudo- substrates” such as perfluoro-n-hexane, per- fluoro-cyclohexane, and 1 ,1, 1 -trichloroethane, (2) partial uncouplers such as hexobarbital, 41 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. benzphetamine, barbiturates, N-acetyl-p-benzo- quinoneimines (NAPQI), and 2,3,5,6-tetra- methylbenzoquinone (TMQ), and (3) the alky- lating agent 2-bromo-4'-nitroacetophenone (Figure 15, Table 2). The fact that the oxygen reductase activity is stimulated by specific com- pounds (Table 2),90-92 that it is inhibited by type I1 ligand inhibitors of P45093 and by carbon monox- ide,88 and that it is dependent on the ratio of P450 to RED in reconstituted systems of purified en- zymesg8 strongly suggest that P450 is indeed the primary source of 0, reduction. F F F F F F F-C-C -C-C-C-C-F F F F F F F I I I I I I I I I I I I Perfluoronated-n- hexane C1 0 I CI - C- CH, I c1 l,l,l-Trichloroethane Hexobarbi tal Benzphetamine 0 II y'Jj F F F F Perfluronated cyclohexane 5-Ethyl-5-alkylsubstituted barbiturates 0 2,3,5,6-Teuamethylbenzoquinone 0 II K H3C . 2-Bromo-44troacetophenone 0 0 N-acetyl-p-benzoquinoneimine 3,s-dimethyl-NAPQI (NAPQI) FIGURE 15. Chemical structures of compounds known to stimulate the oxygen reductase activity of cytochrome P450. 42 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. TABLE 2 Compounds that Stimulate the Oxygen Reductase Activity of Cytochrome P450 Cytochrome P450 Compound P4501A 2,3,5,6-Tetramethylbenzoquinone Kacetyl-pbenzoquinoneirnine 3,5-Dirnethyl-Kacetyl-pbenzquinoneimine 2-Bromo-4’-nitroacetophenone 5-Alkyl-5-ethylbarbiturates Benzphetamine Perfluoro-mhexane Perfluoronated cyclohexane 1,l ,I-Trichloroethane P45011B Hexobarbital P45OCI Norcamphor Camphane 7. Oxygen Reducfase Activity of Cyfochrome P450 Induced by Pseudosubs fra fes Perfl~oro-n-hexane,~~ perfluoro-cyclohex- and 1 , 1 , 1 -tri~hloroethane~~ act as “pseudo- substrates” for P450 (Figure 15). These pseudo- substrates bind to P450 and induce the oxygen reductase activity of this enzyme without under- going monooxygenation themselves. For instance, 1 , 1 , 1 -trichloroethane (Figure 15) has been shown to increase the rate of 0, consumption and, con- comitantly, H,O, prod~ct ion .~~ The stoichiometry of 1 , 1 , 1 -trichloroethane oxidation and 0, con- sumption is about 0.01 1 in rat liver micr0somes,9~ indicating that 1 , 1 , 1 -trichloroethane oxygenation is very slow compared with 0, reduction. Using partially succinylated ferricytochrome c for the detection of 0;. and the ferrithiocyanate method for the determination of H,O,, a stoichiometry of 0 5 . to H,O, close to 2: 1 was obtained during the oxygen reductase activity of P450 stimulated by both perfluoro-n-hexane and perfluoro-cyclohex- 2. Oxygen Reducfase Activity of Cyfochrome P450 Induced by Partial Uncouplers Perhaps the most well-known compound in- ducing the oxygen reductase activity of P450 is Ref. 111 1 08 1 08 112 101 88 90 82 69 94 21 21 hexobarbital (Figure 15). Hexobarbital produces a type I binding spectrum with microsomal P450.95 During the normal monooxygenase cycle of P450, hexobarbital is hydroxylated in the 3‘ position in the cyclohexenyl ring” and epoxidated at the 1’,2’- position.97 When the kinetics of 3’-hydroxy- hexobarbital formation are compared with 0, re- duction to H,O, in liver microsomes of different animals, higher rates of H,O, formation were found in all species tested.98 The observed hexobarbital- induced H202 formation has been attributed to additional oxygen reductase activity of P450.91,99 It has been shown that hexobarbital stimulates H,O, formation in hepatic microsomal incuba- tions in a concentration-dependent manner.91J00 The monooxygenase activity of phenobarbital- induced rat liver microsomes toward (-)- hexobarbital was 1.6-fold lower than toward (+)- hexobarbital.’O’ The production of H202 occurred by a decay of a [hexobarbital-Fe3+-P4501 [O, ‘1 complex, forming OF., or, alternatively, by proto- nation of a [hexobarbital-Fe3+-P4501 [O?] com- plex, giving rise to H,O, f~rmation.~’ In addition, Klinger et al. have shown that H,O, production originates directly from the Fexobarbital-Fe3+- P45O][Oj-] complex upon reduction by a second electron from cytochrome b5.1M However, the exact role of cytochrome b, in hexobarbital-in- duced H,O, formation is unclear. More recently, using a homologous series of 5-ethyl-5-alkylsubstituted barbiturates (R = pro- pyl, butyl, pentyl, 3-methylbutyl, l-methylbutyl, 43 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. 2,3-&methylbutyl, hexyl, heptyl, octyl, and nonyl; Figure 15), Bast et al. have shown that the loga- rithm of the rate of barbiturate-induced H,O, for- mation correlates with the logarithm of the appar- ent partition coefficient (n-octanofiuffer) of these compounds according to a parabolic function.Im Moreover, the statistics of the correlation was improved by applying a bilinear model.103 This bilinear model, which was based on drug trans- port studies in which several compartments were involved, suggests that both transport of the bar- biturates to P450, which is located in the microso- mal membrane, and the interaction with the hy- drophobic catalytic site of P450 are critically involved in the oxygen reductase activity of P450 stimulated by this type of substrate.1o3 Benzphetamine (Figure 15) has also been shown to induce H202 formation in hepatic mi- crosomal preparations by stimulating the oxygen reductase activity of P45O.'O4 Similar to hexo- benzphetamine and benzphetamine analogs produce a type I interaction with P450,'" indicating a Fe3+-P450, to Fe3+-P45OHS shift. However, only 60% of the NADPH consumed accounts directly for the formation of formalde- hyde.lM Moreover, during the metabolism of benzphetamine, 0,. formation was observed that was attributed to the oxygen reductase activity of P45O.'O7 Inasmuch as a stoichiometry of 0,. to H,O, of 2:l was obtained, the formation of 0;. was suggested to occur by dissociation of the [knzphetamine-Fez+-P450][O;] complex,107 as it rules out a two-electron reduction of 0, to 0;- as the main source of H202. Similar to hexobarbital, the rate of benzphetamine-induced H202 produc- tion is especially high in microsomes from phenobarbital-induced rats.g0 The measurement of H,O, does not distin- guish between autooxidative formation of 0 ~ ' and 08 because H,O, is also a product of 0,. dismutation. While a two-electron reduction of 0, to OF was suggested to occur during NAPQI and 3,5-dimethyl NAPQI reductive metabolism by P450 under aerobic conditions,108 a direct one- electron reduction of 0, to 0;. was proposed with benzphetamine as substrate.107 The formation of 0;. has been detectedIw and quantitated in he- patic microsomes from phenobarbital-pretreated rats67 as well as in reconstituted P450 systems.I1O Recently, direct electron spin resonance measure- ments using 5,5'dimethyl-N-pyrroline oxide as a spin trap for OF. have provided clear evidence that 0;. dissociation from the [Fe2+-P450] [O;] complex is the autooxidative mechanism of TMO- induced oxygen reductase activity of P45O.l1' 3. Oxygen Reductase Activity of Cytochrome P450 Induced by Alkylation Thus far, it has been shown that pseudo- substrates and poor substrates for P450-mediated oxygenations that bind to P450 and cause an in- crease in the rate of NADPH oxidation and 0, consumption may be considered as likely stimu- lators of the oxygen reductase activity of P450. These substrates were shown to form reversible P450-substrate complexes. Recently, however, 2-bromo-4'-nitroacetophenone has been shown to induce P450 oxygen reductase activity upon co- valent binding to Cys292 also outside the sub- strate-binding site."* 2-Bromo-4'-nitroaceto- phenone alkylated ten isoenzymes of purified rat liver microsomal P450, but it inhibited monooxy- genase activity of P450IA1 exclusively.112 While examining the mechanism of inactivation of P450IA1 by 2-bromo-4'-nitroacetophenone, it was found that P450IA1 in the alkylated state efficiently oxidized NADPH and catalyzed the reduction of 0, to H,O, via 02..113 Obviously, alkylation of P450IA 1 by 2-brom0-4j-nitro- acetophenone stimulates the oxygen reductase activity of P450 prior to the introduction of the second electron. 113 2-Bromo-4'-nitroacetophenone, however, is fundamentally different from all other known substrates because (1) it does not act as a pseudosubstrate, (2) 2-bromo-4'-nitroaceto- phenone binds covalently to P450 outside the substrate-binding site and induces the oxygen reductase activity of P450IA1, and (3) alkylation does not cause a spin-state tran~ition. '~~ IV. XENOBIOTIC REDUCTASE ACTIVITY OF CTYOCHROME P450 A. Anaerobic Reduction Reactions Catalyzed by Cytochrome P450 Although P450 is generally recognized as a monooxygenation catalyst, it also catalyzes the 44 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. reduction of xenobiotics. Owing to the fact that P450-mediated reactions involve electron trans- fer reactions, either 0, or the xenobiotic can ac- cept these reducing equivalents.60 In the absence of O,, certain xenobiotics may accept electrons directly from the reduced [Fe2+-P4501 complex in the so-called xenobiotic reductase activity of P450. In fact, 0, acts as an inhibitor of P450-mediated xenobiotic reduction by competing for electrons at the catalytic site of P450. Classic P450 inhibi- tors are also potential inhibitors of the xenobiotic reductase activity by either competing with the xenobiotics at the catalytic site of P450 (a type I i n t e r a ~ t i o n ) ~ l ~ J ~ ~ or binding directly to the heme iron (a type I1 intera~tion),"~ thereby inhibiting electron flow to the xenobiotics. The events that commit P450 to reduce xenobiotics is not clear. In the anaerobic reduc- tion of halogenated alkanes, these compounds act as type I substrates, indicating that they bind to the catalytic site of P450. Binding of these usually nonpolar compounds to P450 has been shown not to alter their electronic properties.ll6 Due to the substrate-induced Fe3+-P450, to Fe3+-P450, shift, the [substrate-Fe3+-P450,,] complex is reduced by RED in a one-electron reduction reaction to the [substrate-Fe2+-P4501 complex. During the P450 monooxygenase cycle, 0, would bind to the [substrate-Fe2+-P4501 complex at this stage, form- ing the [substrate-Fe2+-P4501 [O,] intermediate (Figure 10). However, in the absence of 0,, the [ substrate-FeZ+-P450] complex can donate an elec- tron directly to the substrate to yield Fe3+-P450 and a substrate free-radical (Figure 10, pathway IV). Using halogenated alkanes as substrates, it was clearly shown that reduction of these com- pounds is favored by hydrophobic environments such as the catalytic site of P450 is expected to be.117 The substrate free-radical can either leave the catalytic site of P450 or undergo further re- ductive or oxidative metabolism. The toxicity of halogenated alkanes has been attributed to the xenobiotic reductase activity of P450 under anaerobic conditions. l8 Experiments designed with rat liver microsomes and isolated rat hepatocytes have shown that low steady-state 0, partial pressure (PO,) augments haloalkane free radical-induced toxicity. l9 A characteristic of halogenated alkane-induced liver injury is centrilobular necrosis.lm Within the liver lobule, phenobarbital-inducible P450s are predominantly located in the centrilobular area. 121 The PO, in the liver has been shown to vary between 1 and 60 mmHg, with the lowest values around the cen- tral vein of the lobule.122 Thus, the site of liver injury is critically associated with the site of haloalkane reduction by P450 and low PO, values in the liver.119 As shown in Table 3, phenobarbital- inducible P450s are predominantly involved in xenobiotic reduction. However, the role of etha- nol-inducible P450 (P450IIEl) in the xenobiotic reductase activity is also increasingly appreci- ated.'= The presence of P450IIE1 has been asso- ciated with increased hypoxia especially in the centrilobular region, most likely due to increased 0, consumption by the spontaneous oxygen re- ductase activity of P45011E1.123 The hypothesis that the oxygen reductase activity as well as the xenobiotic reductase activity of P450IIE1 in the centrilobular region would lead to regioselective hepatotoxicity due to free radical-mediated dam- age is interesting but needs confirmation. Xenobiotics known to be reduced by P450 (Table 3) can roughly be divided in three classes (1) the halogenated alkanes, (2) the azo dyes, and (3) the nitro compounds. In addition, evidence has been presented for the involvement of P450 in the one-electron reduction of benzoquinone- imineslo8 and The quinones and quinoneimines may, therefore, form a fourth class of compounds. Other compounds shown to be reduced by P450 include gentian violet,129 chromium compounds,13o 13-hydroxyperoxy-9,11- octadecadienoic acid,l3' 15-hydroxyperoxy- 5,8,11,13-eicosatetraenic acid,13' cumenehydro- peroxide,131 and t-b~ty1hydroperoxide.l~~ 1. Carbon Tetrachloride Various halogenated hydrocarbons have been shown to be reductively dehalogenated by P450 under anaerobic conditions. Carbon tetrachloride (CCl,) and halothane (CF3CHC1Br) are the most extensively studied e~amp1es . l~~ Benzyl- halides,133 chloroform,134 l i ~ ~ d a n e , ~ ~ ~ polychlori- nated ethanes,13'j and CC1,Br137 are also reduced by P450. CCl, is a widely used solvent, a former anthelminthic and dry cleaning agent causing centrilobular hepatic necrosis and fatty liver.lm 45 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. TABLE 3 Substrates for the Xenobiotic Reductase Activity of Cytochrome P450 Cytochrome P450 Substrate P45011B P45011E1 P4501A Dimethylaminobenzene Kacetyl-pbenzoquinoneimine 3,5-Dimethyl-Nacetyl-pbenzoquinoneimine 1 -Nitropyrene Danthron Benzylhalides Carbon tetrachloride Halothane Dimethy laminobenzene 2,3,5,6-Tetramethylbenzoquinone Mitomycin C Adriamycin 3-Nitrof luoranthenes Benzyl halides Chloroform Lindane Polychlorinated ethanes Tetrachlorobromide Carbon tetrachloride Chromium (CP) 13-Hydroperoxy-9,ll -octadecadienoic acid 15-Hydroperoxy-5,8,11,13-eicosatetraenic acid Cumenehydroperoxide f-Butylhydroperoxide P45011A1 Dimethy laminobenzene P4501VA Dimethylaminobenzene (I-compounds) The major cytotoxic effects of CCl, are confined to the liver,138 which has been associated with reductive metabolism by P450.139 The first step in the P45O-mediated reductive metabolism of CCl, involves binding to the catalytic site of P450 (Fig- ure 16). CC1, produces a type I interaction with Fek-P450, which indicates binding to the hydro- phobic catalytic site of P450.Il7 Subsequent one- electron reduction of the [Fe3+-P450-CCl4] com- plex by RED, followed by a second electron transfer results in the formation of CCl, upon dissociation of C1- (Figure 16).I4O 'CCI, can either leave the active site of P450 or bind directly to Fe2+-P450 forming, either a [Fez+-P450-Cc1,] (fer- rous-trichloromethyl) or a [Fe3+-P450-CCl3-] (ferric-trichloromethyl anion) The [Fe3+-P450-CCl3-] complex can accept another electron and eliminate a second C1-, thus produc- ing a [Fe2+-P450-CClJ (ferrous-carbene) com- plex. The [Fe2+-P450-CCl,] complex may hydro- Ref. 170 108 108 21 5 127 133 152 158 167 114 124 126 21 4 133 134 135 136 137 150 130 131 131 131 131 1 70 169 lyze to CO and HC1 or react with 0,, forming C1,CO (phosgene), thereby restoring Fe2+-P450. The CCl, radical is highly reactive and can participate in several potentially toxic reactions.141 CCl, is able to abstract a proton from unsaturated membrane lipids,117 thereby initiating the lipid peroxidation process. The formation of a trichloromethyl peroxyradical (CC1,OO) has also been observed after CC1, reduction by P450 (Fig- ure 16), probably formed by a direct reaction of 'CC1, with 0,.142 CCl,OO. can be reduced to trichloromethanol, which rapidly decomposes to yield phosgene.141 Although phosgene formation from CCl, has been detected,',, its toxicological significance is questionable. 144 CCl,OO prima- rily attacks unsaturated membrane lipids, causing lipid peroxidation. Alternatively, CCl,OO binds covalently to critical cellular macromolecules, thereby affecting their normal physiological func- tion.'& Reduction of CCl, by P450 can also lead 46 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. t CC1, I 0 2 4 e' e cc1~coo - 0 2 'CCl:, a L - C1' FIGURE 16. Proposed mechanism for the interaction of carbon tetrachloride (CCL) and CC1,- derived metabolites with cytochrome P450. The anaerobic reduction of CCI, by cytochrome P450 produces CCI,, which can be released (pathway A) or undergo a second one-electron reduction (pathway B). Cytochrome P450 is conveniently indicated as Fe&. (From Luke, B. T., Loew, G. H., and McLean, A. D., J. Am. Chem. SOC., 109, 1307, 1987. With permission.) to irreversible inactivation of the enzyme upon subsequent loss of the prosthetic heme g r 0 ~ p . I ~ ~ P450 probably is inactivated by a free radical intermediate of CCl, in a so-called ''suicidal'' in- activation p r o ~ e s s . ~ ~ ~ , ' ~ ~ The fact that the heme moiety of P450 is both the site and the target of CCl, reductive metabolism most likely explains the observed loss in P450 activity upon CCl, exposure. The hepatotoxicity of CCl, has been shown to increase by acute149 or chroniclS0 pretreatment of rats with ethanol. P450IIE1 (the major etha- nol-inducible P450)123 is known to possess a particularly high affinity for CCI, (Table 3).lS0 This observation may have important toxico- logical consequences, as evidence has been pro- vided for the existence of a human homolog of the ethanol-inducible form of rat liver P450IIE1 that can participate directly in the metabolism of CC1,.lS1 While P450IIE1 has been shown to play a significant role in the reductive bioactivation of CCl,, other isoenzymes of P450 such as the phenobarbital-inducible forms (P450IIB) may be involved in CCl, reduction.lS2 In agreement with a role of P450, inhibitors of P450 such as isoniazid, cimetidine, and tryptamine have been shown to inhibit the reduction and cytotoxicity of CCI,."~ 47 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. 2. Halothane 3. Azo Dyes Another polyhalogenated compound that is reductively bioactivated by P450 is halo- thane (CF,CHClBr), a commonly used vola- tile anaesthetic agent. Halothane is metabo- lized by P450 both oxidatively and reductively. The oxidative metabolism of halothane by P450 results in the formation of trifluoroacetic acid,t53 whereas the reductive metabolism of halothane yields free radicals such as [CF3CHC1]'154 and stable products such as CFzCHC1 and CF,CH2C1.155 CF,CHClBr binds primarily to Fe2+-P450, presumably at the cata- lytic site but not directly to the heme iron under anaerobic conditions.t16 After one-elec- tron reduction of the [Fez+-P450-CF3CHC1Br] complex followed by Br- elimination, a [CF,CHCI]' is formed. [CF,CHCI]' can either leave the active site of P450 or undergo further reductive metabol i~m. '~~ A second electron re- duction of the [Fe2+-P450-CF,CHCl] complex to the [Fez+-P450-CF3CHC1]- complex followed by F- elimination yields [Fez+-P450- CF,CHCl]. The [Fez+-P450-CFzCHC1] complex dissociates under formation of CF2CHCl and Fe2+-P450. 117 In addition, protonation of the [Fe2+-P450-CF3CHC1]- complex and subsequent product dissociation can form CF,CH,Cl. l I 6 [CF,CHCl]' has been shown to destroy rat liver P450 by attacking its prosthetic Human liver P450 also can be reductively inac- tivated by [CF,CHCl]', most likely via a sui- cidal type of mechanism.t32 [CF,CHCl]- can also bind covalently to other proteins, or abstract a proton from unsaturated fatty acids, thereby initiating lipid peroxidation and ultimately cause irreversible cell damage. Interestingly, the cy- totoxicity of CF,CHClBr is species depen- dent.lS7 It has been shown that CF,CHClBr- induced hepatic microsomal lipid peroxidation in guinea pig is higher when compared with the rat, most likely due to the large amount of relevant hepatic P450 isoenzymes in guinea pigs.157 The reduction of CF,CHClBr by P450158 is inducible by both phenobarbital and poly- chlorinated biphenyls but not by 3-methyl- ~ h o l a n t h r e n e . ~ ~ ~ Azo dyes are widely used in textiles, food, drinks, pharmaceuticals, paper, and leather.159 In view of their widespread use, there is a common concern about their cytotoxicity, and more spe- cifically about their carcinogenicity. Metabolic activation of azo dyes has been associated with these toxic effects.I6O Reduction of the azo link- age to aromatic amines under anaerobic condi- tions is catalyzed by mammalian liver microso- cytosolic enzymes162 and colon bacteria.I6, Cytosolic enzymes known to catalyze azo reduc- tions are NADPH:quinone reductase (DT-diapho- rase), catalyzing the reduction of methyl red (2'-carboxy-dirnethylaminoazobenzene),'62 and aldehyde oxidase, which reduces methyl red, amaranth, methyl orange, and dimethylamino- azobenzene under anaerobic conditions (Figures 17 and 18).lW Based on the carbon monoxide sensitivity of dimethylaminoazobenzene reduction, in 1967 Hernandez et al.,165 showed for the first time that the reduction of neoprontosil by rat liver mi- crosomes was attributable to P450. Moreover, dimethylaminoazobenzene reduction was in- creased upon pretreatment of rats with both phenobarbital and 3-methylcholanthrene. Recon- stitution experiments using purified enzymes fur- ther confirmed the role of P450 in the reductive metabolism of dimethylaminoazobenzene.166 P450 has been suggested to be primarily respon- sible for the 0,- and CO sensitive one-electron reduction of amaranth in rat hepatic mi- crosomes. 167 At least two types of P450 have been shown to be involved in dimethylaminoazobenzene re- duction.t68 One type is selectively induced by clofibrate (P450IVA), a hypolipidemic agent,169 and the other types are induced by phenobarbital (P45011B), Pnaphthoflavone (P450IA), isosafrole (P450IA2), and pregnenolone-l6a-carbonitrile (P450IIIA1) as well as clofibrate (P4501VA).170 An unusual characteristic of dimethylamino- azobenzene reduction by clofibrate-inducible P450s is the insensitivity of the one-electron re- duction reaction to CO or Oz.169 The CO insensi- tivity of dimethylaminoazobenzene reduction ar- Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. Methyl orange Na03S Acid yetlow S03Na \ A ma rant h S03Na . . FIGURE 17. Structures of representative azo dyes such as methyl orange, acid yellow, and amaranth. gues against a role of P450, while the observation that known inhibitors of P450, such as SK&F 525-Al7I and a-naphth~flavone,'~~ inhibit azo re- ductase activity by P450IV argues for a role of P450.169 Azo dyes with electron-donating sub- stituents (i.e., amino or hydroxy groups) are re- duced in an 0,- and CO insensitive reaction. These insensitive dyes were designated I-substrates by Levine et d.168 (Figure 18). In contrast, azo dyes with electron-withdrawing substituents (i.e., COOCH,, COOH, SO,H, and As0,HJ have been shown to be reduced in an 0,- and CO sensitive manner and were therefore designated S (sensi- tive)-substrates (Figure 18).168 A previous study has shown that polar elec- tron-donating substituents para to the azo bond are required for P45O-mediated azo reduction,173 and that they are essential for the binding of azo dyes to microsomal P450.I7O Azo dyes lacking these electron-donating groups do not bind to P450 and are not at all reduced by P450. In addi- tion, S-substrates have been shown to be more readily reduced by P450 when compared with I-substrates, which was in agreement with the difference in redox potentials obtained for I- and S-sub~t ra tes .~~~ It has been suggested that in addi- tion to binding to P450, azo reduction also may depend on the redox potential of these com- 49 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. I-substrates (insensitive) Dimethylaminoambenzene (DAB) 4I-Amino-DAB Meth ylaminoazobenzene S-substrates (sensitive) COOH DAB-arsonate FIGURE 18. Classification of azo dyes according to their O2 and CO-sensitivity. The 1 (insensitive) substrates contain only electron donating groups and are reduced by cytochrome P450 in an 0,- and CO-insensitive reaction. The S (sensitive) substrates contain both electron-donating and electron-withdrawing substituents and are reduced by cytochrome P450 in an 0,- and CO-sensitive reaction. (From Levine, W. G., Stoddart, A., and Zbalda, S., Xenobiofica, 22, 1 1 1 1, 1992. With permission.) Using cyclic voltametry, it was clearly shown that both I- and S-substrates exhibit redox potentials ranging from +0.6 to +1.09 V.170J75 Inasmuch as the redox potential of P450 is in the range of -0.350 V for Fe3+-P450, to -0.175 V for Fek-P450,,,50 it was assumed that substrates pos- sessing high redox potentials are prone to un- dergo P450-mediated reduction. Thus, the pres- ence of both electron-donating substituents and positive redox potentials are major factors in- volved in azo reduction by P450.I7O The I- and S-substrates also differ in their sensitivity to 0, during P450-mediated azo re- duction.168 It has been shown that the presence of air readily quenches the one-electron reduced potentials of S-substrates but not that of I-sub- strates. One-electron-reduced free-radical in- termediates of S-substrates can undergo rapid oxidation in the presence of 0, with the formation of 0,.,176 whereas those of I-substrates are stable under aerobic conditions. Addition of a second electron to the free-radical intermediate of I-sub- strates yields a second intermediate (Figure 19).I6O This two-electron-reduced intermediate is rapidly protonated to the hydrazo intermediate.160 The latter protonation is much faster than protonation of the one-electron-reduced free radical (Fig- ure 19).I6O Based on observations with zinc-re- duced dimethylaminoa~obenzene,~~~ the fiial two- electron reduction step has been suggested to occur via disproportionation. 4. Quinones and Quinoneimines Quinones and quinoneimines occur widely in nature and have been extensively studied for their potential antitumor properties. 177~178 The clinically useful anticancer quinones are usually extensively substituted p-benzoquinones such as mitomycin C (MMC) and adriamycin (ADR) (Figure 20). In addition, several other quinones have been syn- thesized and screened for antitumor activity.179 MMC and ADR are clinically effective antineoplastic drugs used in the treatment of a variety of human cancer~ .~~~- '~O These antitumor Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. I +le. ON"-CJ / \ / \ 2' - + 2H' FIGURE 19. Proposed mechanism for the microsomal reduction of azo dyes. One-electron reduction of the azo dye results in the formation of a free radical. A second electron transfer followed by protonation yields the hydrazo intermediate, which is subsequently reduced to primary amines. Protonation of the one-electron reduced free radical also occurs, but slower than protonation of the two-electron reduced form. (Taken from Levine, W. G., Drug. Metab. Rev., 23, 253, 1991. With permission.) compounds require reductive bioactivation prior to eliciting their cytotoxic and antitumor activity. For instance, MMC and ADR can undergo both one-electron reduction to the corresponding semiquinone free radicalsIg1 and two-electron re- duction to the corresponding h y d r o q u i n o n e ~ . ~ ~ ~ . ~ ~ ~ Several enzymes, including RED,lg4 xanthine oxida~e, '~~ and NADH-cytochrome b5 r e d ~ c t a s e , ~ ~ ~ have been implicated in the one-electron reduc- tion of MMC and ADR. However, previous in- vestigations have also clearly shown that P450 is directly involved in the reduction of d a n t h ~ n , ~ ~ ~ 1-piperidinoanthraquinone,128 and M M P (Fig- ure 20). More recently, several lines of evidence have been presented for a role of phenobarbital- inducible P450s in the one-electron reduction of TMQ,'l1JI4 MMC,'" and ADR.126 These quino- nes were shown to bind with a high affinity to the catalytic site of phenobarbital-inducible rat liver microsomal P450s. Furthermore, one-electron reduction of these quinones to their correspond- ing semiquinone free radicals in phenobarbital- induced rat liver microsomes as well as in com- plete reconstituted systems of purified P450IIB 1 and RED were strongly inhibited by SK&F 525- A, metyrapone, and P450IIB antibodies under anaerobic conditions. 114~126 Moreover, quinone- induced H,O, formation, either as a result of re- dox cycling of the quinone moiety with 0, or quinone-induced oxygen reductase activity of P450, was completely inhibited by SK&F 525-A and metyrapone.111JaJ26 The mechanism of one-electron reduction of quinones and quinoneimines by P450 is unclear. Previously, it has been shown that NAPQI is reduced by P450 in a one-electron reduction reac- 51 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. Mitomycin C 2,3,5,6-TetramethyIbenzoquinone Danthron 1 -Piperidinoanthraquinone FIGURE 20. Chemical structures of 2,3,5,6-tetramethylbenzoquinone, danthron, 1 -piperidinoanthraquinone, and the clinically interesting cytostatic quinones mitomycin C and adriamycin. tion under anaerobic conditions.Im It has been suggested that upon binding of NAPQI to P450, the [Fe3+-P450-NAPQIl complex is reduced by RED to [Fe2+-P450-NAPQIl. Subsequently, the [Fe2+-P450-NAPQIl complex transfers one elec- tron to NAPQI under formation of the semiquinone free radical and Fe3+-P450 under anaerobic condi- tions.los A similar mechanism of one-electron reduction of quinones by P450 can be anticipated. In fact, the rate of reduction of quinones by RED has been found to decrease markedly at redox potentials below -250 mV.186 The low redox po- tential of TMQ (-235 mV), MMC (-310 mV), and ADR (-328 mV)179 correlates with the ob- served low rate of quinone one-electron reduction by RED a10ne114J24J26 under anaerobic conditions. A proposal for the mechanism of P45O-mediated one-electron reduction of quinones is that RED alone can reduce TMQ, MMC, and ADR, albeit to a small extent under anaerobic conditions. As far as microsomal reduction of these quinones is concerned, both P450 (notably P450IIl31) and RED possess quinone reductive activity. The high- affinity binding of TMQ,l1'JI4 MMC,124 and ADR126 to phenobarbital-inducible rat liver P450s is likely to induce a conformational change in P450, thereby facilitating electron flow from RED to the preformed [Fe3+-P450-quinone] complex. P450 can then participate more easily in the one- electron reduction reaction by donating electrons directly to the quinones. The undesired cytotoxic properties of these cytostatic quinones are generally thought to be mediated by initial one-electron reduction to the corresponding semiquinone free radicals.Is7 This one-electron reduction pathway is particularly sensitive to oxygen due to reoxidation of the semiquinone free radical in the presence of 0, and concomitant formation of 0;. (Figure 21),lS8 a process frequently referred to as "redox cy- 52 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. - H202 I Cytochrome P450 1 FIGURE 21. Schematic representation of the oneelectron reductive bioactivation of the cytostatic quinone adriamycin. After one-electron reduction of adriamycin by cytochrome P450, the semiquinone free radical is formed under anaerobic conditions. Under aerobic conditions, the serniquinone free radical of adriamycin can redox cycle with molecular oxygen and produce 0;’ and H,OP 0, Cytochrome P450; 0, NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase is able to reduce adriamycin (small arrow); however, cytochrome P450 participates more easily in the one-electron reduction reaction (bold arrow). (From Goeptar, A. R., te Koppele, J. M., Lamme, E. K., Pique, J. M., and Vermeulen, N. P. E., Mol. fharmacol., 44, 1267, 1993. With permission.) cling”. 177~178 The enzymatic or spontaneous dismutation of 0,- can produce H20,,189 and in the presence of trace amounts of certain metal ions (i.e., ferric ions)1go even more deleterious oxygen species such as ‘OH can be for~ned.l~~J~~ ‘OH can cause DNA strand breakage,192J93 antigenic changes in DNA,194 and cell damage by processes such as lipid p e r o x i d a t i ~ n . ~ ~ ~ J ~ ~ Lipid peroxidation is known to cause irreversible modifications of membrane structures and cellular functions.183 In addition, lipid peroxidation can also liberate me- tabolites causing DNA damage197 and/or affect the viability and replication of tumor cells.198 The observation that P450 (notably P450IIB1) is in- volved in the one-electron reductive bioactivation of quinones may have serious consequences for the spectrum of toxicity and antitumor activity of these types of anticancer drugs. 5. Gentian Violet The triphenylmethyl dye gentian violet (hexamethylpararosaniline; Figure 22) as well as structurally related compounds are widely used as 53 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. Carbon-centered free radical 0 2 NADPH-cytochrom 02- P450 reductase I Cytochrome P450 1 FIGURE 22. Proposed one-electron reduction of gentian violet to the carboncentered free radical catalyzed by cytochrome P450. Under aerobic conditions, the free radical intermediate disappears completely, most likely due to direct molecular oxygen reduction under formation of 05.. 0, Cyto- chrome P450; 0, NADPH-cytochrome P450 reductase. (Adapted from Docampo, R. and Moreno, S. N. A, Drug Metab. Rev., 22, 161, 1990.) biological dyes for wood, silk, food, and in cos- metics.'* Gentian violet is a potent mutagen, caus- ing frameshift mutations.200 The mutagenicity of gentian violet is increased in the presence of sub- cellular fractions of rat liver.m It has been sug- gested that gentian violet is one-electron reduced by P450 to a carbon-centered free radical metabo- lite under anaerobic conditions (Figure 22), as demonstrated by the CO and metyrapone sensi- tivity of this reduction reaction.201 Under aerobic conditions, the free-radical intermediate disap- pears completely, most likely due to a direct elec- tron transfer to 0, under concomitant formation of 0, ' and H,O,. However, 0, could also prevent the one-electron reduction of gentian violet by competing directly for electrons at the catalytic site of P450 that is, by the oxygen reductase activity of P450.202 Whether the free-radical intermediate is in- volved in the cytotoxicity and carcinogenicity of gentian violet is not known. Intestinal microflora have been shown to reduce gentian violet under anaerobic conditions.203 Pure cultures of nine gen- era of strict and facultative microflora were clearly shown to convert gentian violet to leucogentian violet.203 This reaction may have toxicological 54 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. significance because the dimethylated derivative leucopararosaniline has also been recognized as a potential carcinogen in 6. Nitro Compounds Nitro compounds also occur widely in the environment. Nitrated polycyclic aromatic hydro- carbons have been detected in the environment as products of incomplete combustion processes, cigarette smoke, and ~ h a r c o a l . ~ ~ J ~ ~ They are also formed in the atmosphere upon reaction of poly- cyclic aromatic hydrocarbons with traces of nitro- gen oxides.2o6 Many of these nitrated polycyclic aromatic hydrocarbons are highly mutagenic and carcinogenic in r ~ d e n t s . ~ . ~ ~ ~ Nitro compounds such as nifurimox208 and benznidazole (N-benzyl- 2-nitro- 1 -imidazole-a~etamid)~ are used as cy- tostatic drugs against hypoxic cells in solid tu- mors. They are used either as an adjunct to radiotherapy or in combination with other It is believed that reduction of the nitro group is necessary for the cytostatic activity and/or cy- totoxic effects.210 Enzymes known to catalyze nitro cyt0statics.~10 reduction include DT-diaphorase,2l0 xanthine oxidase,211 RED,212 and aldehyde oxidase.*13 Evi- dence has been presented that P450 also is in- volved in the reduction of certain nitro com- pounds.211*21"216 Microsomal reduction of nitrobenzene to aniline involves subsequent re- ductive steps (Figure 23).217 The nitro group is reduced in a one-electron reaction to a nitro anion radical (Figure 23), which has been demonstrated by electron spin resonance studies.218 Addition of a second electron and two protons results in the formation of the nitroso compound, which is gen- erally indicated as an intermediate in the reduc- tion of nitro compounds but has rarely been ob- served.219 Subsequent reduction of the nitroso compound yields a hydronitroxide free radical that is detectable by electron spin resonance stud- ies.218 The fourth electron reduction results in the formation of the hydroxylamine. It has been shown that P450 is primarily involved in the phenyl- hydroxylamine reduction to aniline,216 and thus in the last step of nitro reduction. The major metabolite of l-nitropyrene reduc- tion appears to be l -amin~pyrene.~~~ Inhibition, induction, and reconstitution experiments indi- cate that P450IA enzymes are responsible for the H e- e- I R-NO H+ =C R -NO R-NO, - R-NO,' Nitro-compound Nitroanion Nitroso Hydronitroxide free radical t H I .+ e- R-NH, - [ R-NH, R-N-OH Amine Hydroxylamine FIGURE 23. Proposed mechanism for nitro reduction. Nitro compounds are reduced to amines via the nitro anion, nitroso compound, hydronitroxide free radical, and hydroxylamine. Cytochrome P450 is primarily involved in the reduction of hydroxylamines to the amines. (From Mason, R. P. and Josephy, P. S., in Free Radical Mechanism of Nifmeducfase, Rickert, D. E., Ed., Hemisphere, Washington, D.C., 1985. With permission.) 55 Cr iti ca l R ev ie w s i n To xi co lo gy D ow nl oa de d fro m in fo rm ah ea lth ca re .c om b y Qu ee n's U niv ers ity on 09 /26 /13 Fo r p er so na l u se o nl y. observed nitro reduction.215 Interestingly, the re- duction of 1-nitropyrene has been associated with the high-affinity binding of 1 -nitropyrene to P450L41, which is higher than to other forms of P450. With 3-nitrofluoroanthene, the nitro- reductase activity is increased by phenobarbital pretreatment in a reaction inhibited by O,, CO, SK&F 525-A, and n-octylamine, which strongly suggests that P450IIB enzymes are involved in the reduction of 3-nitrofluoroanthene in microso- ma1 fractions of rat liver.214 The P450-mediated reduction of nitro-compounds may have toxico- logical significance because these compounds have been shown to induce bacterial mutations,220 mam- malian mutations,221 sister chromatid and unscheduled DNA ~ynthesis.”~ Moreover, the carcinogenicity of 1 -nitropyrene and 3-nitro- fluoroanthene has been e~tablished.2~~ V. CONCLUSIONS In this review, insights are provided in the mechanism of the oxygen and xenobiotic reduc- tase activities of P450. Inasmuch as P450 is gen- erally recognized as an monooxygenase catalyst, less emphasis has been placed on its role as a reductase. From a historical point of view, early P450 activity was limited to reductive reactions under anaerobic conditions. Thus far, the best documented cases of P450-mediated reduction reactions under anaerobic conditions involves halogenated alkanes. Under aerobic conditions, P450 appears to participate in the one-electron reduction of O,, an activity in this review introduced as “oxygen re- ductase” activity instead of the usual “oxidase” or “uncoupling” activity. The formation of 0, ’ dur- ing the oxygen reductase of P450 is attributed to autooxidation of the [Fe3+-P4501 [Oil complex either spontaneously or in the presence of xenobiotics. When the rate of P450 reduction is greater than the turnover of xenobiotic oxygen- ation, the [Fe3+-P450][0,] complex will accumu- late during steady-state metabolism and conse- quently lead to 0, ’ formation. P450IIl3 enzymes have been shown to exhibit a high oxygen reduc- tase activity. It is also becoming apparent that the oxygen reductase activity may be especially im- portant for P450IE1. During the oxygen reduc- tase activity of P450, reactive oxygen species are formed that can cause irreversible cell damage unless effective means of detoxication are present. There is now also information available on the xenobiotic reductase activity of P450. P450IpB and P450IIE isoenzymes are the major catalyst of xenobiotic reduction reactions. As a result, free- radical intermediates are formed that can lead to irreversible cell damage. A possible role of P450IIE1 in the pathogenesis of some hepatic disorders, particularly alcoholic liver diseases, has been attributed to reduction of xenobiotics and 0, to their respective free-radical intermediate^.'^^ Acetone and other ketones are potent inducers of P450IIE1 and may, at least in part, account for the increased level of this protein after fasting in rats.225 Clearly, the compounds that are reductively activated by P450 have little structural similarity. The precise molecular mechanism describing the driving forces underlying the xenobiotic reduc- tase activity of P450 remains unclear. The de- scription of the molecular mechanisms of xenobiotic and oxygen reduction reactions by P450 is limited by lack of knowledge of the 3D struc- ture of the P450 proteins.226 ACKNOWLEDGMENT The authors thank Dr. J. M. te Koppele (TNO Institute of Ageing and Vascular Research, Leiden, The Netherlands) for valuable discussions. REFERENCES 1 . 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