Essential Role of Singlet Oxygen Species in Cytochrome P450-dependent Substrate Oxygenation by Rat Liver Microsomes

Drug Metab. Pharmacokinet. 20 (1): 14–23 (2005).

Regular Article Essential Role of Singlet Oxygen Species in Cytochrome P450-dependent Substrate Oxygenation by Rat Liver Microsomes Seiko HAYASHI, Hiroyuki YASUI and Hiromu SAKURAI Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University Full text of this paper is available at http://www.jssx.org

Summary: Previously, we reported that singlet oxygen (1O2 ) was involved in rat liver microsomal P450-dependent substrate oxygenations in such reactions as p-hydroxylation of aniline, O-deethylation of 7-ethoxycoumarin, v- and (v-1)-hydroxylations of lauric acid, O-demethylation of p-nitroanisole, and N-demethylation of aminopyrine. In order to conˆrm the generality of 1O2 involvement, we have further investigated which kinds of reactive oxygen species (ROS) are formed during P450-dependent substrate oxygenation in microsomes. We examined CYP2E1-dependent hydroxylation of p-nitrophenol in rat liver microsomes in the presence of some ROS scavengers, because CYP2E1 has been reported to predominantly generate ROS in the hepatic microsomes and to relate with the oxidative stress in the body. The addition of 1O2 quenchers, b-carotene, suppressed the hydroxylation of p-nitrophenol. Furthermore, a nonspeciˆc P450 inhibitor, SKF525A, and a ferric chelator, deferoxamine, both suppressed the hydroxylation. No other ROS scavengers such as superoxide dismutase (SOD), catalase, or mannitol altered the reaction. 1O2 was detectable during the reaction in the microsomes as measured by an electron spin resonance (ESR) spin-trapping method when 2,2,6,6-tetramethyl-4-piperidone (TMPD) was used as a spin-trapping reagent. The 1O2 was quenched by additions of b-carotene, p-nitrophenol, and SKF525A. The reactivity of p-nitrophenol and 1O2 correlated linearly with its hydroxylation rate in the microsomes. On the basis of these results, we conclude that 1O2 contributes to the p-nitrophenol hydroxylation in rat liver microsomes, by adding a new example of 1O2 involvement in the CYP2E1-dependent substrate oxygenations.

Key words: cytochrome P450; CYP2E1; p-nitrophenol hydroxylation; singlet oxygen (1O2); electron spin resonance-spin trapping; rate constant tion of 7-ethoxycoumarin, v- and (v-1)-hydroxylations of lauric acid, O-demethylation of p-nitrosnisole, and N-demethylation of aminopyrine.2,3) CYP2E1 is induced by some chemicals, such as ethanol, acetone, and isoniazid, and under physiological conditions such as diabetes, fasting, or liver disease.4–6) Several groups reported that CYP2E1 from rat liver microsomes elevated the generation of ROS, and thus enhanced the oxidative stress in the body.7,8) Furthermore, some researchers have provided evidence that superoxide anion radical (･O－ 2 ) as well as hydroxyl radical (･OH) were generated in rat liver microsomes including CYP2E1.9–11) However, the P450-dependent reactions in terms of ROS generation have been charac-

Introduction Cytochrome P450 (P450) is a group of enzymes that are responsible for the biotransformation of numerous endogenous and exogenous compounds. A mechanism for the P450-dependent catalytic cycle has been accepted by many researchers, in which an oxo-ferrylporphyrin-p-cation radical is involved as an active oxygen intermediate formed by introducing two electrons.1) However, the precise process of dioxygen activation involved in P450-dependent substrate oxygenations has not been established. We previously proposed the participation of singlet oxygen (1O2 ) in p-hydroxylation of aniline, O-deethyla-

Received; July 2, 2004, Accepted; January 13, 2005 To whom correspondence should be addressed : Prof. Hiromu SAKURAI, Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan. Tel. 81-75-595-4629, Fax. 81-75-595-4753, E-mail: sakurai＠mb.kyoto-phu.ac.jp Abbreviations: P450: cytochrome P450, NADPH: nicotinamide adenine dinucleotide phosphate reduced form, ROS: reactive oxygen species, ESR: electron spin resonance, SOD: superoxide dismutase, DMSO: dimethylsulfoxide, SKF525A: 2-diethylaminoethyl-2,2-diphenylpentanoate, TMPD: 2,2,6,6-tetramethyl-4-piperidone, DMPO: 5,5?-dimethyl-1-pyrroline-N-oxide, 4-oxo-TEMPO: 2,2,6,6-tetramethyl-4-piperidone-N-oxyl

14

Role of Singlet Oxygen in P450-dependent Oxidation

terized insu‹ciently. The purpose of this study was to provide evidence of the contribution of ROS to the P450-dependent substrate oxygenations in rat liver microsomes and to add a new example of 1O2-dependent substrate oxygenation. To achieve this, we used p-nitrophenol as an indicator of CYP2E1 activity12–15) and examined whether ROS were generated in the P450-dependent catalytic oxygenation cycle. The reaction was characterized by using various inhibitors such as hydroxyl radical scavengers, singlet oxygen quenchers, a superoxide scavenger, and a hydrogen peroxide scavenger. In addition, ROS generation during the reaction was examined using an electron spin resonance (ESR) spintrapping method. Material and Methods Materials: Superoxide dismutase (SOD), catalase, deferoxamine, SKF525A, and bovine serum albumin (BSA) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Dithiothreitol, p-nitrophenol, 4nitrocatechol, mannitol, dimethyl sulfoxide (DMSO), Tween-20, and hematoporphyrin were purchased from Wako Pure Chemicals (Osaka, Japan). Sodium azide (NaN3 ) was purchased from Nacalai Tesque (Kyoto, Japan). b-Carotene was obtained from Tokyo Kasei (Tokyo, Japan). 4-Aminophenyl-methanesulfonyl ‰uoride was purchased from Roche Diagnostics (Tokyo, Japan). b-NADPH was purchased from Oriental Yeast (Tokyo, Japan). 5,5-Dimethyl-1pyrroline-N-oxide (DMPO) was purchased from Labotec (Tokyo, Japan). 2,2,6,6-Tetramethyl-4piperidone-N-oxyl (4-oxo-TEMPO), and 2,2,6,6tetramethyl-4-piperidone (TMPD) were purchased from Aldrich (Milwaukee, WI, USA). Rabbit anti-rat CYP2E1 was obtained from Chemicon International, Inc. (Temecula, CA, USA). Peroxidase-conjugated goat anti-rabbit immunoglobulin was purchased from Dakocytomation Co. (Kyoto, Japan). Rat CYP2E1＋ P450 reductase microsomes which are expressed from a transfected rat CYP2E1 cDNA in human lymphoblast was obtained from Gentest Co. (Woburn, MA, USA). All other chemical compounds used were of the highest analytical grade commercially available. Animals: Male Wistar rats (6 weeks old) weighing 180–240 g were purchased from Shimizu Experimental Material Co. (Kyoto, Japan) and maintained on a light W dark cycle in our central animal facility before being used. All animals had free access to diet and tap water prior to the experiments. All experiments were approved by the Experimental Animal Research Committee of Kyoto Pharmaceutical University (KPU) and performed according to the Guidelines for Animal Experimentation of KPU. Microsomes preparations: Microsomes were pre-

15

pared from the livers of male Wistar rats. The animals were fasted overnight prior to being sacriˆced. Under the anesthetized condition by ether, the livers were perfused with an ice-cold 1.15z (w W v) KCl and then homogenized. The nuclei and mitochondria were removed by centrifugation at 12,000×g for 15 min. The supernatant was centrifugated at 105,000×g for 65 min. The obtained microsomal pellet was suspended in 1.15z (w W v) KCl containing 0.25 mM 4-amidinophenyl-methanesulfonyl ‰uoride, and it was centrifugated at 105,000×g for 65 min. The pellet was resuspended in 0.1 M potassium phosphate buŠer, pH 7.4, containing 30z (v W v) glycerol and 1 mM dithiothreitol. The protein concentration was determined by the method of Lowry et al. using BSA as the standard.16) P450 was assayed by the method of Omura and Sato in terms of the CO-reduced P450 complex at pH 7.2.17) Enzyme-linked immunosorbent assay (ELISA)18,19): We loaded 1z (w W v) BSA in phosphate-buŠered saline (PBS) onto 96-well microtiter plates (Nunc, Tokyo, Japan) to block non-speciˆc binding of the proteins. After incubation for 30 min, human lymphoblast microsomes as a standard curve where rat CYP2E1 is well) or rat microexpressed (0.05–1.92 pmol CYP2E1 W somal samples were also plated and then incubated with a primary antibody (anti-rat CYP2E1 diluted 1:500) for 3 h. Subsequently, the plates were incubated with a secondary antibody (anti-rabbit-horseradish peroxidase conjugate diluted 1:2000) for 30 min. All incubations performed were at 309C. Plates were washed three times with PBS including 0.05z (v W v) Tween-20 (PBS-T) between incubations. Next, we added 3, 3?, 5, 5?tetramethyl-benzidine (TMB) as a peroxidase substrate solution (Moss Inc., CA, USA), and ˆnally 0.1 M HCl was added to each well to stop the reaction. The absorbance at 450 nm was measured with a Spectra‰uor Plus (TECAN, Tokyo, Japan). p-Nitrophenol hydroxylation activity: p-Nitrophenol hydroxylation activity in terms of 4-nitrocatecohol formation was determined at 379C in 50 mM phosphate buŠer, pH 7.5, for 10 min. The incubation mixture contained 1.3 nmol W mL P450, 0.1 mM pnitrophenol, and 1 mM NADPH in a total volume of 0.5 mL, and the reaction was initiated by addition of NADPH. The reaction was terminated by addition of v) perchloric acid to the reaction 0.25 mL of 10z (v W mixture. The mixture was then cooled on ice, and the protein was removed by centrifugation at 15,000×g for 5 min. An aliquot (0.3 mL) of the supernatant fraction was added to 30 mL of 10 M NaOH. The absorbance at 540 nm was measured with a Spectra‰uor Plus.12–14) We examined the contribution of ROS in the p-nitrophenol hydroxylation by adding the following compounds to mL SOD, 5000 units W the reaction mixture: 5000 units W

16

Seiko HAYASHI, et al.

mL catalase, 0.2–1 mM mannitol, 0.2–5 mM NaN3, 0.5–1 mM b-carotene, 0.86–85.7 mM ethanol, 0.70–70.5 mM DMSO, 1–20 mM deferoxamine, and 0.05–0.1 mM SKF525A in a total volume of 0.5 mL. ESR spin-trapping of 1O2 in the rat microsomal reaction: ESR spectra were recorded at room temperature on a JEOL JES-RFR30 spectrometer (Tokyo, Japan) using an aqueous quartz ‰at cell (Labotec, Tokyo, Japan). TMPD was used as a 1O2-trapping agent.3,20–23) The incubation mixture contained 50 mM TMPD, 1.3 nmol W mL P450, 1 mM NADPH, and the presence or absence of various concentrations of b-carotene, pnitrophenol, and SKF525A in 50 mM phosphate buŠer (pH 7.5) at 379C in a total volume of 0.5 mL. ESR spectra were measured at 10 min after addition of NADPH as an initiator of the reaction. The ESR signal intensity appearing at the lowest magnetic ˆeld in a triplet spectrum due to a TMPD-1O2 adduct (2,2,6,6tetramethyl-4-piperidone-N-oxyl, 4-oxo-TEMPO) was expressed as the ratio to the third signal intensity from the low magnetic ˆeld due to the external standard, Mn(II) doped in MnO. 1 O2 scavenging activity of b-carotene, p-nitrophenol, and SKF525A in the hematoporphyrin-UVA system: 1 O2 was generated by an ultraviolet light A (UVA) irradiation to the hematoporphyrin (HP) solution.20,21) UVA was irradiated through a UVA ˆlter at a dose of cm2 using a Supercure-203S (San-Ei Electric 800 mW W MFG, Osaka, Japan), which was connected to the ESR cavity. The reaction mixtures contained 62.5 mM HP, 50 mM TMPD, and 0–250 mM p-nitrophenol, 0–500 mM b-carotene, or 0–5 mM SKF525A at room temperature (229C) in a total volume of 0.2 mL of 50 mM phosphate buŠer (pH 7.5). ESR spectra were measured at 30 sec cm2 ) of the reaction mixtures. after the irradiation (24 J W Determination of rate constants for the reaction of 1 O2 and b-carotene or p-nitrophenol: The rate constant, ka, for the physical quenching plus chemical reaction of 1O2 and b-carotene or p-nitrophenol was determined by the ESR spin-trapping method using the following equation (5), Sterm-Volmer plot based on the competitive reaction,24,25) where kd is the rate constant for deactivation of 1O2 in H2 O and kr is the rate constant for reaction of 1O2 and TMPD. The values of kd (5.00× 105 s－1 ) and kr (4.00×107 M－1 s－1 ) were adopted from the literature.25,26) kd

O2''ª3O2

1

(1) kr

O2＋TMPD''ª1O2－TMPD

1

ka (quenching)

O2＋quencher'''''ª O2＋quencher

1

ka (reaction)

3

O2＋quencher''''ªoxidation products

1

I0 W I ＝ 1＋

ka [quencher] kd＋kr [TMPD]

(2) (3) (4) (5)

where I is the ESR signal intensity due to the TMPD-1O2 signal for the system containing the 1O2 generation system, b-carotene or p-nitrophenol, and TMPD, and I0 is that of the control, which contains the 1O2 generating system and TMPD. The ka values were estimated from I vs. [quencher] plots the slopes of straight lines for I0 W with linear regression method, using all quencher concentrations. Substrate-induced diŠerence spectra: Substrateinduced diŠerence spectra were recorded with a Shimadzu MultiSpec-1500 (Kyoto, Japan). The spectral dissociation constant (Ks ) of p-nitrophenol was obtained by a double reciprocal Lineweaver-Burk plot due to the spectral changes associated with additions of increasing concentrations of p-nitrophenol to the liver mL).27) The microsomes microsomes (0.4 nmol P450 W used for the study were suspended in 50 mM phosphate buŠer, pH 7.5. Statistical analysis: All experimental results are expressed as the means±standard deviations (SDs). Statistical signiˆcance was performed by analysis of variance (ANOVA) at a 1z or 5z signiˆcance level of the diŠerence. Results ELISA: In liver microsomes, there exist several molecular forms of cytochrome P450 that diŠer in their primary structure, substrate speciˆcity, and inducibility.28–30) Therefore, an enzyme-linked immunosorbent assay (ELISA) has been used to quantify the individual P450 isoforms in microsomal preparations. In the present study, we determined what percentage of total P450 was CYP2E1, which has been reported to relate with hydroxylation of p-nitrophenol.12,31,32) P450 speciˆc contents and CYP2E1 contents were 0.81±0.05 nmol mg protein and 0.17±0.03 nmol CYP2E1 W nmol P450 W total P450, respectively. CYP2E1 was observed to comprise approximately 17z of the total P450. Substrate oxidations and eŠects of ROS scavengers, a P450 inhibitor, and an iron chelator in the rat liver microsomes: We examined the hydroxylation of pnitrophenol in rat liver microsomes in the presence of scavengers against ROS to determine the ROS involved. Additions of 5000 units W mL SOD (superoxide anion radical scavenger), 5000 units W mL catalase (hydrogen peroxide scavenger), and 0.5 and 2 mM mannitol (hydroxyl radical scavenger) exhibited essentially no eŠects. However, the hydroxylation rate of pnitrophenol was signiˆcantly suppressed by the addition of SKF525A, which is a nonspeciˆc P450 inhibitor.33) In addition, high concentration (20 mM) of deferoxamine, which is an iron chelator, reduced the hydroxylation rate signiˆcantly (Fig. 1). When 1O2 quenchers, b-carotene34–37) and NaN320,23,26) were added to the incubation mixture at 379 C for 10

Role of Singlet Oxygen in P450-dependent Oxidation

min, p-nitrophenol hydroxylation was suppressed in a 1 O2 quencher concentration-dependent manner (Fig. 2), although the higher NaN3 concentrations may inhibit the CYP2E1-dependent substrate oxidation.38) Similarly, addition of ethanol and DMSO, both of which are substrates for CYP2E1, restrained the hydroxylation rate in a concentration-dependent manner (Fig. 3). Detection of 1O2 by ESR spin-trapping in the rat liver microsomes: Because the eŠects of 1O2 quenchers suggested involvement of 1O2 in p-nitrophenol hydroxylation, we next estimated the eŠects of a 1O2 quencher, a P450 substrate and inhibitor in the rat liver microsomal system by an ESR spin-trapping method. Higher con-

Fig. 1. EŠect of ROS scavengers, a P450 inhibitor, and an iron chelator on the hydroxylation of p-nitrophenol in rat liver microsomes. The reaction mixtures contained 1.3 nmol W mL P450, 0.1 mM p-nitrophenol, 1 mM NADPH, and various substances in a ˆnal volume of 0.5 mL of 50 mM phosphate buŠer, pH 7.5, at 379C for 10 min. Concentrations: SOD: 5000 units W mL, catalase: 5000 units W mL, mannitol 1–2: 0.5, 2 mM, SKF525A1–2: 0.05, 0.1 mM, deferoxamine: 20 mM. Signiˆcance: *pº0.01 vs. the control.

17

centrations (À1 mM) of NaN3, a well-known inhibitor of catalase, was reported to inhibit CYP2E1-dependnet ethanol oxidation,38) and thus NaN3 was not used as a 1 O2 quencher in ESR spin-trapping. In addition, to analyze the origin of 1O2 in P450-dependent oxygenations, we examined the eŠect of SKF525A. Detection of 1O2 by ESR spin-trapping was based on the reaction of 1O2 and TMPD, which is a spin-trapping agent selective for 1O2. TMPD generates a stable nitroxide-free radical due to a TMPD-1O2 adduct (2,2,6,6-tetramethyl-4-piperidone-Noxyl; 4-oxo-TEMPO), which is detectable by ESR as shown in the following scheme:

When TMPD was added to the reaction mixture of the microsomes with NADPH, an ESR signal due to 4-oxo-TEMPO with g＝2.0062 and a coupling constant of AN＝1.609 mT was clearly observed, as shown in Fig. 4(A), the parameters being consistent with those reported previously.20,21) No such spectrum was observed without NADPH or microsomes. When p-nitrophenol, b-carotene, or SKF525A was added to the microsomes with NADPH, the signal intensity due to 4-oxo-TEMPO was suppressed in a concentrationdependent manner (Figs. 4(B) and (C), and 5(A)). Detection of ･O－ 2 and ･OH by ESR spin-trapping in the rat liver microsomes: We also investigated whether ･O－ and ･OH contributed to the p-nitrophenol 2 hydroxylation. For this purpose, DMPO was used as a and ･OH. However, spin-trapping agent for ･O－ 2

Fig. 2. EŠect of singlet oxygen quenchers on the hydroxylation of p-nitrophenol in rat liver microsomes. (A): p-Nitrophenol (0.1 mM) was incubated with 1.3 nmol W mL P450, 0.2–5 mM NaN3, and 1 mM NADPH in a total volume of 0.5 mL of 50 mM phosphate buŠer, pH 7.5, at 379C for 10 min. (B): p-Nitrophenol was incubated in the same way as for A with 0.5 and 1 mM b-carotene. Signiˆcance: *pº0.01 vs. the control.

18

Seiko HAYASHI, et al.

Fig. 3. EŠect of other competitive substrates for CYP2E1 on hydroxylation of p-nitrophenol in rat liver microsomes. (A): p-Nitrophenol was incubated in the same way as for Fig. 2 with 0.9–43 mM ethanol. (B): p-Nitrophenol was incubated in the same way as for Fig. 2 with 0.7–35 mM DMSO. Signiˆcance: *pº0.01 vs. the control.

Fig. 4. ESR spectra due to the singlet oxygen adducts of TMPD (4oxo-TEMPO) at room temperature and the eŠects of p-nitrophenol. The reaction mixture contained 50 mM TMPD, 1.3 nmol W mL P450, and 1 mM NADPH in a total volume of 0.5 mL of 50 mM phosphate buŠer, pH 7.5. (A): microsomes＋NADPH＋TMPD, (B): (A) ＋50 mM p-nitrophenol, (C): (A) ＋100 mM p-nitrophenol, (D): microsomes ＋NADPH＋50 mM DMPO, (E): microsomes＋NADPH＋50 mM DMPO＋500 mM p-nitrophenol. Both side signals in the ˆgure correspond to those for the 3rd and 4th lowest signals due to standard Mn(II).

neither ･O－ 2 nor ･OH was detectable (Figs. 4(D) and (E)), suggesting that these ROS were not likely to be generated during the reaction. Detection of 1O2 by ESR spin-trapping in the hematoporphyrin-UVA system: To investigate where 1 O2 is generated and whether the compounds such as a 1 O2 quencher, a P450 substrate and inhibitor react with 1 O2 chemically, we examined the 1O2 quenching activities of b-carotene, p-nitropheol, and SKF525A in the chemical systems by the ESR spin-trapping method. 1 O2 was generated in the HP solution under UVA irradiation. The signal intensity due to 4-oxo-TEMPO decreased following additions of b-carotene and p-nitrophenol in a concentration-dependent manner, indicating that p-nitrophenol reacted chemically with 1 O2. While, SKF525A had no eŠect (Fig. 5(B)). Determination of rate constants for the reaction of 1 O2 and b-carotene or p-nitrophenol: To quantitatively investigate the reactivity of 1O2 and compounds, we determined the rate constants for the reactions (Fig. 6 and Table 1). b-Carotene showed the similar rate constants in the rat liver microsomes (2.70×1010 M－1 s－1 ) and HP-UVA (2.78×1010 M－1 s－1 ) systems, which coincided well with the reported value of 1.4–3.0×1010 M－1 s－1,34,37) indicating that b-carotene equally quenched 1O2 in both systems. p-Nitrophenol showed the higher rate constant of 2.95×1010 M－1 s－1 in the HPUVA system than that of 1.68×1010 M－1 s－1 in the rat liver microsomes, suggesting that p-nitrophenol mainly resulted in physical quenching in the HP-UVA system. However, SKF525A reacted only in the rat liver microsomes with the rate constant of 1.27×1010 M－1 s－1 but hardly in the HP-UVA system. Linear correlation of the relationship between the hydroxylation and 1O2-quenching activity of pnitrophenol in the rat liver microsomes: To understand the relationship between the P450-dependent

Role of Singlet Oxygen in P450-dependent Oxidation

19

Fig. 5. Concentration-dependent scavenging eŠect of b-carotene (), p-nitrophenol (), and SKF525A ($) on both the generated 1O2 in the microsomes-NADPH system (A) and chemically generated 1O2 in HP-UVA system (B). (A): The reaction systems contained 1.3 nmol W mL P450, 50 mM TMPD, 1 mM NADPH, and 0–500 mM b-carotene, 0–250 mM p-nitrophenol, or 0–2 mM SKF525A in a total volume of 0.5 mL of 50 mM phosphate buŠer, pH 7.5 at 379C. ESR spectra were measured 10 min after the addition of NADPH. (B): The systems contained 62.5 mM HP, 50 mM TMPD, 0–500 mM b-carotene, 0–250 mM p-nitrophenol, or 0–2 mM SKF525A in a total volume of 0.2 mL of 50 mM phosphate buŠer, pH 7.5, at room temperature (229C). ESR spectra were measured after 30 sec of UVA irradiation of the system. Signiˆcance: #pº0.05, *pº0.01 vs. the control.

Fig. 6. Sterm-Volmer plot for the quenching of 1O2 by b-carotene (), p-nitrophenol (), and SKF525A ($) on both the generated 1O2 in the microsomes-NADPH system (A) and chemically generated 1O2 in HP-UVA system (B). The experimental procedures were described as Fig. 5. The straight lines were obtained with linear regression, using all concentrations of compounds.

substrate oxidation and 1O2 generation, both the hydroxylation rate of p-nitrophenol and the decreasing ratio of signal intensity due to 4-oxo-TEMPO by addition of p-nitrophenol were examined. The signal intensity decreased signiˆcantly as a result of addition

of p-nitrophenol to the microsome-NADPH system with 50 mM TMPD and 1 mM NADPH. The decrease of the signal intensity due to 4-oxo-TEMPO in the presence of p-nitrophenol correlated linearly (r＝ 0.9991) with the hydroxylation rate of p-nitrophenol,

20

Seiko HAYASHI, et al. Table 1.

Rat liver microsomes HP-UVA

Rate constants for the reactions of 1O2 and several compounds

kp-nitrophenol (M－ 1 s－1)

kb-carotene (M－ 1 s－1)

kSKF525A (M－1 s－1)

kd (s－ 1)

kr (M－1 s－ 1)

1.68×1010 2.95×1010

2.70×1010 2.78×1010

1.27×1010 n.d.

5.00×105

4.00×107

n.d.: not determined. The kd and kr are the rate constants for deactivation of 1O2 in H2O and for reaction of 1O2 and TMPD, respectively.

Discussion

Fig. 7. Relationship between the hydroxylation rate of pnitrophenol and its reactivity to 1O2 as evaluated from the decrease of the signal intensity due to 4-oxo-TEMPO in rat liver microsomes. mL P450, The reaction mixture contained 50 mM TMPD, 1.3 nmol W and 1 mM NADPH, with or without p-nitrophenol (0–250 mM), in a total volume of 0.5 mL of 50 mM phosphate buŠer, pH 7.5, at 379C. After ESR measurement, the samples were recovered and subsequently the quantities of 4-nitrocatechol were determined. The correlation coe‹cient for the linear regression was estimated to be 0.9991.

indicating that p-nitrophenol hydroxylation in the microsomes-NADPH system depends on the consumption of 1O2 generated during the P450 catalytic cycle (Fig. 7). Substrate-induced diŠerence spectra: The spectral change in p-nitrophenol-induced diŠerence was characterized by the appearance of a trough at 380 nm and a peak at 430 nm. This spectrum due to the interaction of p-nitrophenol with P450 indicated that the substrate directly coordinated to the heme site of P450.27) On the other hand, the spectral change was not observed in the case of b-carotene, indicating that the interaction of b-carotene with P450 was unnoticed. We examined the substrate-binding features of P450 with respect to the spectral dissociation constant (Ks ) calculated from a reciprocal plot in substrate-induced diŠerence spectra, and estimated Ks of p-nitrophenol for the rat microsomes to be 0.28 mM, indicating that the binding of p-nitrophenol was not so strong compared with that of SKF525A (Ks＝0.5–1.0 mM).27,39)

The mean ratio of P450 isoforms in human liver microsomes was reported, in which CYP3A4, CYP2C, CYP1A2, CYP2E1, CYP2A6, and CYP2D6 accounted for 28.8, 18.2, 12.7, 6.6, 4.0, and 1.5 percent, respectively, of the total P450.40) In the present study, we found CYP2E1 of the rat microsomes to account for approximately 17z of the total P450, suggesting a higher contribution of CYP2E1 in substrate oxygenation. CYP2E1 was reported to predominantly generate ROS and to relate the oxidative stress.7–11) However, the contribution of ROS to the P450-dependent reactions has been characterized insu‹ciently. Then, we examined whether ROS were generated in the P450-dependent catalytic oxygenation cycle, using p-nitrophenol as an indicator of CYP2E1 activity.12–15) The p-nitrophenol hydroxylation rate was not altered by addition of SOD, catalase, or mannitol (Fig. 1), indicating that neither superoxide (･O－ 2 ) nor hydroxyl radical (･OH) was involved in the P450-dependent substrate oxygenation mechanism. Additions of 1O2 quenchers, b-carotene and NaN3, suppressed signiˆcantly the substrate oxygenation (Fig. 2), demonstrating the great involvement of 1O2 in the rat liver microsomal substrate oxygenation, by supporting our previous ˆndings in diŠerent types of substrate.2,3) However, there remained to be another possibility that NaN3 over 1 mM inhibited the CYP2E1-dependent p-nitrophenol hydroxylation,38) and that NaN3 might aŠect the binding of a type II substrate such as p-nitrophenol with P450.2) Therefore, NaN3 should be avoided to use as a 1O2 quencher when examining the P450-dependent substrate oxygenation. The p-nitrophenol hydroxylation rate was suppressed by addition of CYP2E1 competitive substrates, ethanol and DMSO (Fig. 3), demonstrating the great contribution of CYP2E1 to the hydroxylation of p-nitrophenol. IC50 values of ethanol and DMSO on the p-nitrophenol hydroxylation were estimated to be 2.61 and 4.81 mM, respectively (Fig. 3). While, that of b-carotene was estimated to be 1.06 mM (Fig. 2), indicating that a strong 1O2 quencher suppressed more e‹ciently the reaction than CYP2E1 competitor. These results suggested that the involvement of 1O2 is general in the rat liver P450 substrate oxygenations including CYP2E1. To conˆrm the participation of 1O2 in the present

Role of Singlet Oxygen in P450-dependent Oxidation

system, we applied the ESR spin-trapping method by using TMPD as a 1O2-trapping agent. In the system containing microsomes and NADPH, 1O2 was distinctly detected (Fig. 4). Additions of b-carotene, p-nitrophenol, or SKF525A to the system each suppressed the generation of 1O2 in a concentration-dependent manner (Fig. 5(A)). These results indicated two possibilities: either the substances reacted with 1O2 directly or inhibited the process of 1O2 generation. Consequently, we evaluated which process is predominant in the system. For this purpose, we used a chemical system, in which 1 O2 was generated in the hematoporphyrin (HP) solution under UVA irradiation, and then compared the rate constants to 1O2 estimated between the systems. When the compounds were added to the HP-UVA system, diŠerent tendencies were observed. Addition of b-carotene to the system decreased the amount of 1O2 spin-adduct in a concentration-dependent manner (Fig. 5(B)), with almost the same rate constant of 2.78 ×1010 M－1 s－1 to 1O2 as that of 2.70×1010 M－1 s－1 estimated in the microsomes (Fig. 6 and Table 1). While, no change of 1O2 generation was observed following the addition of SKF525A (Fig. 5(B)). These results indicated that 1O2 reacted with b-carotene based on physical quenching but not with SKF525A. This is because SKF525A, which inhibits P450 by strong interaction with the protein moiety of P450 active site (Ks＝0.5–1.0 mM),27,39) suppressed the amount of 1O2 spin-adduct in the microsomes by inhibiting the process of 1O2 generation. When addition of p-nitrophenol to the system decreased the amount of 1O2 spin-adduct in a concentration-dependent manner (Fig. 5(B)), the rate constant of 2.95×1010 M－1 s－1 to 1O2 in the HP-UVA system was higher than that of 1.68×1010 M－1 s－1 in the microsomes (Fig. 6 and Table 1). p-Nitrophenol was found to have equivalent reactivity to b-carotene, a potent 1O2 quencher, in the HP-UVA system. In terms of the a‹nity with P450, the binding of p-nitrophenol with P450 was conˆrmed to be very weak (Ks＝0.28 mM) compared with that of SKF525A, but that of b-carotene was almost ignored. From these results, we concluded that p-nitrophenol reacted with 1O2 more like it did to b-carotene in the microsomal system. This is probably why the suppressive eŠect of b-carotene on the p-nitrophenol hydroxylation was not strong in spite of having the equivalent rate constant with p-nitrophenol (Fig. 2 (B)). Thus, it was indicated that 1O2 was generated in P450-dependent substrate oxygenations. However, the rate constant of 1O2 and p-nitrophenol estimated in the HP-UVA system was considerably higher than those of 1O2 and other phenolic compounds such as 2, 6-diisopropylphenol previously reported (0.2–2.7×105 M－1 s－1 ),41–43) suggesting the diŠerent mechanism on the reaction of 1O2 and p-nitrophenol from that of 1O2 and other phenolic compounds. Several

21

researchers have shown that the phenoxide anions as the deprotonated forms have much higher reactivity to 1O2 in the basic high pH solution than phenols in the acidic or neutral pH solution,44–46) where the rate constants of 1 O2 and phenoxide anions in the physical quenching manner were estimated to be 1–2×109 M－1 s－1 in H2 O at room temperature. This is because pKa values of phenolic compounds usually range from 8.5 to 10.3.47) While, p-nitrophenol has a pKa value of 7.15 in the aqueous solution, and thus exists 69z as the phenoxide anion form in the neutral pH solution (pH 7.5).47,48) This is the major reason why p-nitrophenol showed the higher rate constant to 1O2 in the experimental systems (pH 7.5) compared with other phenols. The kinetic analysis of photooxidation reported that the phenoxide anion of p-chlorophenol interacted with the chemically generated 1O2 in both the physical quenching and chemical reaction manners.46) The higher rate constant (2.9×1010 M－1 s－1 ) of 1O2 and p-nitrophenol than those of 1O2 and the phenoxide anions of trimethylphenol45) and p-chlorophenol44,46) indicated that the interaction between 1O2 and p-nitrophenol is mainly due to the chemical reaction. Actually, we observed the formation of the p-nitrophenol-derived phenoxy radical in the HP-UVA system by ESR measurement (data not shown), but did not ˆnd predominantly the hydroxylation of p-nitrophenol in the chemical generation system. The detailed reaction pathway including other products will be needed to investigate in the chemical system.49) On the other hand, we tried to detect ･OH and ･O－ 2 in terms of DMPO-OH and DMPO-OOH adducts, respectively, in the rat liver microsomes-NADPH system by the ESR spin-trapping using DMPO as a trapping agent, but we observed no signals in either the absence or presence of p-nitrophenol (Fig. 4). Accordingly, ･OH and ･O－ 2 were not likely to contribute to the P450-dependent substrate oxygenations. Because ROS such as ･OH and ･O－ 2 generated due to the uncoupling and autoxidation in P450 catalytic cycle was already reported not to contribute to the P450dependent substrate reactions and its production was found to be unchanged in the presence and absence of substrates, that is, ROS due to the uncoupling was not consumed during the P450 substrate oxygenations,50,51) at last we examined the linear relationship between hydroxylation rate of p-nitrophenol and the consumption of 1O2 in the P450 reaction pathway. The detection of 1O2 by the ESR spin-trapping method with TMPD is based on the speciˆc reaction of 1O2 and TMPD, and thus the ESR signal intensity due to the spin-adduct, 4-oxo-TEMPO, is linearly proportional to the total generation of 1O2 in the system.20,21) The signal intensity due to 4-oxo-TEMPO was decreased signiˆcantly by addition of p-nitrophenol in comparison with that without p-nitrophenol in the system. This ˆnding is

22

Seiko HAYASHI, et al.

agreement with the previous results on aniline, 7-ethoxycoumarin and p-nitroanisole.2,3) Extent of the decrease of the signal intensity was linearly correlated (r＝ 0.9991) with the hydroxylation rate of p-nitrophenol without TMPD (Fig. 7), indicating that 1O2 generated in P450 catalytic cycle directly contributes to or partially participates in P450-dependent substrate oxygenations. This last question will be needed to examine in the future. In conclusion, the detection of 1O2 generation during the P450-dependent oxygenations and the suppression of p-nitrophenol hydroxylation by 1O2 quenchers strongly indicated that 1O2 played an essential role in the hepatic microsomal substrate oxygenation system, which is similar to our previous ˆndings.2,3) These results indicate that 1O2 may be generated as a heme moiety and that involvement of 1O2 during the P450-dependent oxygenations occurs not only in type I substrates but also in the substrates of the type that bind to the heme site like p-nitrophenol. Further study will be needed to clarify the possible contribution of CYP2E1 to substrate hydroxylation, for example, by using microsomes that contain a large amount of CYP2E1. This investigation is underway.

9) 10)

11)

12)

13)

14)

15)

References 1)

2)

3)

4)

5) 6)

7)

8)

Kellner, D. G.; Hung, S.-C.; Weiss, K. E. and Sligar, S. G.: Kinetic characterization of compound I formation in the thermostable cytochrome P450 CYP119. J. Biol. Chem., 277: 9641–9644 (2002). Osada, M., Ogura, Y., Yasui, H. and Sakurai, H.: Involvement of singlet oxygen in cytochrome P450dependent substrate oxidations. Biochem. Biophys. Res. Commun., 263: 392–397 (1999). Yasui, H., Deo, K., Ogura, Y., Yoshida, H., Shiraga, T., Kagayama, A. and Sakurai, H.: Evidence for singlet oxygen involvement in the rat and human cytochrome P450-dependent substrate oxidations. Drug Metabol. Pharmacokin., 17: 416–426 (2002). Hu, Y., Ingelman-Sundberg, M. and Lindros, K. O.: Induction mechanisms of cytochrome P450 2E1 in liver: interplay between ethanol treatment and starvation. Biochem. Pharmacol., 50: 155–161 (1995). Lieber, C. S.: Cytochrome P-4502E1: Its physiological and pathological role. Physiol. Rev., 77: 517–544 (1997). Isayama, F., Froh, M., Bradford, B. U., McKim, S. E., Kadiiska, M. B., Connor, H. D., Mason, R. P., Koop, D. R., Wheeler, M. D. and Arteel, G. E.: The CYP inhibitor 1-aminobenzotriazole does not prevent oxidative stress associated with alcohol-induced liver injury in rats and mice. Free Rad. Biol. Med., 35: 1568–1581 (2003). Navasumrit, P., Ward, T. H., Dodd, N. J. F. and O'Connor, P. J.: Ethanol-induced free radicals and hepatic DNA strand breaks are prevented in vivo by antioxidants: eŠects of acute and chronic ethanol exposure. Carcinogenesis, 21: 93–99 (2000). Khan, S. and O'Brien, P. J.: Role of the cellular redox state in modulating acute ethanol toxicity in isolated

16)

17)

18)

19)

20)

21) 22)

23)

24)

hepatocytes. Clin. Biochem., 32: 585–589 (1999). McDonough, K. H.: Antioxidant nutrients and alcohol. Toxicology, 189: 89–97 (2003). Lewis, D. F. V.: Oxidative stress: the role of cytochromes P450 in oxygen activation. J. Chem. Tech. Biotech., 77: 1095–1100 (2002). Krikun, G., Lieber, C. S. and Cederbaum, A. I.: Increased microsomal oxidation of ethanol by cytochrome P450 and hydroxyl radical-dependent pathways after chronic ethanol consumption. Biochem. Pharmacol., 33: 3306–3309 (1984). Reinke, L. A. and Moyer, M. J.: p-Nitrophenol hydroxylation. A microsomal oxidation which is highly inducible by ethanol. Drug Metab. Dispos., 13: 548–552 (1985). Hargreaves, M. B., Jones, B. C., Smith, D. A. and Gescher, A.: Inhibition of p-nitrophenol hydroxylase in rat liver microsomes by small aromatic and heterocyclic molecules. Drug Metab. Dispos., 22: 806–810 (1994). Koop, D. R.: Hydroxylation of p-nitrophenol by rabbit ethanol-inducible cytochrome P450 isozyme 3a. Mol. Pharmacol., 29: 399–404 (1986). Tassaneeyakul, W., Veronese, M. E., Birkett, D. J. and Miners, J. O.: High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes. J. Chromatogr. Biomed. Appl., 616: 73–78 (1993). Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the folin phenol reagent. J. Biol. Chem., 243: 265–275 (1951). Omura, T. and Sato, R.: The carbon monoxide-binding pigment of liver microsomes. J. Biol. Chem., 239: 2370–2378 (1964). Paye, M., Beaune, P., Kremers, P., Guengerich, F. P., Letawe-Goujon, F. and Gielen, J.: Quantiˆcation of two cytochrome P-450 isoenzymes by an enzyme-linked immunosorbent assay (ELISA). Biochem. Biophys. Res. Commun., 122: 137–142 (1984). Roe, A. L., Poloyac, S. M., Howard, G., Shedlofsky, S. I. and Blouin, R. A.: The eŠect of endotoxin on hepatocyte nuclear factor 1 nuclear protein binding: potential implications on CYP2E1 expression in the rat. J. Pharm. Pharmacol., 53: 1365–1371 (2001). Lion, Y., Delmelle, M. and Van de Vorst, A.: New method of detecting singlet oxygen production. Nature, 263: 442–443 (1976). Moan, J. and Wold, E.: Detection of singlet oxygen production by ESR. Nature, 279: 450–451 (1979). Dzwigaj, S. and Pezerat, H.: Singlet oxygen-trapping reaction as a method of 1O2 detection: role of some reducing agents. Free Rad. Res. Commun., 23: 106–115 (1995). Ando, T., Yoshikawa, T., Tanigawa, T., Kohno, M., Yoshida, N. and Kondo, M.; Quantiˆcation of singlet oxygen from hematoporphyrin derivative by electron spin resonance. Life Sci., 61: 1953–1959 (1997). Saito, I., Matsuura, T. and Inoue, K.: Formation of superoxide ion via one-electron transfer from electron donors to singlet oxygen. J. Am. Chem. Soc., 105:

Role of Singlet Oxygen in P450-dependent Oxidation

25)

26)

27)

28) 29)

30)

31)

32)

33)

34)

35)

36)

37)

38)

39)

3200–3206 (1983). Tanaka, K., Miura, T., Umezawa, N., Urano, Y., Kikuchi, K., Higuchi, T. and Nagano, T.: Rational design of ‰uorescein-based ‰uorescence probes. Mechanism-based design of a maximum ‰uorescence probe for singlet oxygen. J. Am. Chem. Soc., 123: 2530–2536 (2001). Lion, Y., Gandin, E. and Van de Vorst, A.: On the production of nitroxide radicals by singlet oxygen reaction: an EPR study. Photochem. Photobiol., 31: 305–309 (1980). Schenkman, J. B., Remmer, H. and Estabrook, R. W.: Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol., 3: 113–123 (1967). Hankinson, O.: The aryl hydrocarbon receptor complex. Annu. Rev. Pharmacol. Toxicol., 35: 307–340 (1995). Sueyoshi, T. and Negishi, M.: Phenobarbital response elements of cytochrome P450 genes and nuclear receptors. Annu. Rev. Pharmacol. Toxicol., 41: 123–143 (2001). Goodwin, B., Redinbo, M. R. and Kliewer, S. A.: Regulation of CYP3A gene transcription by the pregnane X receptor. Annu. Rev. Pharmacol. Toxicol., 42: 1–23 (2002). Reinke, L. A., Sexter, S. H. and Rikans, L. E.: Comparison of ethanol and imidazole pretreatments on hepatic monooxygenase activities in the rat. Res. Commun. Chem. Pathol. Pharmacol., 47: 97–106 (1985). Koop, D. R., Laethem, C. L. and Tierney, D. J.: The utility of p-nitrophenol hydroxylation in P450IIE1 analysis. Drug Metab. Rev., 20: 541–551 (1989). Buening, M. K. and Franklin, M. R.: SKF 525-A inhibition, induction, and 452-nm complex formation. Drug Metab. Dispos., 4: 244–255 (1976). Mascio, P. D., Kaiser, S. and Sies, H.: Lycopene as the most e‹cient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys., 274: 532–538 (1989). Fukuzawa, K., Inokami, Y., Tokumura, A., Terao, J. and Suzuki, A.: Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and alpha-tocopherol in liposomes. Lipids, 33: 751–756 (1998). Cantrell, A., McGarvey, D. J., George T. T., Rancan, F. and Bohm, F.: Singlet oxygen quenching by dietary carotenoids in a model membrane environment. Arch. Biochem. Biophys., 412: 47–54 (2003). Mascio, P. D., Devasagayam, T. P. A., Kaiser, S. and Sies, H.: Carotenoids, tocopherols and thiols as biological singlet molecular oxygen quenchers. Biochem. Soc. Tran., 18: 1054–1056 (1990). Salmela, K. S., Tsyrlov, I. B. and Lieber C. S.: Azide inhibits human cytochrome P-4502E1, 1A2, and 3A4. Alcohol. Clin. Exp. Res., 25: 253–260 (2001). Topham, J. C.: Relation between diŠerence spectra and metabolism. Barbiturates, drug interaction, and species

40)

41)

42)

43)

44)

45)

46)

47)

48)

49)

50)

51)

23

diŠerence. Biochem. Pharmacol., 19: 1695–1701 (1970). Shimada, T., Yamazaki, H., Mimura, M., Inui, Y. and Guengerich, F. P.: Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J. Pharmacol. Exp. Ther., 270: 414–423 (1994). Scurlock, R., Rougee, M. and Bensasson, R. V.: Redox properties of phenols, their relationships to singlet oxygen quenching and to their inhibitory eŠects on benzo(a)pyrene-induced neoplasia. Free Rad. Res. Commun., 8: 251–258 (1990). Garcia, N. A.: Singlet-molecular-oxygen-mediated photodegradation of aquatic phenolic pollutants. J. Photochem. Photobiol. B: Biology, 22: 185–196 (1994). Heyne, B., Kohnen, S., Brault, D., Mouithys-Mickalad, A., Tˆbel, F., Hans, P., Fontaine-Aupart, M. P. and Hoebeke, M.: Investigation of singlet oxygen reactivity towards propofol. Photochem. Photobiol. Sci., 2: 939–945 (2003). Palumbo, M. C. and Garcia, N. A.: On the mechanism of quenching of singlet oxygen by chlorinated phenolic pesticides. Toxicol. Environm. Chem., 17: 103–116 (1988). Tratnyek, P. G. and Hoigne, J.: Photooxidation of 2, 4, 6-trimethylphenol in aqueous laboratory solutions and natural waters: kinetics of reaction with singlet oxygen. J. Photochem. Photobiol., A: Chemistry, 84: 153–160 (1994). Ozoemena, K., Kuznetsova, N. and Nyokong, T.: Photosensitized transformation of 4-chlorophenol in the presence of aggregated and non-aggregated metallophthalo-cyanines. J. Photochem. Photobiol., A: Chemistry, 139: 217–224 (2001). Liptak, M. D., Gross, K. C., Seybold, P. G., Feldgus, S. and Shields, G. C.: Absolute pKa determination for substituted phenols. J. Am. Chem. Soc., 124: 6421–6427 (2002). Hung, H. C. and Chang, G. G.: Partitioning of 4nitrophenol in aerosol-OT reverse micelles. J. Chem. Soc., Perkin Trans 2, 2177–2182 (1999). Briviba, K., Devasagayam, T. P. A., Sies, H. and Steenken, S.: Selective para hydroxylation of phenol and aniline by singlet molecular oxygen. Chem. Res. Toxicol., 6: 548–553 (1993). Bell-Parikh, L. C. and Guengerich, F. P.: Kinetic of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde. J. Biol. Chem., 274: 23833–23840 (1999). Nguyen, N. S. D., Cottet-Marie, F., Buetler, T. M., Russo, A. L., Krauskopf, A. S., Armstrong, J. M., Vickers, A. E. M., Mace, K. and Ruegg, U. T.: Metabolism-dependent stimulation of reactive oxygen species and DNA synthesis by cyclosporin A in rat smooth muscle cells. Free Rad. Biol. Med., 27: 1267–1275 (1999).