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Hydroperoxide specificity of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines
Biochimica et Biophysica Acta 1475 (2000) 256^264
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Hydroperoxide speci¢city of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines Carl G. Hover, Arun P. Kulkarni * Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, MDC-56, University of South Florida, 13201 Bruce B. Downs Boulevard, Tampa, FL 33612-3805, USA Received 30 December 1999; received in revised form 18 April 2000; accepted 20 April 2000
Abstract Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117 þ 12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9 þ 0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106 þ 11 nmol HCHO/min/mg SLO, and 3.2 þ 0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Soybean lipoxygenase ; Human placental lipoxygenase ; Chlorpromazine; Phenothiazine; Organic hydroperoxide ; Xenobiotic co-oxidation ; N-Demethylation
1. Introduction Following the discovery of peroxygenase activity of cytochrome P450 (P450), there has been an increasing use of organic hydroperoxides such as cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) in contemporary research dealing with di¡erent issues in biochemical pharmacology/toxicology. These studies have provided valuable insights into the areas such as tissue injury due to oxidative stress, mechanism of oxygen activation, and me-
Abbreviations : LO, lipoxygenase ; HTPLO, human term placental lipoxygenase; SLO, soybean lipoxygenase ; CPZ, chlorpromazine; TBHP, tert-butyl hydroperoxide ; CHP, cumene hydroperoxide ; HCHO, formaldehyde; Con A, concanavalin A-Sepharose 4B; NDGA, nordihydroguaiaretic acid ; BHA, butylated hydroxyanisole ; BHT, butylated hydroxytoluene ; GSH, reduced glutathione; LAOOH, linoleic acid hydroperoxide * Corresponding author. Fax: +1-813-974-4986; E-mail : [email protected]
tabolism of xenobiotics. It is now well established that organic peroxides support xenobiotic oxidation mediated by di¡erent heme-containing enzymes [1,2]. However, the available information on this subject also reveals that each enzyme exhibits a unique speci¢city toward peroxide. Thus, many reports have documented that CHP and TBHP, the two most commonly employed organic peroxides, e¤ciently support a variety of xenobiotic oxidation reactions catalyzed by cytochrome P450 (P450) [1,2]. However, both CHP and TBHP are almost ine¡ective with prostaglandin synthase [3]. Fatty acid hydroperoxides serve as the preferred cofactor and couple xenobiotic oxidation with ease when the reactions are mediated by prostaglandin synthase [3], but not by P450 [4]. Many reports have established that H2 O2 is highly e¡ective in supporting chemical oxidation mediated by horseradish peroxidase [5], lactoperoxidase [6] and myeloperoxidase [7]. In contrast, H2 O2 was reported to be several orders of magnitude less e¡ective than alkyl hydroperoxides in xenobiotic oxidation catalyzed by di¡erent forms of P450 [8,9].
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However, as an exception, H2 O2 e¤ciently couples the activation of 2-amino-3-methylimidazo[4,5-f]quinoline by the P450 isozyme CYP 1A2 [4]. Lipoxygenases (LOs) constitute a family of non-heme iron-containing proteins which exhibit dioxygenase and co-oxidase activities [10]. The dioxygenase activity of LO is characterized by the stereospeci¢c insertion of molecular oxygen into a 1,4-cis-cis-pentadiene moiety of a polyunsaturated fatty acid, leading ultimately to the formation of corresponding stable lipid hydroperoxide [11]. The intermediate peroxyl radical and/or hydroperoxide of polyunsaturated fatty acid support the co-oxidase activity of LO toward di¡erent xenobiotics. At present, our understanding of the peroxide speci¢city for co-oxidase activity of lipoxygenase is poor. Thus far, investigators have employed 5-hydroperoxyeicosatetraenoic acid [12], 15-hydroperoxyeicosatetraenoic acid [13], and/or 13-hydroperoxyoctadecadienoic acid [14^16] in studies on the LOmediated xenobiotic oxidation. Kulkarni and Cook [17] demonstrated that H2 O2 can be substituted for the lipid hydroperoxide to support co-oxidase activity of soybean LO (SLO), a model plant LO, toward various xenobiotics. Subsequent reports have con¢rmed and extended these observations [18^21]. Whether plant and animal tissue LO can utilize CHP and TBHP to support xenobiotic oxidation is unknown at present. Therefore, the primary objective of this study was to examine whether SLO and human term placental LO (HTPLO), a model human tissue LO, can utilize CHP and TBHP. For the following reasons, N-demethylation of phenothiazines was employed to test the hypothesis. N-dealkylation represents a common metabolic reaction of xenobiotics. Earlier, puri¢ed P450 [22] and other hemeproteins, such as horseradish peroxidase [23] and prostaglandin synthase [24] have been shown to dealkylate xenobiotics in the presence of H2 O2 or organic hydroperoxides. Recent studies have documented that SLO can mediate N-dealkylation of N,N-dimethylaniline and related compounds [25], several pesticides [26], and aminopyrine [27,28] in the presence of H2 O2 . Chlorpromazine (CPZ), one of the widely prescribed phenothiazine drugs, is oxidized by SLO [18,19] and undergoes N-demethylation [29] in the presence of H2 O2 [29] or linoleic acid [30]. The evidence presented here for the ¢rst time demonstrates that both SLO and HTPLO can e¡ect N-demethylation of selected phenothiazines in the presence of either TBHP or CHP. 2. Materials and methods 2.1. Materials SLO, type-V (760 000 Sigma U/mg protein), concanavalin A-Sepharose 4B (Con A), cumene hydroperoxide 70% (CHP), t-butyl hydroperoxide 80% (TBHP), chlorproma-
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zine (CPZ), promazine, promethazine, trimeprazine, nordihydroguaiaretic acid (NDGA), gossypol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbate, glutathione (GSH) and dithiothreitol (DTT) were purchased from Sigma, St. Louis, MO. All other chemicals were of reagent grade. 2.2. Human term placentas Human term placentas obtained from healthy women with no known history of smoking, drug or alcohol abuse, or pathological/physiological problems were used in this study. Placentas, obtained from a local hospital immediately after normal delivery, were kept on ice until use. The University of South Florida's Institutional Review Board governing the policies and procedures for research involving human subjects approved the use of human term placentas in this study. HTPLO was puri¢ed by a¤nity chromatography using Con A according to the method described by Joseph et al. [31]. Protein content of enzyme preparations was determined according to Bradford [32]. 2.3. Enzyme assay The standard incubation medium (1.0 ml ¢nal volume) contained, 5.0 mM CPZ (or desired substrate), 20 Wg SLO in 50 mM phosphate bu¡er, pH 7.0 or 100 Wg HTPLO in 50 mM phosphate bu¡er, pH 6.5 and 0.5 mM CHP or TBHP. A control (incubation medium containing all components except the enzyme) was run for each set of experiments. In each case, the reaction was initiated by the addition of the desired organic hydroperoxide. After incubation at 37³C for 30 min, the reaction was terminated by the addition of 1 ml of cold 10% trichloroacetic acid. The N-demethylase activity of SLO and HTPLO was estimated from the amount of formaldehyde accumulated in the reaction medium. Double strength Nash reagent [33] was used for the formaldehyde determination. The data reported are corrected for non-enzymatic reaction. Each experiment was repeated v4 times and the experimental data are presented as mean þ S.E.M. 3. Results CPZ was selected as a prototypic phenothiazine drug to examine the formation of the cation radical. The optical di¡erence spectra exhibited a distinct peak with an absorption maximum at V525 nm when CPZ was incubated in 50 mM citrate bu¡er, pH 3.5, with SLO in the presence of CHP or TBHP (Fig. 1A). Similar results were obtained when CPZ was incubated in 50 mM citrate bu¡er, pH 3.5, with either HTPLO and CHP or HTPLO and TBHP (Fig. 1B). Due to instability, the CPZ cation radical was undetectable when experiments were conducted in 50 mM phosphate bu¡er at the pH that is optimal for N-de-
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Fig. 1. Optical di¡erence spectra obtained during lipoxygenase-mediated N-demethylation of chlorpromazine (CPZ) in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP). The reaction media (1.0 ml ¢nal volume) contained speci¢ed enzyme (absent in the reference cuvette), 5 mM CPZ, and 0.5 mM CHP or TBHP in 50 mM citrate bu¡er, pH 3.5. The spectrum was recorded 5 min after initiation of the reaction by the addition of the organic hydroperoxide. (A) SLO. (B) HTPLO.
methylation of CPZ, i.e. at pH 6.5, for HTPLO and pH 7.0 for SLO. Pilot experiments, designed to establish optimal assay conditions for N-demethylation, clearly indicated that formaldehyde accumulates in the reaction medium when either HTPLO or SLO was incubated with CPZ or other phenothiazines in the presence of either TBHP or CHP. Using optimal assay conditions, the e¡ect of di¡erent experimental parameters on CPZ N-demethylation was studied. As shown in Fig. 2A^D, the pH optimum for CPZ N-demethylation was approximately pH 6.5 for HTPLO and pH 7.0 for SLO regardless of whether CHP or TBHP was used. At 7.4 pH, a signi¢cant rate of the reaction was observed in the incubations with either SLO or HTPLO. The enzymatic formaldehyde production from CPZ was also dependent on the incubation time. The CHP-dependent reaction was linear for over 40 min with HTPLO (Fig. 3A) as well as with SLO (Fig. 4A). Similar results were obtained with TBHP (Figs. 5A and 6A). In view of this, all other experiments were terminated at 30 min. The rate of formaldehyde accumulation increased proportionally with an increase in the amount of HTPLO (Figs. 3B and 5B) or SLO (Figs. 4B and 6B). When experiments were conducted in the presence of varying concentrations of the organic hydroperoxide, the highest speci¢c activity was noted when 0.5 mM TBHP or CHP was used with HTPLO (Figs. 3C and 5C) and SLO (Figs. 4C and 6C). A further increase in the concentration of organic hydroperoxide ( s 0.5 mM) resulted in a progressive decrease in the rate of formaldehyde production from CPZ by HTPLO or SLO. This e¡ect is most likely due to inactivation of the enzyme protein by denaturation. The rate
Fig. 2. E¡ect of pH on the formaldehyde production during lipoxygenase-mediated N-demethylation of chlorpromazine (CPZ) in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP). The reaction media (1.0 ml ¢nal volume) contained speci¢ed enzyme (absent in the control) 5 mM CPZ, and 0.5 mM CHP or TBHP in 50 mM phosphate bu¡er at the indicated pH. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Materials and Methods for further details. (A,C) SLO. (B,D) HTPLO.
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Fig. 3. Formaldehyde production during HTPLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time ; (B) HTPLO concentration ; (C) CHP concentration ; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
of formaldehyde production also demonstrated a marked dependence on the concentration of CPZ in the incubations containing HTPLO (Figs. 3D and 5D) or SLO (Fig. 4D and 6D). The analysis of enzyme kinetic data by Lineweaver^ Burk plots for the TBHP supported reaction yielded an apparent KM value of 2.6 mM for CPZ and an apparent Vmax value of 147 þ 15 nmol of HCHO/min/mg SLO. The estimated KM value for TBHP in this reaction was 0.23 mM. Similar data analysis for the SLO-mediated reaction
in the presence of CHP resulted in an apparent KM value of 2.2 mM for CPZ and an apparent Vmax value of 133 þ 13 nmol of HCHO/min/mg SLO. The KM value for CHP in this reaction was determined to be 0.22 mM. Similar experiments with HTPLO in the presence of TBHP yielded an apparent KM value of 2.0 mM for CPZ and an apparent Vmax value of 5.0 þ 0.9 nmol of HCHO/min/mg HTPLO. The estimated KM value for TBHP in this reaction was 0.26 mM. The analysis for the reaction in the presence of CHP resulted in an appar-
Fig. 4. Formaldehyde production during SLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time; (B) SLO concentration ; (C) CHP concentration; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
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Fig. 5. Formaldehyde production during HTPLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time ; (B) HTPLO concentration; (C) TBHP concentration ; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
ent KM value of 2.6 mM for CPZ and an apparent Vmax value of 4.5 þ 0.8 nmol of HCHO/min/mg HTPLO. The estimated KM for CHP in this reaction was determined to be 0.29 mM. A signi¢cant dose-dependent inhibitory e¡ect was observed when NDGA, a classical LO inhibitor, was added to the incubation medium containing either SLO or HTPLO (Table 1). A similar inhibitory response was noted when gossypol, another known LO inhibitor, was
included in the reaction medium. The free radical scavengers, BHA or BHT, in the incubation medium also decreased the rate of production of formaldehyde in a concentration-dependent manner suggesting the involvement of free radicals in the reaction. Similarly, the inclusion of a reducing agent, such as ascorbate, GSH, or DTT in the reaction medium caused a signi¢cant suppression of the reaction. Other phenothiazines were also N-demethylated by
Fig. 6. Formaldehyde production during SLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time ; (B) SLO concentration; (C) TBHP concentration ; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
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C.G. Hover, A.P. Kulkarni / Biochimica et Biophysica Acta 1475 (2000) 256^264 Table 1 E¡ect of modi¢ers on SLO-mediated N-demethylation of CPZ in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP) Modi¢er
Concentration (WM)
CPZ N-demethylation (relative activity) SLO
^ NDGA
Gossypol
BHA BHT Ascorbate
Glutathione
DTT
^ 50 100 250 5 10 20 100 250 100 250 100 250 500 100 250 500 50 100 200
HTPLO
CHP
TBHP
CHP
TBHP
100 66 47 20 73 63 46 87 52 73 48 65 36 17 64 56 41 90 76 62
100 73 56 25 74 62 48 80 47 70 47 62 33 15 65 58 44 88 73 64
100 84 61
100 81 60
78 68 51 84 50 72 54 66 35 21 69 59 47 92 77 68
79 70 53 83 48 71 50 65 37 20 71 62 50 91 75 69
The incubation media (1.0 ml ¢nal volume) contained 20 Wg SLO or 100 Wg HTPLO (enzyme absent in the control), 0.5 mM CHP or TBHP, 5 mM CPZ, and the indicated concentration of modi¢er in 50 mM phosphate bu¡er pH 7.0 for SLO and pH 6.5 for HTPLO. The modi¢er was preincubated with the speci¢ed modi¢er for 2 min prior to the addition of other components. All incubations were performed at 37³C for 30 min. The amount of formaldehyde produced was estimated by Nash reagent. The speci¢c activity values in the controls without a modi¢er were 106 þ 11 nmol HCHO/min/mg SLO with CHP and 117 þ 12 nmol formaldehyde/min/mg SLO with TBHP and 3.2 þ 0.35 nmol HCHO/min/mg HTPLO with CHP and 3.9 þ 0.40 nmol formaldehyde/min/mg HTPLO with TBHP.
HTPLO and SLO in the presence of CHP or TBHP (Table 2). The observations indicate that promethazine served as the best substrate for both the enzymes, while other phenothiazines yielded relatively low speci¢c activities when compared with CPZ. Additional experiments performed (data not shown) with SLO in the presence of di¡erent concentrations of CHP or TBHP indicated that drugs,
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such as ca¡eine or nicotine, which have been demonstrated to undergo N-demethylation by cytochrome P450, yield no detectable formaldehyde accumulation in the reaction media. 4. Discussion Previous studies have demonstrated the ability of CHP to support the N-dealkylation of xenobiotics in the presence of P450 [22]. Recently, it has been shown that SLO is also capable of this reaction in the presence of H2 O2 [25^ 29]. The evidence suggests that this property is also shared by HTPLO [25,29]. Lipid peroxidation is a normal biochemical process that occurs constantly in various tissues in the body. The process is either non-enzymatic, associated with the free redox active metals or enzymatic, catalyzed by prostaglandin synthase or LO. Available reports [12^16] suggest that the variety of lipid hydroperoxides generated during peroxidation of free polyunsaturated fatty acids can support co-oxidation of xenobiotics via the LO pathway. In theory, other hydroperoxides, derived from complex membrane-bound lipids, such as phospholipids, can also support these reactions. However, due to various technical di¤culties in the isolation of individual species of lipid hydroperoxide in su¤cient quantities and/ or prohibitive costs, their utilization in the investigation of hydroperoxidase activity of LO has been extremely limited. On the other hand, synthetic organic peroxide, such as TBHP and CHP, if found to be useful, may allow full exploration of this aspect of the enzyme. Therefore, the primary objective of this investigation was to evaluate whether TBHP and CHP can be substituted for H2 O2 or polyunsaturated fatty acid hydroperoxides in supporting the LO-mediated co-oxidation of xenobiotics. Our observations clearly indicate that the hydroperoxidase activity of LO from both plant (SLO) and human tissue (HTPLO) does not exhibit strict substrate speci¢city towards hydroperoxides and they can e¤ciently utilize both CHP and TBHP in the enzymatic reaction leading to the oxidation of phenothiazines. The pH optimum for the SLO-mediated N-dealkylation of chlorpromazine was found to be 7.0, while that for
Table 2 Lipoxygenase-mediated N-demethylation of phenothiazines in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP) Phenothiazine
CPZ N-demethylation [speci¢c activity (nmol HCHO/min)] Per mg SLO
Chlorpromazine Promazine Promethazine Trimeprazine
Per mg HTPLO
CHP
TBHP
CHP
TBHP
106 þ 11 57 þ 6 294 þ 27 151 þ 14
117 þ 12 68 þ 7 356 þ 31 157 þ 15
3.2 þ 0.35 2.0 þ 0.22 11.3 þ 1.44 5.0 þ 0.56
3.9 þ 0.40 2.3 þ 0.24 15.8 þ 1.55 7.9 þ 0.80
The incubation media (1.0 ml ¢nal volume) contained 20 Wg SLO or 100 Wg HTPLO (enzyme absent in the control), 0.5 mM CHP or TBHP, 5.0 mM speci¢c substrate in 50 mM phosphate bu¡er pH 7.0 for SLO and pH 6.5 for HTPLO. All incubations were performed at 37³C for 30 min. The amount of formaldehyde produced was estimated by Nash reagent. Values are mean þ S.E.M. (nv4).
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HTPLO was 6.5 in the presence of either organic hydroperoxide (Fig. 2). The consistent dependency of the reaction mediated by SLO and HTPLO on the incubation time, organic hydroperoxide, substrate, and enzyme concentrations (Figs. 3^6) clearly suggests an enzymatic nature of the reaction. The suppression of the rate of formaldehyde production by NDGA or gossypol (Table 1) implicates LO as the catalyst involved in the reaction. Although several studies have been carried out on the peroxidative oxidation of CPZ by di¡erent protein catalysts [18,19,34^38], they are limited to the demonstration of the formation of initial cation radical, a relatively stable species that can be detected spectrally as a peak with an absorption maximum at V525 nm. Thus far, only two studies [29,30] have reported the LO-mediated N-demethylation of phenothiazines. The authors [29,30] proposed that the ¢rst step in the H2 O2 -supported LO-catalyzed N-demethylation of phenothiazines involves the formation of a cation radical as the initial intermediate. The data presented in Fig. 1A,B clearly indicate that the formation of the cation radical from CPZ does occur when the reaction media containing either SLO or HTPLO are supplemented with either CHP or TBHP. The optimal pH of the reaction medium to detect the CPZ radical generated either by SLO or HTPLO was 3.5. This may be due to the instability of the radical species at higher pH. The requirement of an acidic environment to detect the CPZ cation radical noted in this study is in accordance with the observations reported by others with various enzyme systems [18,19,29,34^38]. Clearly, it appears that the instability of the species in the media with higher pH re£ects the characteristic of the cation radical itself and is totally unrelated to the enzyme catalyst responsible for its generation. We believe that the free radical mechanism similar to that proposed for the H2 O2 -dependent LO-mediated N-demethylation of aminopyrine [27,28], CPZ [29] and other compounds [25,26] may be operational in the CPZ N-demethylation studied here in the presence of CHP and TBHP. This contention is supported by the facts that in addition to the formation CPZ cation radical (Fig. 1), the expected signi¢cant suppression of the formaldehyde accumulation was noted when free radical scavengers, such BHT and BHA, as well as reducing agents, such as ascorbate, GSH or DTT, were included in the reaction media (Table 1). The present ¢ndings raise the question of the identity of the one electron oxidant of CPZ generated by LO in the presence of CHP and TBHP. However, the experiments performed in this investigation cannot shed any light on this aspect. Based on the known chemistry of radical decomposition of hydroperoxide [39], the generation of cumyloxy radical from CHP by LO can be anticipated. Further decomposition to the methyl radical and acetophenone is also known [39]. Thus, it is possible that the cumyloxy and/or methyl radical participate in the CHP-dependent CPZ oxidation. Similarly, the alkoxyl radical formed by homolytic cleavage of TBHP is pre-
sumed to initiate CPZ N-demethylation by SLO and HTPLO. A comparison of the speci¢c activity rates of SLO and HTPLO-mediated N-demethylation of phenothiazines observed here with the organic peroxides as opposed to the previous report that employed H2 O2 [29], shows a remarkable similarity. In the previous study [29], the apparent Vmax value for the H2 O2 -dependent reaction was determined to be 132 þ 15 nmol of HCHO produced/min/mg SLO. The apparent Vmax values for the TBHP- and CHP-dependent reactions were 147 þ 15 nmol and 133 þ 13 nmol of HCHO produced/min/mg SLO, respectively. The Km values ranging between 0.2 and 0.3 mM for CHP and TBHP noted in this study for SLO and HTPLO are comparable with the previously reported values of 0.3 and 0.1 mM for the H2 O2 -supported CPZ N-demethylation catalyzed by SLO and HTPLO, respectively [29]. Taken together, these observations suggest that both CHP and TBHP are nearly as e¡ective as H2 O2 in supporting the N-demethylase activity of SLO and HTPLO toward CPZ. The relatively higher rate of reaction observed with the SLO-mediated, as opposed to HTPLO-mediated CPZ N-demethylation in the presence of CHP and TBHP can be attributed to the expression of speci¢c activity on per milligram protein basis, rather than normalizing the data on equal amounts of functional enzyme based on units of activity (1 unit equals the amount of LO that produces 1 Wmol of LAOOH/min when dioxygenase activity is assayed using linoleic acid). The SLO utilized in these experiments contained 760 000 units of enzyme per milligram protein. The HTPLO was found to contain an average of 188 þ 21 units per milligram protein. An inverse picture emerges when the speci¢c activity data are re-calculated on the basis of per unit of dioxygenase activity (0.14 and 17.0 pmol/min/unit of enzyme, for SLO and HTPLO, respectively) suggesting that HTPLO is V120 times more e¤cient in utilizing these hydroperoxides than SLO. Although N-demethylation of phenothiazines was employed in this study as a model reaction for xenobiotic oxidation, it is possible that CHP and TBHP may support other reactions of xenobiotic oxidation via the LO pathway. Further studies are needed to better understand the hydroperoxidase activity of LOs. The typical characteristics of cellular oxidative stress noted by many investigators include a rise in the cytosolic calcium, a signi¢cant depletion of the GSH and pyridine nucleotide pools, the generation of free radicals, reactive oxygen species and an increasing accumulation of lipid peroxidation products [40]. TBHP is commonly used to induce cellular oxidative stress in liver [41], brain [42] lung [43] and other tissues. The present ¢ndings that HTPLO can utilize tertiary hydroperoxides as cofactors may be relevant in the understanding of the hydroperoxide-induced cellular oxidative stress considering the facts that easily measurable LO activity occurs in liver [44], brain [45], lungs [46] and other tissues. Furthermore, LO
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exhibits a remarkable ability to oxidize GSH [47^49], NAD(P)H [50] and generate concomitantly superoxide anions. An elevation of cytoplasmic calcium is known to activates LO in intact cells while an in vitro calcium addition to the reaction media stimulates peroxidation of polyunsaturated fatty acids via the dioxygenase activity of puri¢ed HTPLO [31] and other LOs. Thus, besides GSH peroxidase and P450, this study implicates, for the ¢rst time, that LO pathway as an active participant contributing directly to the genesis of oxidative stress in hydroperoxide exposed cells. Further studies are needed to con¢rm or refute this postulate.
References [1] M.J. Coon, A.D. Vaz, L.L. Bestervelt, Peroxidative reactions of diversozymes, FASEB J. 10 (1996) 428^434. [2] P.J. O'Brien, Hydroperoxides and superoxides in microsomal oxidations, Pharmacol. Ther. A2 (1978) 517^536. [3] W.L. Smith, L.J. Marnett, Prostaglandin endoperoxide synthase: structure and catalysis, Biochim. Biophys. Acta 1083 (1991) 1^17. [4] M.R. Anari, P.D. Josephy, T. Henry, P.J. O'Brien, Hydrogen peroxide supports human and rat cytochrome P450 1A2-catalyzed 2-amino-3-methylimidazo[4,5-f]quinoline bioactivation to mutagenic metabolites: signi¢cance of cytochrome P450 peroxygenase, Chem. Res. Toxicol. 10 (1997) 582^588. [5] Y. Tsuruta, P.D. Josephy, A.D. Rahimtula, P.J. O'Brien, Peroxidasecatalyzed benzidine binding to DNA, and other macromolecules, Chem.-Biol. Interact. 54 (1985) 143^158. [6] H. Kohler, H. Jenzer, Interaction of lactoperoxidase with hydrogen peroxide. Formation of enzyme intermediate and generation of free radicals, Free Radic. Biol. Med. 6 (1989) 323^339. [7] V. Fischer, J.A. Haar, L. Greiner, R.V. Lloyd, R.P. Mason, Possible role of free radical formation in clozapine (clozaril)-induced agranulocytosis, Mol. Pharmacol. 40 (1991) 846^853. [8] G.D. Nordblom, R.E. White, M.J. Coon, Studies of hydroperoxidedependent substrate hydroxylation by puri¢ed liver microsomal cytochrome P450, Arch. Biochem. Biophys. 175 (1976) 524^533. [9] R. Penneberg, F. Scheller, K. Ruckpaul, J. Pirrwitz, P. Mohr, NADPH and H2 O2 -dependent reactions of cytochrome P450LM compared with peroxidase catalysis, FEBS Lett. 96 (1978) 349^353. [10] A.P. Kulkarni, Role of xenobiotic metabolism in developmental toxicity, in: R. Hood (Ed.), Handbook of Developmental Toxicology, CRC Press, Boca Raton, FL, 1996, pp. 383^421. [11] S. Yamamoto, Mammalian lipoxygenases : molecular structures and functions, Biochim. Biophys. Acta 1128 (1992) 117^131. [12] C. Cucurou, J.P. Battioni, R. Daniel, D. Mansuy, Peroxidase-like activity of lipoxygenases : Di¡erent substrate speci¢city of potato 5-lipoxygenase and soybean 15-lipoxigenase and particular a¤nity of vitamin E derivatives for the 5-lipoxygenase, Biochim. Biophys. Acta 1081 (1991) 99^105. [13] M.F. Hughes, W. Chamulitrat, R.P. Mason, T.E. Eling, Epoxidation of 7,8-dihydrobenzo[a]pyrene via a hydroperoxide-dependent mechanism catalyzed by lipoxygenase, Carcinogenesis 10 (1989) 2075^2080. [14] D. Mansuy, C. Cucurou, B. Biatry, J.P. Battioni, Soybean lipoxygenase-catalyzed oxidations by linoleic acid hydroperoxide : di¡erent reducing substrates and dehydrogenation of phenidone and BW 755C, Biochem. Biophys. Res. Commun. 151 (1988) 339^346. [15] D. Riendeau, J.P. Falgueyret, J. Guay, N. Ueda, S. Yamamoto, Pseudoperoxidase activity of 5-lipoxygenase stimulated by potent benzofuranol and N-hydroxyurea inhibitors of the lipoxygenase reaction, Biochem. J. 274 (1991) 287^292.
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C.G. Hover, A.P. Kulkarni / Biochimica et Biophysica Acta 1475 (2000) 256^264 kocytes: induction of covalent binding of chlorpromazine to nucleic acids and proteins, Chem. Biol. Int. 79 (1991) 15^30. L.H. Piette, G. Bulow, J. Yamazaki, Electron-paramagnetic resonance studies of the chlorpromazine free radical formed during enzymatic oxidation by peroxidase-hydrogen peroxide, Biochim. Biophys. Acta 88 (1964) 120^129. A. Vazquez, J. Tudela, R. Varon, F. Garcia-Canovas, Determination of the molar absorptivities of phenothiazine cation radicals generated by oxidation with hydrogen peroxide/peroxidase, Anal. Biochem. 202 (1992) 245^248. M.S. Kharasch, A. Fono, W. Nudenberg, The chemistry of hydroperoxides. III. The free radical decomposition of hydroperoxides, J. Organ. Chem. 15 (1950) 763^774. H. Sies, Oxidative Stress, Academic Press, London 1985. K. Yamamoto, J.L. Farber, Metabolism of pyridine nucleotides in cultured rat hepatocytes intoxicated with tert-butyl hydroperoxide, Biochem. Pharmacol. 43 (1992) 1119^1126. K. Abe, H. Saito, Characterization of t-butyl hydroperoxide toxicity in cultured rat cortical neurones and astrocytes, Pharmacol. Toxicol. 83 (1998) 40^46. I.S. Farrukh, J.R. Michael, S.P. Peters, A.M. Sciuto, N.F. Atkinson, H.S. Freeland, A. Paky, E.W. Spannhake, W.R. Summer, G.H. Gurtner, The role of cycloooxygenase and lipoxygenase mediators in oxidant-induced lung injury, Am. Rev. Resp. Dis. 137 (1988) 1343^1349.
[44] S. Roy, A.P. Kulkarni, Isolation and some properties of dioxygenase and co-oxidase activities of adult human liver cytosolic lipoxygenase, J. Biochem. Toxicol. 11 (1997) 161^174. [45] A.K. Naidu, A.K. Naidu, A.P. Kulkarni, Dioxygenase and hydroperoxidase activities of rat brain cytosolic lipoxygenase, Res. Commun. Chem. Pathol. Pharmacol. 75 (1992) 347^356. [46] P. Roy, A.P. Kulkarni, Cooxidation of acrylonitrile by soybean lipoxygenase and partially puri¢ed human lung lipoxygenase, Xenobiotica 29 (1999) 511^531. [47] P. Roy, M. Sajan, A.P. Kulkarni, Lipoxygenase mediated glutathione oxidation and superoxide generation, J. Biochem. Toxicol. 10 (1995) 111^120. [48] A.P. Kulkarni, M. Sajan, A novel mechanism of glutathione conjugate formation by lipoxygenase. A study with ethacrynic acid, Toxic. Appl. Pharmacol. 143 (1997) 179^188. [49] A.P. Kulkarni, M. Sajan, Lipoxygenase ^ another pathway for glutathione conjugation of xenobiotics. A study with human term placental lipoxygenase and ethacrynic acid, Arch. Biochem. Biophys. 371 (1999) 220^227. [50] P. Roy, S. Roy, A. Mitra, A.P. Kulkarni, Superoxide generation by lipoxygenase in the presence of NADH and NADPH, Biochim. Biophys. Acta 1214 (1994) 171^179.
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Hydroperoxide speci¢city of plant and human tissue lipoxygenase: an in vitro evaluation using N-demethylation of phenothiazines Carl G. Hover, Arun P. Kulkarni * Florida Toxicology Research Center, Department of Environmental and Occupational Health, College of Public Health, MDC-56, University of South Florida, 13201 Bruce B. Downs Boulevard, Tampa, FL 33612-3805, USA Received 30 December 1999; received in revised form 18 April 2000; accepted 20 April 2000
Abstract Since hydroperoxide specificity of lipoxygenase (LO) is poorly understood at present, we investigated the ability of cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) to support cooxidase activity of the enzyme toward the selected xenobiotics. Considering the fact that in the past, studies of xenobiotic N-demethylation have focused on heme-proteins such as P450 and peroxidases, in this study, we investigated the ability of non-heme iron proteins, namely soybean LO (SLO) and human term placental LO (HTPLO) to mediate N-demethylation of phenothiazines. In addition to being dependent on peroxide concentration, the reaction was dependent on enzyme concentration, substrate concentration, incubation time, and pH of the medium. Using Nash reagent to estimate formaldehyde production, the specific activity under optimal assay conditions for the SLO mediated N-demethylation of chlorpromazine (CPZ), a prototypic phenothiazine, in the presence of TBHP, was determined to be 117 þ 12 nmol HCHO/min/mg protein, while that of HTPLO was 3.9 þ 0.40 nmol HCHO/min/mg protein. Similar experiments in the presence of CHP yielded specific activities of 106 þ 11 nmol HCHO/min/mg SLO, and 3.2 þ 0.35 nmol HCHO/min/mg HTPLO. As expected, nordihydroguaiaretic acid and gossypol, the classical inhibitors of LOs, as well as antioxidants and free radical reducing agents, caused a marked reduction in the rate of formaldehyde production from CPZ by SLO in the reaction media fortified with either CHP or TBHP. Besides chlorpromazine, both SLO and HTPLO also mediated the N-demethylation of other phenothiazines in the presence of these organic hydroperoxides. ß 2000 Elsevier Science B.V. All rights reserved. Keywords : Soybean lipoxygenase ; Human placental lipoxygenase ; Chlorpromazine; Phenothiazine; Organic hydroperoxide ; Xenobiotic co-oxidation ; N-Demethylation
1. Introduction Following the discovery of peroxygenase activity of cytochrome P450 (P450), there has been an increasing use of organic hydroperoxides such as cumene hydroperoxide (CHP) and tert-butyl hydroperoxide (TBHP) in contemporary research dealing with di¡erent issues in biochemical pharmacology/toxicology. These studies have provided valuable insights into the areas such as tissue injury due to oxidative stress, mechanism of oxygen activation, and me-
Abbreviations : LO, lipoxygenase ; HTPLO, human term placental lipoxygenase; SLO, soybean lipoxygenase ; CPZ, chlorpromazine; TBHP, tert-butyl hydroperoxide ; CHP, cumene hydroperoxide ; HCHO, formaldehyde; Con A, concanavalin A-Sepharose 4B; NDGA, nordihydroguaiaretic acid ; BHA, butylated hydroxyanisole ; BHT, butylated hydroxytoluene ; GSH, reduced glutathione; LAOOH, linoleic acid hydroperoxide * Corresponding author. Fax: +1-813-974-4986; E-mail : [email protected]
tabolism of xenobiotics. It is now well established that organic peroxides support xenobiotic oxidation mediated by di¡erent heme-containing enzymes [1,2]. However, the available information on this subject also reveals that each enzyme exhibits a unique speci¢city toward peroxide. Thus, many reports have documented that CHP and TBHP, the two most commonly employed organic peroxides, e¤ciently support a variety of xenobiotic oxidation reactions catalyzed by cytochrome P450 (P450) [1,2]. However, both CHP and TBHP are almost ine¡ective with prostaglandin synthase [3]. Fatty acid hydroperoxides serve as the preferred cofactor and couple xenobiotic oxidation with ease when the reactions are mediated by prostaglandin synthase [3], but not by P450 [4]. Many reports have established that H2 O2 is highly e¡ective in supporting chemical oxidation mediated by horseradish peroxidase [5], lactoperoxidase [6] and myeloperoxidase [7]. In contrast, H2 O2 was reported to be several orders of magnitude less e¡ective than alkyl hydroperoxides in xenobiotic oxidation catalyzed by di¡erent forms of P450 [8,9].
0304-4165 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 0 ) 0 0 0 7 4 - X
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However, as an exception, H2 O2 e¤ciently couples the activation of 2-amino-3-methylimidazo[4,5-f]quinoline by the P450 isozyme CYP 1A2 [4]. Lipoxygenases (LOs) constitute a family of non-heme iron-containing proteins which exhibit dioxygenase and co-oxidase activities [10]. The dioxygenase activity of LO is characterized by the stereospeci¢c insertion of molecular oxygen into a 1,4-cis-cis-pentadiene moiety of a polyunsaturated fatty acid, leading ultimately to the formation of corresponding stable lipid hydroperoxide [11]. The intermediate peroxyl radical and/or hydroperoxide of polyunsaturated fatty acid support the co-oxidase activity of LO toward di¡erent xenobiotics. At present, our understanding of the peroxide speci¢city for co-oxidase activity of lipoxygenase is poor. Thus far, investigators have employed 5-hydroperoxyeicosatetraenoic acid [12], 15-hydroperoxyeicosatetraenoic acid [13], and/or 13-hydroperoxyoctadecadienoic acid [14^16] in studies on the LOmediated xenobiotic oxidation. Kulkarni and Cook [17] demonstrated that H2 O2 can be substituted for the lipid hydroperoxide to support co-oxidase activity of soybean LO (SLO), a model plant LO, toward various xenobiotics. Subsequent reports have con¢rmed and extended these observations [18^21]. Whether plant and animal tissue LO can utilize CHP and TBHP to support xenobiotic oxidation is unknown at present. Therefore, the primary objective of this study was to examine whether SLO and human term placental LO (HTPLO), a model human tissue LO, can utilize CHP and TBHP. For the following reasons, N-demethylation of phenothiazines was employed to test the hypothesis. N-dealkylation represents a common metabolic reaction of xenobiotics. Earlier, puri¢ed P450 [22] and other hemeproteins, such as horseradish peroxidase [23] and prostaglandin synthase [24] have been shown to dealkylate xenobiotics in the presence of H2 O2 or organic hydroperoxides. Recent studies have documented that SLO can mediate N-dealkylation of N,N-dimethylaniline and related compounds [25], several pesticides [26], and aminopyrine [27,28] in the presence of H2 O2 . Chlorpromazine (CPZ), one of the widely prescribed phenothiazine drugs, is oxidized by SLO [18,19] and undergoes N-demethylation [29] in the presence of H2 O2 [29] or linoleic acid [30]. The evidence presented here for the ¢rst time demonstrates that both SLO and HTPLO can e¡ect N-demethylation of selected phenothiazines in the presence of either TBHP or CHP. 2. Materials and methods 2.1. Materials SLO, type-V (760 000 Sigma U/mg protein), concanavalin A-Sepharose 4B (Con A), cumene hydroperoxide 70% (CHP), t-butyl hydroperoxide 80% (TBHP), chlorproma-
257
zine (CPZ), promazine, promethazine, trimeprazine, nordihydroguaiaretic acid (NDGA), gossypol, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbate, glutathione (GSH) and dithiothreitol (DTT) were purchased from Sigma, St. Louis, MO. All other chemicals were of reagent grade. 2.2. Human term placentas Human term placentas obtained from healthy women with no known history of smoking, drug or alcohol abuse, or pathological/physiological problems were used in this study. Placentas, obtained from a local hospital immediately after normal delivery, were kept on ice until use. The University of South Florida's Institutional Review Board governing the policies and procedures for research involving human subjects approved the use of human term placentas in this study. HTPLO was puri¢ed by a¤nity chromatography using Con A according to the method described by Joseph et al. [31]. Protein content of enzyme preparations was determined according to Bradford [32]. 2.3. Enzyme assay The standard incubation medium (1.0 ml ¢nal volume) contained, 5.0 mM CPZ (or desired substrate), 20 Wg SLO in 50 mM phosphate bu¡er, pH 7.0 or 100 Wg HTPLO in 50 mM phosphate bu¡er, pH 6.5 and 0.5 mM CHP or TBHP. A control (incubation medium containing all components except the enzyme) was run for each set of experiments. In each case, the reaction was initiated by the addition of the desired organic hydroperoxide. After incubation at 37³C for 30 min, the reaction was terminated by the addition of 1 ml of cold 10% trichloroacetic acid. The N-demethylase activity of SLO and HTPLO was estimated from the amount of formaldehyde accumulated in the reaction medium. Double strength Nash reagent [33] was used for the formaldehyde determination. The data reported are corrected for non-enzymatic reaction. Each experiment was repeated v4 times and the experimental data are presented as mean þ S.E.M. 3. Results CPZ was selected as a prototypic phenothiazine drug to examine the formation of the cation radical. The optical di¡erence spectra exhibited a distinct peak with an absorption maximum at V525 nm when CPZ was incubated in 50 mM citrate bu¡er, pH 3.5, with SLO in the presence of CHP or TBHP (Fig. 1A). Similar results were obtained when CPZ was incubated in 50 mM citrate bu¡er, pH 3.5, with either HTPLO and CHP or HTPLO and TBHP (Fig. 1B). Due to instability, the CPZ cation radical was undetectable when experiments were conducted in 50 mM phosphate bu¡er at the pH that is optimal for N-de-
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Fig. 1. Optical di¡erence spectra obtained during lipoxygenase-mediated N-demethylation of chlorpromazine (CPZ) in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP). The reaction media (1.0 ml ¢nal volume) contained speci¢ed enzyme (absent in the reference cuvette), 5 mM CPZ, and 0.5 mM CHP or TBHP in 50 mM citrate bu¡er, pH 3.5. The spectrum was recorded 5 min after initiation of the reaction by the addition of the organic hydroperoxide. (A) SLO. (B) HTPLO.
methylation of CPZ, i.e. at pH 6.5, for HTPLO and pH 7.0 for SLO. Pilot experiments, designed to establish optimal assay conditions for N-demethylation, clearly indicated that formaldehyde accumulates in the reaction medium when either HTPLO or SLO was incubated with CPZ or other phenothiazines in the presence of either TBHP or CHP. Using optimal assay conditions, the e¡ect of di¡erent experimental parameters on CPZ N-demethylation was studied. As shown in Fig. 2A^D, the pH optimum for CPZ N-demethylation was approximately pH 6.5 for HTPLO and pH 7.0 for SLO regardless of whether CHP or TBHP was used. At 7.4 pH, a signi¢cant rate of the reaction was observed in the incubations with either SLO or HTPLO. The enzymatic formaldehyde production from CPZ was also dependent on the incubation time. The CHP-dependent reaction was linear for over 40 min with HTPLO (Fig. 3A) as well as with SLO (Fig. 4A). Similar results were obtained with TBHP (Figs. 5A and 6A). In view of this, all other experiments were terminated at 30 min. The rate of formaldehyde accumulation increased proportionally with an increase in the amount of HTPLO (Figs. 3B and 5B) or SLO (Figs. 4B and 6B). When experiments were conducted in the presence of varying concentrations of the organic hydroperoxide, the highest speci¢c activity was noted when 0.5 mM TBHP or CHP was used with HTPLO (Figs. 3C and 5C) and SLO (Figs. 4C and 6C). A further increase in the concentration of organic hydroperoxide ( s 0.5 mM) resulted in a progressive decrease in the rate of formaldehyde production from CPZ by HTPLO or SLO. This e¡ect is most likely due to inactivation of the enzyme protein by denaturation. The rate
Fig. 2. E¡ect of pH on the formaldehyde production during lipoxygenase-mediated N-demethylation of chlorpromazine (CPZ) in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP). The reaction media (1.0 ml ¢nal volume) contained speci¢ed enzyme (absent in the control) 5 mM CPZ, and 0.5 mM CHP or TBHP in 50 mM phosphate bu¡er at the indicated pH. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Materials and Methods for further details. (A,C) SLO. (B,D) HTPLO.
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Fig. 3. Formaldehyde production during HTPLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time ; (B) HTPLO concentration ; (C) CHP concentration ; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
of formaldehyde production also demonstrated a marked dependence on the concentration of CPZ in the incubations containing HTPLO (Figs. 3D and 5D) or SLO (Fig. 4D and 6D). The analysis of enzyme kinetic data by Lineweaver^ Burk plots for the TBHP supported reaction yielded an apparent KM value of 2.6 mM for CPZ and an apparent Vmax value of 147 þ 15 nmol of HCHO/min/mg SLO. The estimated KM value for TBHP in this reaction was 0.23 mM. Similar data analysis for the SLO-mediated reaction
in the presence of CHP resulted in an apparent KM value of 2.2 mM for CPZ and an apparent Vmax value of 133 þ 13 nmol of HCHO/min/mg SLO. The KM value for CHP in this reaction was determined to be 0.22 mM. Similar experiments with HTPLO in the presence of TBHP yielded an apparent KM value of 2.0 mM for CPZ and an apparent Vmax value of 5.0 þ 0.9 nmol of HCHO/min/mg HTPLO. The estimated KM value for TBHP in this reaction was 0.26 mM. The analysis for the reaction in the presence of CHP resulted in an appar-
Fig. 4. Formaldehyde production during SLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time; (B) SLO concentration ; (C) CHP concentration; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
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Fig. 5. Formaldehyde production during HTPLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time ; (B) HTPLO concentration; (C) TBHP concentration ; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
ent KM value of 2.6 mM for CPZ and an apparent Vmax value of 4.5 þ 0.8 nmol of HCHO/min/mg HTPLO. The estimated KM for CHP in this reaction was determined to be 0.29 mM. A signi¢cant dose-dependent inhibitory e¡ect was observed when NDGA, a classical LO inhibitor, was added to the incubation medium containing either SLO or HTPLO (Table 1). A similar inhibitory response was noted when gossypol, another known LO inhibitor, was
included in the reaction medium. The free radical scavengers, BHA or BHT, in the incubation medium also decreased the rate of production of formaldehyde in a concentration-dependent manner suggesting the involvement of free radicals in the reaction. Similarly, the inclusion of a reducing agent, such as ascorbate, GSH, or DTT in the reaction medium caused a signi¢cant suppression of the reaction. Other phenothiazines were also N-demethylated by
Fig. 6. Formaldehyde production during SLO-mediated N-demethylation of CPZ under di¡erent experimental conditions. The assay conditions are speci¢ed in the inserts. E¡ect of (A) incubation time ; (B) SLO concentration; (C) TBHP concentration ; and (D) CPZ concentration. The amount of formaldehyde accumulated in the reaction media was estimated using Nash reagent. See Section 2 for further details.
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Concentration (WM)
CPZ N-demethylation (relative activity) SLO
^ NDGA
Gossypol
BHA BHT Ascorbate
Glutathione
DTT
^ 50 100 250 5 10 20 100 250 100 250 100 250 500 100 250 500 50 100 200
HTPLO
CHP
TBHP
CHP
TBHP
100 66 47 20 73 63 46 87 52 73 48 65 36 17 64 56 41 90 76 62
100 73 56 25 74 62 48 80 47 70 47 62 33 15 65 58 44 88 73 64
100 84 61
100 81 60
78 68 51 84 50 72 54 66 35 21 69 59 47 92 77 68
79 70 53 83 48 71 50 65 37 20 71 62 50 91 75 69
The incubation media (1.0 ml ¢nal volume) contained 20 Wg SLO or 100 Wg HTPLO (enzyme absent in the control), 0.5 mM CHP or TBHP, 5 mM CPZ, and the indicated concentration of modi¢er in 50 mM phosphate bu¡er pH 7.0 for SLO and pH 6.5 for HTPLO. The modi¢er was preincubated with the speci¢ed modi¢er for 2 min prior to the addition of other components. All incubations were performed at 37³C for 30 min. The amount of formaldehyde produced was estimated by Nash reagent. The speci¢c activity values in the controls without a modi¢er were 106 þ 11 nmol HCHO/min/mg SLO with CHP and 117 þ 12 nmol formaldehyde/min/mg SLO with TBHP and 3.2 þ 0.35 nmol HCHO/min/mg HTPLO with CHP and 3.9 þ 0.40 nmol formaldehyde/min/mg HTPLO with TBHP.
HTPLO and SLO in the presence of CHP or TBHP (Table 2). The observations indicate that promethazine served as the best substrate for both the enzymes, while other phenothiazines yielded relatively low speci¢c activities when compared with CPZ. Additional experiments performed (data not shown) with SLO in the presence of di¡erent concentrations of CHP or TBHP indicated that drugs,
261
such as ca¡eine or nicotine, which have been demonstrated to undergo N-demethylation by cytochrome P450, yield no detectable formaldehyde accumulation in the reaction media. 4. Discussion Previous studies have demonstrated the ability of CHP to support the N-dealkylation of xenobiotics in the presence of P450 [22]. Recently, it has been shown that SLO is also capable of this reaction in the presence of H2 O2 [25^ 29]. The evidence suggests that this property is also shared by HTPLO [25,29]. Lipid peroxidation is a normal biochemical process that occurs constantly in various tissues in the body. The process is either non-enzymatic, associated with the free redox active metals or enzymatic, catalyzed by prostaglandin synthase or LO. Available reports [12^16] suggest that the variety of lipid hydroperoxides generated during peroxidation of free polyunsaturated fatty acids can support co-oxidation of xenobiotics via the LO pathway. In theory, other hydroperoxides, derived from complex membrane-bound lipids, such as phospholipids, can also support these reactions. However, due to various technical di¤culties in the isolation of individual species of lipid hydroperoxide in su¤cient quantities and/ or prohibitive costs, their utilization in the investigation of hydroperoxidase activity of LO has been extremely limited. On the other hand, synthetic organic peroxide, such as TBHP and CHP, if found to be useful, may allow full exploration of this aspect of the enzyme. Therefore, the primary objective of this investigation was to evaluate whether TBHP and CHP can be substituted for H2 O2 or polyunsaturated fatty acid hydroperoxides in supporting the LO-mediated co-oxidation of xenobiotics. Our observations clearly indicate that the hydroperoxidase activity of LO from both plant (SLO) and human tissue (HTPLO) does not exhibit strict substrate speci¢city towards hydroperoxides and they can e¤ciently utilize both CHP and TBHP in the enzymatic reaction leading to the oxidation of phenothiazines. The pH optimum for the SLO-mediated N-dealkylation of chlorpromazine was found to be 7.0, while that for
Table 2 Lipoxygenase-mediated N-demethylation of phenothiazines in the presence of cumene hydroperoxide (CHP) and t-butyl hydroperoxide (TBHP) Phenothiazine
CPZ N-demethylation [speci¢c activity (nmol HCHO/min)] Per mg SLO
Chlorpromazine Promazine Promethazine Trimeprazine
Per mg HTPLO
CHP
TBHP
CHP
TBHP
106 þ 11 57 þ 6 294 þ 27 151 þ 14
117 þ 12 68 þ 7 356 þ 31 157 þ 15
3.2 þ 0.35 2.0 þ 0.22 11.3 þ 1.44 5.0 þ 0.56
3.9 þ 0.40 2.3 þ 0.24 15.8 þ 1.55 7.9 þ 0.80
The incubation media (1.0 ml ¢nal volume) contained 20 Wg SLO or 100 Wg HTPLO (enzyme absent in the control), 0.5 mM CHP or TBHP, 5.0 mM speci¢c substrate in 50 mM phosphate bu¡er pH 7.0 for SLO and pH 6.5 for HTPLO. All incubations were performed at 37³C for 30 min. The amount of formaldehyde produced was estimated by Nash reagent. Values are mean þ S.E.M. (nv4).
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HTPLO was 6.5 in the presence of either organic hydroperoxide (Fig. 2). The consistent dependency of the reaction mediated by SLO and HTPLO on the incubation time, organic hydroperoxide, substrate, and enzyme concentrations (Figs. 3^6) clearly suggests an enzymatic nature of the reaction. The suppression of the rate of formaldehyde production by NDGA or gossypol (Table 1) implicates LO as the catalyst involved in the reaction. Although several studies have been carried out on the peroxidative oxidation of CPZ by di¡erent protein catalysts [18,19,34^38], they are limited to the demonstration of the formation of initial cation radical, a relatively stable species that can be detected spectrally as a peak with an absorption maximum at V525 nm. Thus far, only two studies [29,30] have reported the LO-mediated N-demethylation of phenothiazines. The authors [29,30] proposed that the ¢rst step in the H2 O2 -supported LO-catalyzed N-demethylation of phenothiazines involves the formation of a cation radical as the initial intermediate. The data presented in Fig. 1A,B clearly indicate that the formation of the cation radical from CPZ does occur when the reaction media containing either SLO or HTPLO are supplemented with either CHP or TBHP. The optimal pH of the reaction medium to detect the CPZ radical generated either by SLO or HTPLO was 3.5. This may be due to the instability of the radical species at higher pH. The requirement of an acidic environment to detect the CPZ cation radical noted in this study is in accordance with the observations reported by others with various enzyme systems [18,19,29,34^38]. Clearly, it appears that the instability of the species in the media with higher pH re£ects the characteristic of the cation radical itself and is totally unrelated to the enzyme catalyst responsible for its generation. We believe that the free radical mechanism similar to that proposed for the H2 O2 -dependent LO-mediated N-demethylation of aminopyrine [27,28], CPZ [29] and other compounds [25,26] may be operational in the CPZ N-demethylation studied here in the presence of CHP and TBHP. This contention is supported by the facts that in addition to the formation CPZ cation radical (Fig. 1), the expected signi¢cant suppression of the formaldehyde accumulation was noted when free radical scavengers, such BHT and BHA, as well as reducing agents, such as ascorbate, GSH or DTT, were included in the reaction media (Table 1). The present ¢ndings raise the question of the identity of the one electron oxidant of CPZ generated by LO in the presence of CHP and TBHP. However, the experiments performed in this investigation cannot shed any light on this aspect. Based on the known chemistry of radical decomposition of hydroperoxide [39], the generation of cumyloxy radical from CHP by LO can be anticipated. Further decomposition to the methyl radical and acetophenone is also known [39]. Thus, it is possible that the cumyloxy and/or methyl radical participate in the CHP-dependent CPZ oxidation. Similarly, the alkoxyl radical formed by homolytic cleavage of TBHP is pre-
sumed to initiate CPZ N-demethylation by SLO and HTPLO. A comparison of the speci¢c activity rates of SLO and HTPLO-mediated N-demethylation of phenothiazines observed here with the organic peroxides as opposed to the previous report that employed H2 O2 [29], shows a remarkable similarity. In the previous study [29], the apparent Vmax value for the H2 O2 -dependent reaction was determined to be 132 þ 15 nmol of HCHO produced/min/mg SLO. The apparent Vmax values for the TBHP- and CHP-dependent reactions were 147 þ 15 nmol and 133 þ 13 nmol of HCHO produced/min/mg SLO, respectively. The Km values ranging between 0.2 and 0.3 mM for CHP and TBHP noted in this study for SLO and HTPLO are comparable with the previously reported values of 0.3 and 0.1 mM for the H2 O2 -supported CPZ N-demethylation catalyzed by SLO and HTPLO, respectively [29]. Taken together, these observations suggest that both CHP and TBHP are nearly as e¡ective as H2 O2 in supporting the N-demethylase activity of SLO and HTPLO toward CPZ. The relatively higher rate of reaction observed with the SLO-mediated, as opposed to HTPLO-mediated CPZ N-demethylation in the presence of CHP and TBHP can be attributed to the expression of speci¢c activity on per milligram protein basis, rather than normalizing the data on equal amounts of functional enzyme based on units of activity (1 unit equals the amount of LO that produces 1 Wmol of LAOOH/min when dioxygenase activity is assayed using linoleic acid). The SLO utilized in these experiments contained 760 000 units of enzyme per milligram protein. The HTPLO was found to contain an average of 188 þ 21 units per milligram protein. An inverse picture emerges when the speci¢c activity data are re-calculated on the basis of per unit of dioxygenase activity (0.14 and 17.0 pmol/min/unit of enzyme, for SLO and HTPLO, respectively) suggesting that HTPLO is V120 times more e¤cient in utilizing these hydroperoxides than SLO. Although N-demethylation of phenothiazines was employed in this study as a model reaction for xenobiotic oxidation, it is possible that CHP and TBHP may support other reactions of xenobiotic oxidation via the LO pathway. Further studies are needed to better understand the hydroperoxidase activity of LOs. The typical characteristics of cellular oxidative stress noted by many investigators include a rise in the cytosolic calcium, a signi¢cant depletion of the GSH and pyridine nucleotide pools, the generation of free radicals, reactive oxygen species and an increasing accumulation of lipid peroxidation products [40]. TBHP is commonly used to induce cellular oxidative stress in liver [41], brain [42] lung [43] and other tissues. The present ¢ndings that HTPLO can utilize tertiary hydroperoxides as cofactors may be relevant in the understanding of the hydroperoxide-induced cellular oxidative stress considering the facts that easily measurable LO activity occurs in liver [44], brain [45], lungs [46] and other tissues. Furthermore, LO
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exhibits a remarkable ability to oxidize GSH [47^49], NAD(P)H [50] and generate concomitantly superoxide anions. An elevation of cytoplasmic calcium is known to activates LO in intact cells while an in vitro calcium addition to the reaction media stimulates peroxidation of polyunsaturated fatty acids via the dioxygenase activity of puri¢ed HTPLO [31] and other LOs. Thus, besides GSH peroxidase and P450, this study implicates, for the ¢rst time, that LO pathway as an active participant contributing directly to the genesis of oxidative stress in hydroperoxide exposed cells. Further studies are needed to con¢rm or refute this postulate.
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