Introduction

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Aspirin (O-acetylsalicylic acid) is widely used as an analgesic drug, often for self-medication, and as an anti-inflammatory agent in the treatment of rheumatoid arthritis in humans. Furthermore, there is also a great interest in the use of aspirin as a prophylactic agent against thrombotic vascular diseases. After ingestion, a substantial amount of aspirin is hydrolyzed to salicylic acid (SA)¹ by esterases in the gastrointestinal tract, in the liver, and to a smaller extent, in serum (Leonards, 1962; Coudray et al., 1995). SA is further metabolized by conjugation to glycine to form salicyluric acid, by microsomal hydroxylases to form gentisic acid [2,5-dihydroxybenzoic acid (2,5-DHBA)] and by conjugation to glucuronic acid. About 60% of SA remains unmodified and can undergo the attack of highly reactive hydroxyl radical (^·OH) to produce 2,3-dihydroxybenzoic acid (2,3-DHBA) (Grootveld and Halliwell, 1988; Halliwell et al., 1991; Halliwell and Kaur, 1997), and to a smaller extent, catechol, both of which have not been reported as products of enzymatic metabolism. It has been suggested that formation of 2,3-DHBA from salicylate is a mean of monitoring ^·OH formation in vivo (Coudray et al., 1995; Halliwell et al., 1991; Halliwell and Kaur, 1997; Thome et al., 1997).

In contrast, 2,5-DHBA may arise from metabolism of salicylate by enzymes of endoplasmic reticulum (Ingelman-Sundberg et al., 1991). But the cytochrome(s) P-450 involved in the 5-hydroxylation of SA have not been characterized yet in humans. Therefore, this article deals with the demonstration of involvement of both P-450 2E1 and P-450 3A4, which are quantitatively important in the human liver (Guengerich and Turvy, 1991; Shimada et al., 1994) in the 5-hydroxylation of salicylate.

	Materials and Methods

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All reagents were of analytical grade. SA, 2,3-, and 2,5-dihydrobenzoic acids, 3,4-dihydroxybenzoic acid (3,4-DHBA), NADPH, diethyldithiocarbamate (DEDTC), troleandomycin (TAO), desferrioxamine (DFO) were from Sigma-Fluka-Aldrich (Saint-Quentin Fallavier, France).

In Vitro Studies. Microsomal samples. Human liver microsomes belong to a microsome bank set up in our laboratory for many years (Berthou et al., 1991). Their specific content and monooxygenase activities have been previously characterized, especially for P-450 2E1 [4-nitrophenol-2-hydroxylation and chlorzoxazone 6-hydroxylation (Zerilli et al., 1997), N-nitrosodimethylamine N-demethylation (Bellec et al., 1996), butanol oxidation (Lucas et al., 1993)] and P-450 3A4 [nifedipine oxidation, buprenorphine N-dealkylation (Iribarne et al., 1997), tamoxifen N-demethylation (Jacolot et al., 1991)].

Hydroxylation of SA by microsomal P-450s. The K_m of P-450 for 5-hydroxylation of salicylate was evaluated in microsomal human liver samples with salicylate concentrations ranging from 100 to 3000 µM and was calculated using the Eadie-Hofstee representation.

Chemical inhibitions. DEDTC and TAO are known to be mechanism-based inhibitors of P-450 2E1 (Guengerich et al., 1991) and P-450 3A (Rodrigues, 1994; Newton et al., 1995), respectively. DEDTC was preincubated at a concentration of 0.3 mM with microsomal proteins and NADPH 1 mM for 10 min before the addition of SA (600 µM), whereas TAO was used in the same conditions at a concentration of 50 µM. Metabolic rates were compared with corresponding controls including preincubation steps.

In Vivo Studies. SA 2,3, and 2,5-DHBA were measured in the plasma of control (n = 15) and alcoholic subjects (n = 12) 2 h after a single oral dose of 1 g of lysine acetylsalicylate (Aspegic, Synthélabo, de Plessis-Robinson, France). Alcoholic patients (daily consumption of alcohol 148 ± 44 g) were entering the hospital for a detoxification period. They were tested at the beginning of their stay in hospital after overnight ethanol abstinence (day 0) to avoid the presence of ethanol in blood and at 1 week (day 7) of ethanol withdrawal. This protocol was approved by the ethical committee of the Centre Hospitalier Universitaire of Brest (France) and all subjects gave their informed consent.

HPLC Analysis. Determination of 2,3- and 2,5-DHBA. These compounds were separated by HLPC using a Ultraspher ODS column (particle diameter 5 µm, 250 × 4.6 mm; Beckman, Gagny, France) after injection of 20 µl of the samples. The mobile phase consisted of 30 mM sodium citrate/27.7 mM sodium acetate, pH adjusted to 3.55 with orthophosphoric acid/methanol (95:5, v/v). The flow rate was 1 ml/min. The HLPC system was equipped with a BAS-LC-4A electrochemical amperometric detector (Bioanalytical Systems, West Lafayette, IN) equipped with a glassy carbon working electrode operating at +0.7 V against a Ag/AgCl reference electrode and a detection range of 10 nA.

Determination of SA. Samples (20 µl) were applied on a Nucleosil C18 column (particle diameter 5 µm, 250 × 4.6 mm, Interchim, Montluçon, France). SA was detected using an UV detector at 236 nm. The mobile phase consisted of 30 mM sodium citrate/27.7 mM sodium acetate, adjusted at pH 3.5 with orthophosphoric acid/methanol (82:18, v/v). The flow rate was 1 ml/min. Quantification was achieved using standard curves constructed from measurements of peak area ratios.

Statistical Analysis. Correlation coefficients were calculated using an ANOVA table by the least-squares regression analysis from the raw data (Stat-View, Alsyd, Meylan, France). Because a quite normal Gaussian distribution in the panel of 15 human liver microsomal preparations was observed (skewness = 1.00), correlation coefficients were calculated by including all the samples.

	Results

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Kinetic Studies. Two hydroxylated metabolites of salicylate were detected by HPLC when incubated in presence of NADPH and human liver microsomes (Fig. 1). These metabolites were identified by their retention times and their electrochemical properties as 2,3- and 2,5-DHBA. The formation of 2,5-DHBA was dependent upon NADPH. In contrast, production of 2,3-DHBA was very low and could not be clearly related to an enzymatic reaction involving NADPH.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   HPLC analysis of product formation of salicylate 0.6 mM with 1 mg human liver microsomes (FH3 sample) without (A) or with (B) NADPH 1 mM in the presence of DFO 0.1 mM

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Kinetics of salicylate 5-hydroxylation by human liver microsomes (Br031 sample). A, plot of metabolic rate of 2,5-DBHA formed (pmol/min/mg) from salicylate versus substrate concentration (S). B, Eadie-Hofstee plot of metabolic rate v versus v/(S).

Correlation Studies and Chemical Inhibitions. Salicylate was hydroxylated into 2,5-DHBA by human liver microsomes in the presence of NADPH, but this hydroxylation was dramatically decreased when NADPH was omitted. Using 2.5 and 0.6 mM salicylate as substrate concentrations, the mean rate formations of 2,5-DHBA were 47 ± 26 and 21.7 ± 8.5 pmol/min/mg, respectively, for the 15 liver microsomal samples. These two metabolic rates were significantly correlated between them (r = 0.86; p < .001). Formation of 2,3-DHBA also occurred in small amounts (9.1 ± 3.6 and 13.8 ± 3.4 pmol/min/mg, respectively, for salicylate 0.6 and 2.5 mM) despite the use of DFO but was not affected by the absence of NADPH, which suggests a formation via free radicals.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Interindividual variations of metabolic rate of 2,5-DHBA formed from salicylate 0.6 mM () and following preincubation with 0.3 mM DEDTC () in 15 human liver microsomal preparations.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Correlation between percent of salicylate 5-hydroxylation activity inhibited following preincubation with TAO 50 µM and nifedipine oxidation in a panel of 13 human liver microsomes.

Metabolism of SA by Heterologously Expressed Human P-450s. Incubation of SA with microsomes of human or insect cells genetically engineered for stable expression of seven human P-450s demonstrated that the formation of 2,5-DHBA involved mainly two P-450s, 2E1 and 3A4 (Table 3). Preincubation of 0.3 mM DEDTC or 50 µM TAO, respectively, with microsomal preparations of cells genetically engineered for expressing human P-450 2E1 and P-450 3A4, led to inhibition of the salicylate 5-hydroxylation up to 78% and 98%, respectively, versus control incubation. Such a strong inhibition validated the selectivity of these two inhibitors, namely DEDTC and TAO, as mechanism-based inhibitors of P-450 2E1 and 3A4, respectively.

In Vivo Studies. Plasma 2,5-DHBA and SA were measured in 15 controls and in 12 alcoholic patients 2 h after intake of 1 g of aspirin. Due to individual variations in the pharmacokinetics of aspirin, the plasma levels of SA vary largely from one individual to another (42-696 µM; 295 ± 165 µM for 15 controls). Consequently, the concentrations of 2,5-DHBA were corrected for this variation and 2,5-DHBA/SA ratios were reported (Fig. 5). No differences were observed for these ratios between men and women (data not shown). The (2,5-DHBA/SA)-1000 ratio (see Materials and Methods, HPLC Analysis) was dramatically increased in alcoholic patients when entering the hospital: 23.9 ± 10.3 versus 7.82 ± 4.2 in controls (p < .001). This 2,5-DHBA/SA ratio decreased significantly after 1 week of withdrawal: 14.9 ± 10.1 versus 23.9 ± 10.3 (p < .02) (Fig. 5). These results confirm the involvement of P-450 2E1, inducible by alcohol, in the 5-hydroxylation of salicylate.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Variation of metabolic ratio 2,5-DHBA/salicylate measured in plasma of eight alcoholic patients given 1 g acetylsalicylate after an overnight ethanol abstinence and after 7 days of ethanol withdrawal.

	Discussion

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Salicylate is metabolized by P-450 enzymes of human microsomal preparations to give 2,5-DHBA. Our results confirm previous findings reported by Ingelman-Sundberg et al. (1991), in rat and rabbit liver microsomal fractions. In contrast, formation of 2,3-DHBA was not NADPH dependent, although it was not totally inhibited by DFO, which inhibits iron-dependent ^·OH generation. Therefore, the rate of 2,3-DHBA was not correlated with P-450 activities. The turnover number was below 0.01 min¹ for seven heterologously expressed human P-450s (data not shown). The affinity of human liver microsomes for salicylate was fairly low, K_m about 0.6 mM; this value was in agreement with the value reported for rat liver microsomes, K_m about 0.4 mM (Ingelman-Sundberg et al., 1991). This value is in the range of plasmatic salicylate concentration (Leonards, 1962; Day et al., 1988). Another low-affinity K_m could be measured but its high value was out of the physiological range. Recombinant human P-450 3A4 and P-450 2E1 hydroxylated SA followed a monophasic Michaelis-Menten kinetics, characterized by apparent K_m of 513 and 280 µM, respectively. These K_m values are in the same range as the high-affinity K_m of 600 µM measured with human liver microsomes.

The nature of P-450 isoform(s) involved in the 5-hydroxylation of salicylate has not been characterized yet in humans. In a previous study (Ingelman-Sundberg et al., 1991), it has been suggested that there was not great specificity in 2,5-DHBA production in rat. Our study demonstrated that both P-450 3A4 and 2E1 are involved in this production in humans. Such a conclusion is based on four kinds of results: correlation studies, P-450 3A4 and 2E1-selective inhibitors, metabolism by heterologously expressed humans P-450, and in vivo results. Because the salicylate hydroxylation presented two apparent K_m, the metabolic rates were measured at the high-affinity constant, specifically 0.6 mM, i.e., in the range of K_m measured with recombinant human P-450 3A4 and P-450 2E1.

The contribution of P-450 2E1 to the 5-hydroxylation of salicylate was suggested by its inhibition by DEDTC, a mechanism-based inhibitor selective to P-450 2E1. The fraction of inhibition, namely 36 ± 14%, was more highly significantly correlated than the total activity with four catalytic activities specific to P-450 2E1. Moreover, the residual activity following DEDTC preincubation was significantly correlated with three P-450 3A4 activities. The fraction of 5-hydroxylation activity inhibited by TAO (30 ± 12%), a mechanism-based inhibitor selective to P-450 3A4, was correlated with nifedipine oxidation. The selectivity of these two inhitors, DEDTC and TAO, as mechanism-based inhibitors of P-450 2E1 and 3A4 was checked by incubation of recombinant P-450 2E1 and P-450 3A4, respectively. Preincubation of human liver microsomes with 0.3 mM DEDTC plus 50 µM TAO inhibited the formation of 2,5-DHBA by 69 ± 6% (n = 4 samples), whereas the formation of 2,3-DHBA was not significantly modified. Because this double inhibition represented approximately the addition of DEDTC and TAO inhibitions, the contribution of the two P-450s inhibited by DEDTC and TAO, P-450 2E1 and 3A4, was confirmed.

The use of seven heterologously expressed P-450 enzymes allowed the determination of which isoforms are involved in salicylate 5-hydroxylation. Two P-450s were significantly involved in the reaction, specifically P-450 2E1 and 3A4, with mean turnover numbers of 0.45 and 0.34 min¹, respectively. On the basis of the relative levels of these P-450s in human liver microsomes (22 and 96 pmol/mg protein of P-450 2E1 and 3A4, respectively; Guengerich and Turvy, 1991; Shimada et al., 1994), it can be concluded that the mean metabolic rate of salicylate 5-hydroxylation would be approximately 40 pmol/min/mg. This calculated value is in good agreement with the experimental value determined at 0.6 mM substrate, 21.7 ± 8.5 pmol/min/mg. Furthermore, the contribution of P-450 2E1 and P-450 3A4 can be estimated to be equivalent, confirming the results of chemical inhibitions. Such an extrapolation, however, is limited by some caveats. First, estimates of P-450 isoform content in human liver are based on immunodetectable proteins and not on active P-450 enzyme content (Guengerich and Turvy, 1991; Shimada et al., 1994). Second, it must be borne in mind that the rough estimates of salicylate 5-hydroxylation activity calculated by the formula (turnover number × liver P-450 isoform content) represent only an average.

Finally, the in vivo results confirm the involvement of P-450 2E1 in the 5-hydroxylation of salicylate. Indeed the 2,5-DHBA/SA metabolic ratio was significantly increased in alcoholic patients and decreased significantly after 1 week of ethanol withdrawal. The magnitude of a 3-fold increase of the metabolic ratio was in the same range as that obtained when chlorzoxazone was used as in vivo probe of P-450 2E1 (Girre et al., 1994). This increase reflects the liver P-450 2E1 induction by ethanol. However, because alcohols, especially isopentanol (Sinclair et al., 1998), are known to induce P-450 3A4, which displays high salicylate hydroxylation and is present in high amounts in human liver (between 30-50% of total P-450), the salicylate metabolic ratio should reflect both P-450 2E1 and P-450 3A4 activities. Consumption of alcoholic beverages has been shown to increase P-450 3A4 activity measured by the formation of 6-hydroxycortisol (Hoshino and Kawasaki, 1995). Moreover, the finding that 5-hydroxylation activity was still increased by 2-fold after 1 week of alcohol abstinence suggests that the decrease time of P-450 3A4 after alcohol induction was longer than for P-450 2E1. Indeed, the half-lives of P-450 2E1 and 3A4 have been shown to be approximately 6 to 8 h (Roberts et al., 1995) and 40 h (Muntane-Relat et al., 1995) in absence of substrate, respectively. P-450 2E1 activity was shown to return to normal level in less than 3 days after alcohol abstinence (Lucas et al., 1995).