The mechanism of the rat liver cytochrome P4502E1 inhibition by the synthetic prostanoids of A-type

Prostaglandins & other Lipid Mediators 89 (2009) 16–19

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Prostaglandins and Other Lipid Mediators

The mechanism of the rat liver cytochrome P450 2E1 inhibition by the synthetic prostanoids of A-type A.I. Hubich a,∗ , F.A. Lakhvich b , M.V. Sholukh a a b

Belarussian State University, Minsk, Belarus Institute of Bioorganic Chemistry, National Academy of Sciences, Minsk, Belarus

a r t i c l e

i n f o

Article history: Received 29 December 2008 Received in revised form 27 January 2009 Accepted 20 February 2009 Available online 9 March 2009 Keywords: Prostaglandin A2 analogs Cytochrome P450 2E1 Cytoprotection Rat liver microsomes

a b s t r a c t Aim: The elucidation of mechanism of A-type synthetic prostanoids inhibitory action on microsomal cytochrome P450 2E1 (CYP2E1) from rat liver activity was carried out. Results: Analogs U-34 and U-26 in a final concentration of 1 × 10−5 M inhibited CYP2E1 activity by 93% and 46%, respectively; however natural prostaglandins had no effect. These synthetic prostanoids of Atype (5 × 10−8 to 10−4 M) inhibited chlorzoxazone hydroxylation in a dose-dependent manner while IC50 = 7.1 × 10−7 M and 8.0 × 10−7 M for U-26 and U-34, respectively. The curves of CYP2E1 activity in the presence of different concentrations of chlorzoxazone and varying concentration of prostanoids were hyperbolic. Double-reciprocal plots of these results 1/V = f(1/S) indicated that prostanoids inhibit CYP2E1 through a competitive mechanism with particular effect. Conclusion: CYP2E1 is a target for A-type prostanoids, possessing 2-oxo-4-amino-oct-3(E)-enyl in ␣- or ␻-chain, which are able to inhibit its action through a competitive mechanism. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Prostaglandins (PGs) are biologically active molecules arising from arachidonic acid via the cyclooxygenase pathway and regulating different cellular function under physiological and pathological conditions [1]. In accordance to their chemical structure and molecular mechanisms of action prostaglandins are divided into two groups: cyclopentanone (E, F, and D) and cyclopentenone (A, B, and C) [1]. Prostaglandins of the A-series contain an ␣,␤-unsaturated carbonyl group at the five-member ring and are derived from E-type prostaglandins by dehydration in plasma or aqueous solution [2]. Biological action of PGAs includes the modulation of stress reaction, cell cycle inhibition, virus replication suppression, regulation of cellular differentiation, induction of thermotolerance, cell immunity, and neuroprotective action [3–7]. It is established that some of PGAs effects (e.g. hypotension) are mediated by low affinity binding to the known G-proteincoupled prostanoid receptors of EP-, DP- and FP-type [5]. However, the involvement of “classic” PGs receptors in anti-inflammatory, antitumor, and antiviral effects of PGA was not determined [5,8]. The majority of PGAs effects are provided with plasma membrane receptor-independent regulation of stress-inducible gene

∗ Corresponding author at: Department of Biochemistry, Biological Faculty, Independence Ave., 4, Belarussian State University, 220030 Minsk, Belarus. Tel.: +375 17 209 58 97; fax: +375 17 209 58 08. E-mail address: Hubich [email protected] (A.I. Hubich). 1098-8823/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2009.02.003

expression (e.g. genes encoding with heat shock proteins, ␥glutamyl-cysteine synthetase, collagen, and heme oxygenase) [5,6]. Several biological effects of PGA may be realized at least in part through reaction with cysteine residues of many cellular proteins [5,8]. Studies have also revealed the existence of specific receptor for PGA on rat intestine plasma membranes [9]. Nevertheless, the list of protective effects, possible mechanisms and cellular targets for their realization for PGAs and their synthetic derivatives is not complete and is under investigation nowadays. Earlier we have analyzed the cytoprotective effects of novel collection of synthetic PGA2 analogs against CCl4 on the cellular model of a liver damage [10]. It was determined that two prostanoids, possessing 2-oxo-4-amino-oct-3(E)-enyl in ␣-chain, as well as natural PGA2 and PGI2 are able to decrease a toxic action of this agent. The extent of prostanoids protective activity correlated with a decrease of triene conjugate formation in cellular membranes and a stabilization of quantity of intracellular SH-groups in isolated hepatocytes after CCl4 treatment [10]. It is known that CCl4 metabolism to its free radical species by microsomal CYP2E1 is the main reason for CCl4 -induced liver injury [11,17,18]. So, it was suggested that cytoprotective action of prostanoids given was realized by their inhibitory effect on this cytochrome [19]. Despite to the fact that natural PGAs are not very good inhibitors for xenobiotic oxidation catalysed by recombinant P450 enzymes as well as their derivatives [19], the protective properties of the novel prostanoids were closely connected with their ability to suppress CYP2E1 activity in rat liver microsomes in presence and absence of CCl4 [10]. However the biochemical mechanism of this phenomenon was not studied.

A.I. Hubich et al. / Prostaglandins & other Lipid Mediators 89 (2009) 16–19

The present work was undertaken to study the mechanism of inhibitory action of prostanoids with modifications in ␣- and ␻chains on CYP2E1 activity in rat liver microsomes. 2. Materials and methods 2.1. Materials PGE1 , PGE2 and PGA2 , HEPES, NADPH, chlorzoxazone, 6hydroxychlorzoxazone and arachidonic acid were obtained from Sigma Chemical; BSA and EDTA were obtained from DiaM. Ordinary salts (analytical grade) were obtained from various sources. All reagents were prepared with bidistilled water. Six synthetic prostanoids of A-group were synthesized in the Laboratory of Prostaglandins Chemistry (National Academy of Sciences, Minsk, Belarus). The structure of the prostanoids was confirmed by the methods of nuclear magnetic resonance, massspectrometric identification and infrared spectroscopy [12,13]. Prostanoids’ structural formulas and systematic names are shown in Table 1. Stock solutions of prostanoids were prepared using 96% ethanol. The necessary concentrations of analogs were obtained from their initial solutions by diluting with 25 mM Hepes–NaOH (pH 7.4). 2.2. Methods 2.2.1. Isolation of rat liver microsomal fraction Isolation of microsomal fraction was performed as described by Niwa et al. [14]. Briefly, rat livers (10 g) were homogenized using a motor-driven Teflon glass homogenizator for 1.5 min in a buffer containing 25 mM Tris–HCl (pH 7.5), 1 mM EDTA and 0.25 M sucrose. The homogenate was centrifuged at 18,000 × g for 20 min. The supernatant was then centrifuged at 100,000 × g for 60 min. The resulting pellet was resuspended in 0.1 M sodium phosphate buffer (pH 7.5) containing 20% glycerol and stored at −86 ◦ C until used. Protein concentration was determined by the method of Peterson [15] using BSA as a standard. 2.2.2. Determination of CYP2E1 activity Determination of CYP2E1 activity was performed by the method of Lucas et al [16]. Briefly, samples (0.1 mL) containing 200 ␮M of chlorzoxazone (specific cytochrome P4502E1 substrate), 100 ␮g of microsomal protein, appropriate concentration of prostanoids solution, 500 ␮M of NADPH and 0.5% of CCl4 and 50 mM of Hepes NaOH (pH 7.4) were incubated for 30 min at 37 ◦ C. Proteins were then precipitated with 0.4 mL of 0.6 M perchloric acid. After centrifugation for 10 min at 3500 × g, chlorzoxazone and 6-hydroxychlorzoxazone were extracted from the supernatant with two 0.4 mL volumes of ethyl acetate by shaking for 10 min. Following centrifugation for 10 min at 4 ◦ C, the organic phases were evaporated to dryness under a stream of nitrogen. The residues were dissolved in 250 ␮L of acetonitrile–0.5% acetic acid (mobile phase) and 20 ␮L samples were used for the HPLC determination of chlorzoxazone and 6-hydroxychlorzoxazone on liquid chromatography mass-spectrometer (Shimadzu LCMS-QP 8000x; Japan). The HPLC column C18 (150 mm × 4.6 mm) was used, and the flow-rate was 0.75 mL/min. Chlorzoxazone and 6hydroxychlorzoxazone were detected at 287 nm. Peak area measurements were calculated for quantification and compared with standard solutions (0.5–20 ␮g/mL) of chlorzoxazone and 6-hydroxychlorzoxazone. 2.2.3. Statistical analysis Statistical analysis was performed by Stadia 6.0 Software. The results were presented as means ± S.E.M. The significance of differ-

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ences between groups was established by the Student’s t-test. The value of P < 0.05 was considered significant. 3. Results and discussion At the first stage of current investigation we have compared effect of natural prostaglandins E1 , E2 , A2 , I2 and arachidonic acid with the action of synthetic prostanoids of A-type (U-26 and U-34) on CYP2E1 activity in rat liver microsomes (Fig. 1). Each of compounds was used in a final concentration of 10 ␮M. It was found that arachidonic acid inhibited 6-hydroxylation of chlorzoxazone by 47%. The effects of natural prostaglandins were unreliable that comes to a good agreement with the data of other authors [19]. However, PGAs analogs U-26 and U-34 were observed to be the strong inhibitors of cytochrome activity. Then, in presence of analog U-34 a suppression of enzyme activity reached 93%; prostanoid U-26 decreased 6-hydroxychlorzoxazone formation by 46% (Fig. 1). To establish the quantitative characteristic of inhibition the IC50 value (inhibition constant) for analogs tested was determined. So, we investigated the dependence of cytochrome activity from prostanoid’s concentration (10−10 to 10−4 M) (Fig. 2). It was found that prostanoids suppressed 6-hydroxychlorzoxazone formation in a concentration-dependent manner. The maximal effective concentration for prostanoids tested was 1 × 10−4 M. The value of half maximal concentration (IC50 ) was 7.1 × 10−7 M for U-26 and 8.3 × 10−7 M for U-34 (Table 2). It was recognized that CYP2E1 action could be regulated by direct interaction with different compounds [19,20]. There are at least four important mechanisms underlying P450 direct inhibition, namely competitive inhibition, reversible inhibition by nitrogen heterocycles, inhibition through formation of metabolite intermediate complexes and mechanism-based inhibition [19,20]. To clarify the mechanism of the prostanoids tested action, we have performed kinetic analysis. It was shown that curves of cytochrome activity in the presence of different concentration of chlorzoxazone and varying concentrations of U-34 were hyperbolic (Fig. 3). Doublereciprocal plots of these results (1/V = f(1/S)) have demonstrated that the prostanoids changed Michael’s constant of the reaction but did not influence on Vmax value (Fig. 4). However the inhibition was not complete even when extremely high doses of these substances

Fig. 1. The effect of arachidonic acid, natural prostaglandins and synthetic prostanoids of A-type on microsomal cytochrome P450 2E1 activity. CYP2E1 basal activity was measured in the medium containing 25 mM Hepes–NaOH (pH 7.4) with 0.96% ethanol. The final concentration of all substances tested was 10 ␮M, ethanol—0.96%. *P ≤ 0.05, compared to basal cytochrome activity (n = 3).

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Table 1 The list of prostanoids used. Code of prostanoid

Structural formula

Systematic name

PGA2

15(S)-hydroxy-9-oxo-5-cys,10,13-trans-prostanic acid

U-26

3-Isobutoxy-2-methyl-5-(2-oxo-4-amino-oct-3(E)-enyl)-4-(hydroxyhept-2-(Z)enyl)-2-cyclopenten-1-on

U-34

3-Isobutoxy-2-methyl-5-(2-oxo-4-amino-oct-3(E)-enyl)-4-(6-hydroxyhex-2-(Z)enyl)-2-cyclopenten-1-on

Table 2 Kinetic parameters of inhibition of cytochrome P450 2E1 activity by prostanoids in rat liver microsomes (n = 3). Analog U-26 U-34

Vmax , ␮M of 6-hydroxychlorzoxazone/␮g of protein × 30 min X ± Sx 0.168 ± 0.06 0.175 ± 0.04

Vmin , ␮M of chlorzoxazone/␮g of protein × 30 min X ± Sx

IC50 , ×10−7 M X ± Sx

0.084 ± 0.02 0.038 ± 0.01

7.1 ± 0.5 8.0 ± 0.2

The data presented were performed by kinetic analysis of results from Fig. 2. CYP2E1 activity was determined in rat liver microsomes as described in Section 2.

were used. The same kinetic behavior was determined for analog U-26. Taken together, these data indicate that competitive mechanism with particular effect takes place in case of CYP2E1 inhibition by the prostanoids. The regulation of CYP2E1 activity has an important pharmacological and toxicological significance due to its biological role. It is known that the biotransformation of a number of commonly used endogenous substrates and xenobiotics (volatile anesthetics (e.g. halothane and enflurane), organic solvents (e.g. CCl4 , chloroform, vinylidine chloride), therapeutic agents (e.g. acetominophen) and procarcinogens) by CYP2E1 is responsible for bioactivation

Fig. 2. Influence of different prostanoids’ concentration on cytochrome P450 2E1 activity in rat liver microsomes. Determination of CYP2E1 activity was performed by the method of Lucas et al. as described in Section 2. Each point is the mean of triplicate determinations.

and the organ toxicity of these compounds [21]. When unchecked, this toxicity eventually results in different pathological possesses: inflammation, fatty liver diseases, hepatic steatosis, and fibrosis [22,23]. On the other hand, CYP2E1 is also well known as an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide and may produce powerful oxidants such as the hydroxyl radical that can initiate lipid peroxidation [24,25]. CYP2E1-derived reactive oxygen species have been

Fig. 3. Dose-dependent inhibition of cytochrome P450 2E1 activity by analog U-34. Basal cytochrome activity was used as a control and was measured in the medium containing 25 mM Hepes–NaOH (pH 7.4) with 0.96% ethanol. The necessary concentrations of analogs were obtained from their ethanol solutions by diluting with 25 mM Hepes–NaOH (pH 7.4). The analog U-34 was used in the following concentrations: () 5 × 10−6 M, () 10−6 M, () 10−7 M, () 0 M. Determination of cytochrome P450 2E1 activity was performed by the method of Lucas et al. as described in Section 2. Each point is the mean of triplicate determinations.

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References

Fig. 4. Double-reciprocal plot of cytochrome P450 2E1 inhibition by U-34 in rat liver microsomes. V, velocity of reaction in ␮M of 6-hydroxychlorzoxazone/␮g of protein per 30 min. The analog U-34 was used in the following concentrations: () 5 × 10−6 M, () 10−6 M, () 10−7 M, () 0 M. The necessary concentrations of analog were obtained from its initial ethanol solutions by diluting with 25 mM Hepes–NaOH (pH 7.4). Activity of cytochrome P450 2E1 in the absence of prostanoid was used as a control. Each point is the mean of triplicate determination.

implicated as the main reason of hepatotoxic effects of ethanol and cisplatin-induced cytotoxicity [22,26]. It is also considered that inhibition of CYP2E1 may be a novel approach for the prevention of renal tubular injury caused by oxidative stress [27]. The list of established modulators of CYP2E1 activity is not long. It includes disulfiram, which may be useful in preventing CYP2E1-mediated xenobiotic toxicity by mechanism-based enzyme inhibition, and some organosulfur compounds (allyl sulfide and its structural derivatives), that exhibit selective suppression of CYP2E1 activity and expression [28,29]. The ability of 50 ␮M arachidonic acid to inhibit CYP2E1 activity both in human liver microsomes and in recombinant enzyme system is also well known [19]. Dixon plot of this effect indicated that this substance suppressed the enzyme activity directly through a competitive mechanism [19]. Natural cyclopentenone prostaglandins are not good inhibitors of xenobiotic oxidations catalysed by CYP enzymes. Data obtained suggested that some derivatives of PGA2 , possessing 2-oxo-4-amino-oct-3(E)-enyl in ␣-chain, can modify CYP2E1 activity in vitro. At the same time prostanoid with OH-group in ␻-chain was less potent inhibitor than analog containing terminal carboxyl-group. These substances show the possibility of direct competitive pathway of CYP2E1 inhibition. So they could be used for the elucidation of the role of this enzyme in xenobiotic metabolism and for the prevention of toxic effects caused by oxidative stress and xenobiotic biotransformation. Acknowledgments This work was supported by the Belarussian State Programme “Biological active compounds” (Grant 20063147) and INTAS (Grant 03-51-4813).

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