Introduction

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Cytochrome P450 (CYP) is a superfamily of enzymes that catalyze oxidative transformation of a wide variety of organic compounds (Nelson et al., 1993; Rendic and Di Carlo, 1997). Among several CYP enzymes involved in the metabolism of xenobiotics in humans (Guengerich, 1990, 1994, 1995; Smith and Jones, 1992), the members of the 3A subfamily, with their ability to oxidize a wide array of clinically and toxicologically important agents, are most abundant (Guengerich, 1990, 1995).

The primary role of CYP enzymes is to affect metabolic clearance and detoxification of xenobiotics by conversion to more hydrophilic molecules. However, these enzymes are also responsible for generating reactive intermediates, including reactive oxygen species, by oxidative metabolism of xenobiotics. Indeed, the catalytic cycle of CYP involves activation of molecular oxygen to reactive oxygen species, such as superoxide anion (O₂), hydrogen peroxide (H₂O₂), and hydroxyl radical (OH^·) (Karuzina and Archakov, 1994; Bernhardt 1995).

Cyclosporine, a cyclic undecapeptide with potent immunosuppressive activity, has been shown to be predominantly metabolized by CYP3A in liver (Maurer, 1985; Berthault-Peres et al., 1987; Watkins, 1990; Vickers et al., 1992) and in intestinal epithelium (Watkins, 1990; Tjia et al., 1991; Kolars et al., 1992; Vickers et al., 1992). Cyclosporine is known to cause hepatotoxicity in humans and rodents by an unknown mechanism. The colocation of the site of cyclosporine toxicity and its oxidative metabolism by CYP3A suggests a relationship between metabolism and toxicity (Ahmed et al., 1993). Lipid peroxidation of cell membranes, mediated by cyclosporine metabolism, has been considered as one likely mechanism for the toxicity. Increased lipid peroxidation by cyclosporine was observed in kidney transplant recipients as evidenced by increased formation of malondialdehyde (Kasiske, 1998). To determine whether reactive oxygen species were involved in CYP-mediated lipid peroxidation and toxicity caused by cyclosporine, formation of adrenochrome was used as a measure of oxygen radical formation during the reaction (Ahmed et al., 1993, 1995).

Efforts to develop an assay for oxygen radicals dates back to the early 1970s (Valerino and McCormack, 1971; Misra and Fridovich, 1972), demonstrating that epinephrine can be oxidized to adrenochrome by superoxide anions generated during xanthine oxidase-mediated reactions. Indeed, the adrenochrome reaction has been widely employed as a standard assay for reactive oxygen species production (Valerino and McCormack, 1971; Loschen et al., 1974; Cadenas et al., 1977; Boveris, 1984; Knobeloch et al., 1990; Ahmed et al., 1993, 1995). The principle for this assay can be described as follows. Reactive oxygen species are generated during an enzyme-catalyzed reaction such as CYP-mediated oxidation of a substrate, which can oxidize epinephrine to adrenochrome (absorbance maximum at 485 nm). Thus, the assay is dependent on the change of absorbance at 485 nm to monitor and measure formation of reactive oxygen species.

However, it has been reported that epinephrine and adrenochrome can be metabolized by hepatic microsomes (McKillop and Powis, 1976; Powis, 1979). Adrenochrome can also be metabolized by NADPH-CYP reductase (Baez and Segura-Aguilar, 1995). Because of the metabolic instability of epinephrine and adrenochrome under the experimental conditions typically employed in its use, we suspected the validity of using adrenochrome reaction to determine reactive oxygen species for CYP-mediated metabolism of xenobiotics. In the present report, we show that measurement of adrenochrome cannot be used as an index of reactive oxygen species generated during CYP-mediated metabolism of xenobiotics because adrenochrome and its precursor interact with the CYP enzyme system as substrates and inhibitors.

	Experimental Procedures

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Materials

Insect microsomes containing CYP3A4 and CYP reductase (CYP3A4OR) were a gift from GlaxoSmithKline (Research Triangle Park, NC). Testosterone, 6-hydroxytestosterone, and 11-hydroxyprogesterone were obtained from Steraloids, Inc. (Wilton, NH). ()-Epinephrine [sodium (+)-bitartrate], adrenochrome (3-hydroxy-1-methyl-5,6-indolinedione), hypoxanthine, and NADPH were obtained from Sigma Chemical Co. (St. Louis, MO). Xanthine oxidase was purchased from Worthington Biochemical Corp. (Lakewood, NJ). All other chemicals and reagents were the highest grade available from commercial sources.

Oxidation of Epinephrine to Adrenochrome During CYP3A4-Mediated Metabolism of Testosterone

Epinephrine (200 µM) was added to a reaction mixture containing testosterone (200 µM), CYP3A4OR (0.5 mg/ml, 48 pmol of CYP/mg of protein) in phosphate buffer (50 mM, pH 7.4). The reaction was initiated by adding NADPH (1 mM). Control incubations contained all the above components (complete system) except testosterone. The UV-visible spectra (200-600 nm) of the incubation mixture were obtained at several time points. Samples from the above incubations were also subjected to HPLC analysis for determination of 6-hydroxytestosterone, a metabolite produced specifically by CYP3A4-mediated oxidation of testosterone (Brian et al., 1990; Yamazaki et al., 1999).

Oxidation of epinephrine to adrenochrome by xanthine oxidase-mediated oxidation of hypoxanthine (Valerino and McCormack, 1971) was used as a positive control. Epinephrine (50 µM) was added to a system that contained hypoxanthine (57 µM) and xanthine oxidase (0.026 U/ml). The reaction was initiated by adding NADPH (1 mM), and the UV-visible absorption spectra (200-600 nm) were recorded at several time points.

The spectra obtained from both xanthine oxidase and CYP3A4 systems were compared with those of standard adrenochrome in the phosphate buffer system. This standard adrenochrome solution was also used to determine the absorption maximum of adrenochrome in the visible region at pH 7.4 (485 nm). All incubations were performed at ambient temperature.

Stability of Adrenochrome

Chemical Stability. Adrenochrome was dissolved in HCl (0.1 N) as a stock solution (200 µM). An aliquot (50 µl) of this solution was added to a 1-ml phosphate buffer (50 mM, pH 7.4). Absorbance was measured at 485 nm (_max) as a function of time for up to 60 min.

Stability in the Presence of CYP3A4. Adrenochrome (50 µl, 200 µM) was added to an incubation mixture that contained testosterone (200 µM), CYP3A4OR (0.5 mg/ml), and NADPH (1 mM) in phosphate buffer (50 mM, pH 7.4). In addition, an aliquot of adrenochrome was also added to a reaction mixture that contained no NADPH or testosterone. UV-visible spectra (200-600 nm) were recorded at several time points.

Stability in the Presence of Xanthine Oxidase. Stability of adrenochrome (10 µM) toward xanthine oxidase was determined by incubation with xanthine oxidase (0.026 U/ml), hypoxanthine (56 µM), and NADPH (1 mM) in phosphate buffer (50 mm, pH 7.4). UV-visible spectra (200-600 nm) were recorded at several time points.

Effect of Epinephrine and Adrenochrome on CYP3A4-Mediated Metabolism of Testosterone

Epinephrine (200 µM) or adrenochrome (20 or 200 µM) was added to a reaction mixture containing testosterone (200 µM), CYP3A4OR (0.5 mg/ml), and NADPH (1 or 5 mM) in phosphate buffer (50 mM, pH 7.4). Control incubations contained no epinephrine or adrenochrome. All incubations were performed in triplicate and at ambient temperature for 10 min. Formation of 6-hydroxytestosterone under these conditions was determined by HPLC as described below.

Measurement of 6-Hydroxytestosterone

An HPLC method adapted from Lee et al. (1995) was used to measure 6-hydroxytestosterone. Briefly, methylene chloride (2 ml) containing the internal standard (10 µl of 1 mg/ml 11-hydroxyprogesterone) was added to an incubation mixture (0.5 ml) at the end of the incubation period as described previously, and the solution was mixed and centrifuged at 10,000g for 5 min. The organic layer was removed and evaporated under a nitrogen stream. The residue was reconstituted with 0.5 ml of mobile phase (40% CH₃CN and 60% H₂O). Chromatographic separation was achieved using a Hewlett Packard (Palo Alto, CA) 1100 series HPLC system with a Phenomenex (Torrence, CA) SphereClone 5 µ C₆ column (250 × 4.5 mm). Testosterone, 6-hydroxytestosterone, and 11-hydroxyprogesterone were resolved and eluted isocratically (40% CH₃CN and 60% H₂O). The eluant was monitored by UV absorption at 230 nm. The retention times for 6-hydroxytestosterone, 11-hydroxyprogesterone, and testosterone were 4.81, 11.05, and 16.20 min, respectively.

Measurement of Adrenochrome

Adrenochrome was measured by UV-visible spectrophotometry (_max at 485 nm;  4020 liters mol¹ cm¹) with a UV2101PC spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD). The absorbance at 485 nm was recorded with a slit width of 5 nm. Spectra were obtained using a spectrum acquisition mode with a fast scanning speed and a slit width of 2 nm.

	Results

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Lack of Formation of Adrenochrome from Epinephrine During CYP3A4-Mediated Metabolism of Testosterone. To determine whether epinephrine was converted to adrenochrome during a CYP3A4-catalyzed reaction (Ahmed et al., 1993, 1995, 1996), epinephrine was added to the reaction mixture in which testosterone was being oxidized by CYP3A4 to 6-hydroxytestosterone, a known CYP3A4-catalyzed reaction (Brian et al., 1990; Yamazaki et al., 1999). As the enzymatic reaction proceeded, aliquots were withdrawn from the reaction mixture at several time points, and UV-visible spectra were obtained (Fig. 1a). The spectrum at 1 min was similar to that of epinephrine in pH 7.4 buffer (_max at 285 nm). The spectra obtained at subsequent time points showed that epinephrine was being consumed rapidly to produce a compound with a broad absorption band around 340 nm. However, the product formed was not adrenochrome, as evidenced by the absence of an absorption band at 485 nm observed in the spectrum of a standard adrenochrome solution in pH 7.4 buffer (Fig. 1b).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Lack of formation of adrenochrome during CYP3A4-mediated metabolism of testosterone. UV-visible spectra (a) of the incubation mixture that contained CYP3A4OR (0.5 mg/ml), testosterone (200 µM), and epinephrine (200 µM) at 1, 2, 5, 10, 20, 40, and 60 min post addition of NADPH (1 mM) or (b) of authentic sample of adrenochrome in pH 7.4 phosphate buffer.

Degradation of Adrenochrome by a CYP3A4-Meditated Reaction. To determine the stability of adrenochrome during a CYP3A4-mediated oxidation of testosterone, the reaction mixture was spiked with adrenochrome, and UV spectra were obtained as a function of time. Adrenochrome was rapidly degraded in the incubation medium as evidenced by a decline in the UV peak at 485 nm (Fig. 2). More than 65% adrenochrome was degraded within 2 min, and more than 90% was degraded at 5 min, in the incubation mixture that contained all components required for CYP3A4-mediated hydroxylation of testosterone (Fig. 2). Adrenochrome was also degraded in the incubation mixture that contained CYP3A4OR and NADPH but no testosterone; however, it was much more stable in the absence of NADPH (only 5% degradation occurred over a 2 min period in the absence of NADPH as compared with over 50% degradation in the presence of NADPH) (Fig. 2). Less than 10% adrenochrome was degraded in pH 7.4 phosphate buffer over a 10-min period (pseudo first order rate constant of 0.00868 min¹). The stability of adrenochrome in the xanthine oxidase system was comparable with that in pH 7.4 buffer (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Degradation of adrenochrome during CYP3A4OR-mediated oxidation of testosterone. a, UV-visible spectra of the incubation mixture containing testosterone (200 µM) and CYP3A4OR (0.5 mg/ml) at 0.5, 1, 2, 5, and 10 min post addition of adrenochrome. b, time course of degradation of adrenochrome by CYP3A4OR. The incubation mixture contained CYP3A4OR (0.5 mg/ml), testosterone (200 µM), NADPH (1 mM), and adrenochrome () or contained all the components as described above but no testosterone () or NADPH (). The stability of adrenochrome in 0.1 N HCl () is shown for comparison.

Inhibition of CYP3A4 by Epinephrine and Adrenochrome. Effect of epinephrine and adrenochrome on CYP3A4-catalyzed formation of 6hydroxytestosterone from testosterone is shown in Fig. 3, a and b, respectively. Formation of 6-hydroxytestosterone amounted to only ~4% of the control in the presence of 200 µM epinephrine. An increase in NADPH concentration from 1 to 5 mM did not cause significant increase in formation of 6-hydroxytestosterone, indicating that the inhibition of CYP3A4 activity was not due to nonenzymatic degradation of NADPH by epinephrine. Adrenochrome was also inhibitory to CYP3A4, with potency very similar to that observed with epinephrine (Fig. 3b).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Inhibition of CYP3A4-mediated metabolism of testosterone by epinephrine or adrenochrome. The bar graph depicts formation of 6-hydroxytestosterone (at 10 min) in the absence (control; 92 nmol of product/min/nmol of CYP) or presence of epinephrine (200 µM) (a) or adrenochrome (20 or 200 µM) (b). The incubation mixture contained testosterone (200 µM), CYP3A4OR (0.5 mg/ml), and NADPH (1 or 5 mM). Experiments were performed in triplicate at ambient temperature. Significant difference (***, p < 0.001) was observed in formation of 6-hydroxytestosterone between incubation performed in the presence or absence of either epinephrine or adrenochrome.

	Discussion

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Conversion of epinephrine to adrenochrome (Matthews et al., 1985) has been used to determine reactive oxygen species in various preparations. For example, epinephrine was converted to adrenochrome in the xanthine oxidase-mediated oxidation of xanthine or hypoxanthine (Valerino and McCormack, 1971; Boveris, 1984). More recently, adrenochrome reaction was used as an indicator of the formation of reactive oxygen species during metabolism of cyclosporine by rat or human liver microsomes (Ahmed et al., 1993, 1995, 1996). However, it has been reported that epinephrine was metabolized by hepatic microsomal mixed-function oxidase(s) in the rat (McKillop and Powis, 1976; Powis, 1979). More recently, it was suggested that adrenochrome was metabolized by CYP reductase (Baez and Segura-Aguilar, 1995), an enzyme required for CYP-catalyzed reactions.

The present study was, therefore, conducted to evaluate the adrenochrome assay with a model CYP3A4 reaction system involving testosterone metabolism to 6-hydroxytestosterone. Initial experiments were conducted to examine whether adrenochrome was formed during CYP3A4-mediated metabolism of testosterone after epinephrine was added to the reaction mixture. Although epinephrine was consumed, the adrenochrome peak was not observed during CYP3A4-mediated metabolism of testosterone over a period of 1 min up to 1 h (Fig. 1a), despite clear evidence for the formation of 6-hydroxytestosterone from testosterone. In contrast, adrenochrome peak was observed (positive control) for xanthine oxidase-mediated oxidation of hypoxanthine (data not shown). These results suggested that adrenochrome was either not formed during CYP3A4-mediated metabolism of testosterone or that it was formed but degraded so rapidly under the experimental condition that the amount remaining in the medium was below the detection limit of the spectrometric method employed.

Indeed, adrenochrome underwent very rapid degradation during CYP3A4-mediated hydroxylation of testosterone (Fig. 2). Furthermore, adrenochrome also degraded rapidly in the presence of CYP3A4OR (CYP3A4 and NADPH-CYP reductase) and NADPH but not in the presence of CYP3A4OR alone (Fig. 2b). These results demonstrated that adrenochrome is a substrate for CYP3A4 enzyme and, at least in part, explained why adrenochrome peak was not observed in CYP3A4OR-mediated oxidation of testosterone.

While examining formation of adrenochrome during CYP3A4-mediated metabolism of testosterone, we observed that less 6-hydroxytestosterone was formed in the presence of epinephrine than in its absence. Therefore, experiments were conducted to examine the potential inhibitory effect of epinephrine and adrenochrome on CYP3A4-mediated metabolism of testosterone. Results showed that both epinephrine and adrenochrome inhibited CYP3A4-mediated metabolism of testosterone with comparable potency (Fig. 3).

In summary, the present study provides evidence that epinephrine and adrenochrome are substrates and/or inhibitors of CYP3A4. Therefore, use of adrenochrome reaction for measurement of reactive oxygen species formed during CYP-mediated biotransformation is inappropriate.