Introduction

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Before approximately day 60 in humans, the conceptus is referred to as an embryo and subsequently is referred to as a fetus. Commonly, the embryonic period is regarded as the most sensitive in terms of the teratogenic effects of drugs and chemicals and the potential for cytochrome P-450 monooxygenase (CYP)¹-dependent bioactivation as a mechanistic component of chemical teratogenicity is now well established. With the recognition of unique CYP isoforms in human prenatal tissues (e.g., CYP3A7) and the ontogenic changes that occur in CYP isoform content and activity, it thus seems of high importance to investigate the ontogenesis of CYP expression during the embryonic stages of development. Questions pertaining to the extent to which CYP expression is preserved during this developmental period and whether "isoform switching" may occur appear to be of particular interest and importance.

In this study, we used imipramine biotransformation as a probe for the investigation of these questions. Imipramine is an attractive model substrate because it is widely used as an antidepressant agent, can be metabolized by a variety of CYP isoforms, and has been extensively investigated in adult hepatic tissues (e.g., Sequeira and Strobel, 1995; Brosen et al., 1996). Reportedly, CYP1A2 and 3A4 are highly active in catalyzing imipramine biotransformation reactions (Lemooine et al., 1993; Madsen et al., 1997; Simon et al., 1997). Thus, it is logical to expect that embryonically expressed CYP isoforms of the same families/subfamilies such as CYP3A7 and CYP1A1 (reviewed by Juchau et al., 1998) would catalyze the same reactions. We therefore investigated the usefulness of imipramine biotransformation by human embryonic hepatic particulate suspensions as a probe for investigating CYP expression during human embryogenesis. Selective CYP inhibitors and cDNA-expressed human CYP isoenzymes (CYP SUPERSOMES) were used for comparisons and to assist in identification of individual CYP isoforms involved.

	Materials and Methods

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Chemicals and Enzymes. Furafylline was purchased from Ultrafine Chemicals (Manchester, UK) and -naphthoflavone (ANF) was purchased from Aldrich (Milwaukee, WI). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Reagents and solvents used were of the highest purity commercially available. CYP SUPERSOMES, used widely in recent studies (e.g., Gallagher et al., 1994; Wang and Lu, 1997), and adult human liver microsomes were purchased from Gentest Corp. (Woburn, MA).

Preparations of Human Embryonic Hepatic Tissue Particulate Suspension. Human embryonic and fetal hepatic tissues were obtained within 3 h of delivery from the Birth Defects Research Center at the University of Washington (Department of Pediatrics) and were immediately stored in liquid N₂ (-120°C). Handling of human tissues was in accordance with the guidelines of the Human Subjects Review Committee of the University of Washington. The tissue particulate was prepared following a procedure described previously (Chen and Juchau, 1997).

Demethylation of Imipramine. Imipramine demethylation was conducted in reaction vessels containing potassium phosphate buffer (0.1 M, pH 7.4) and 0.1 mM imipramine as substrate. Human embryonic/fetal hepatic particulate suspensions, human liver microsomes, or CYP SUPERSOMES were preincubated with imipramine at 37°C for 3 min. Final volumes of the incubation mixtures were 0.5 ml. Reactions were initiated by the addition of NADPH (1 mM) and were continued for 20 or 40 min. The reactions were terminated by freezing, and imipramine metabolites were extracted according to the methods described by Sequeira and Strobel (1995).

HPLC Analyses of Metabolites of Imipramine Oxidation. The HPLC system consisted of two model 100 A dual piston Beckman (Berkeley, CA) pumps, a Shimadzu (Columbia, MD) SPD-10A UV detector (set at a wavelength of 214 nm) and a Shimadzu C-R5A Chromatopac data processor. Separation, identification, and quantitation of desipramine were conducted with a Supelco LC-PCN column (5 µm, 250 × 4.6 mm) (Bellefonte, PA) following the procedures described by Sequeira and Strobel (1995). The identity of desipramine generated was confirmed by electrospray mass spectroscopy using a Micromass Quattro II tandem quadrupole mass spectrometer (Micromass Inc., Manchester, UK) located at the Mass Spectrometry Center, Department of Medicinal Chemistry, University of Washington.

Protein Determinations. The method of Lowry et al. (1951) was used for quantitative determinations of protein concentrations in hepatic microsomal particulates. BSA was used as a standard protein for the quantitation.

	Results and Discussion

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As shown in Table 1, human embryonic hepatic tissues exhibited significant demethylase activities even at a very early developmental stage (gestational days <60). For purposes of control and comparison, demethylase activities of human fetal hepatic tissues and adult liver microsomes were also measured. Importantly, the embryonic demethylase activities were NADPH dependent and inhibited by both heat inactivation (100°C for 3 min) and carbon monoxide (CO/O₂ = 80:20 versus N₂/O₂ = 80:20), suggesting that CYP-dependent monooxygenation was the primary catalytic mechanism. These studies indicate that functionally active CYP isoforms might catalyze embryonic xenobiotic oxidation in humans and play an important pharmacological/toxicological role during embryogenesis.

Selective CYP inhibitors (1 µM) were used to identify individual embryonic enzymes primarily responsible for catalyzing imipramine demethylation. As shown in Fig. 1A, triacetyloleandomycin (TAO) and ANF (selective for members of the CYP3A subfamily and CYP family 1, respectively) inhibited the reaction by approximately 90 and 70%, respectively, suggesting that CYP3A7 and members of CYP family 1 could play major catalytic roles. Although CYP1A2 effectively catalyzes imipramine demethylation in adult human liver (Lemoine et al., 1993), the active enzyme is apparently not present in embryonic hepatic tissues based on the lack of inhibition by furafylline, which is highly selective for CYP1A2. Rather, ANF may influence the reaction through inhibition of the CYP1A1 and/or CYP1B1 isoforms. Diethyldithiocarbamate (selective for CYP2E1) also failed to inhibit the reaction significantly, suggesting that CYP2E1 did not contribute to catalysis of demethylation of imipramine in human embryonic hepatic tissues. This was further supported by the low demethylase activities measured when CYP2E1 SUPERSOMES were incubated with imipramine as substrate.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Inhibition of embryonic hepatic tissue-dependent demethylation of imipramine by various inhibitors (1 µM; open columns, control; filled columns, control plus inhibitor) (A), inhibition of CYP1A1/1B1 SUPERSOMES-dependent demethylation of imipramine by TAO (open columns, CYP1A1 SUPERSOMES; , CYP1B1 SUPERSOMES) (B), and inhibition of CYP3A7 SUPERSOMES-dependent demethylation of imipramine by ANF (C). Imipramine (0.1 mM) was preincubated with suspensions of human embryonic tissue particulates (0.05-0.1 mg protein) or CYP SUPERSOMES (0.025 nmol) plus inhibitors at 37°C for 3 min. Reactions were initiated by additions of NADPH (1 mM) and were continued for 20 min. For the mechanism-based inhibitors TAO and furafylline, suspensions of tissue particulate or CYP SUPERSOMES were preincubated with the inhibitors plus NADPH at 37°C for 15 min. Reactions were initiated by additions of imipramine (0.1 mM) and were continued for 20 min. Values for controls in Fig. 1B were 945 ± 54 (for CYP1A1 SUPERSOMES) and 102 ± 32 (for CYP1B1 SUPERSOMES) pmol/min/nmol CYP. Value for control in Fig. 1C was 112 ± 26 pmol/min/nmol CYP. Results are means ± S.D. of three to four measurements.

The strong inhibition produced by both TAO and ANF shown in Fig. 1A indicated that the inhibitory effects of TAO and ANF may overlap. To further clarify the relative roles of CYP3A7, CYP1A1, and CYP1B1, CYP3A7 SUPERSOMES were incubated with imipramine plus ANF, and CYP1A1 or CYP1B1 SUPERSOMES were incubated with imipramine plus TAO. The importance of this experiment was 2-fold. Firstly, the results verified that each of these isoforms could catalyze the reaction efficiently, as demonstrated by the significant demethylase activities (Fig. 1, B and C). Secondly, the results verified the importance of CYP3A7 versus CYP1A1/1B1 by investigating the specificity of inhibition by TAO and ANF in human embryonic hepatic tissues. TAO did not exhibit statistically significant inhibitory effects on CYP1A1 or CYP1B1 SUPERSOMES-catalyzed reactions at any of the concentrations tested (Fig. 1B) whereas ANF strongly inhibited demethylase activity of CYP3A7 SUPERSOMES (Fig. 1C). This observation was consistent with earlier reports that ANF could also inhibit members of the CYP3A subfamily to a certain degree, depending on the concentrations used (Chang et al., 1994). Therefore, the effect of ANF appeared to be the sum of inhibition of CYP1A1/1B1 and CYP3A7. These results strongly support that the demethylation of imipramine in human embryonic hepatic tissues is catalyzed primarily by CYP3A7. As reported in previous studies with fetal tissues, CYP3A7 is expressed extensively in human fetal hepatic tissues (Kitada et al., 1991) and catalyzes a variety of xenobiotic biotransformation reactions (Ohmori et al., 1998). These findings along with our studies suggest that CYP3A7 may play important pharmacological and toxicological roles prenatally.

Localized embryonic bioactivation of xenobiotics undoubtedly plays an important role in specific embryotoxicities. Several CYP isoforms, including CYP3A7, CYP1A1, 1B1, and 2E1, are expressed in rodent and human prenatal tissues (Omiecinski et al., 1990; Chapman et al., 1994; Schuetz et al., 1994; Carpenter et al., 1996, 1997; Miller et al., 1996; Shimada et al., 1996). CYP3A7 appears to be expressed primarily in human hepatic tissues, expression of CYP1A1 is largely confined to extrahepatic tissues (in humans), and CYP1B1 appears to be quite ubiquitously expressed. Recent studies have shown that CYP2E1 is expressed not only in human fetal liver (Carpenter et al., 1996, 1997) but also in human prenatal brain (Juchau et al., 1998). To understand the roles of these CYP isoforms and their significance in embryotoxicity, it is of great importance to study the quantitative aspects of their catalytic capacities and functional properties for biotransformation of xenobiotics in human embryonic tissues. Imipramine is a prototype of tricyclic antidepressants that is metabolized predominantly in hepatic tissues of adults to the active metabolite desipramine via N-demethylation of the side chain (Brosen et al., 1996). N-demethylation of imipramine is reportedly catalyzed by several CYP isoforms such as CYP1A2, CYP2C19, and CYP3A4 (Lemoine et al., 1993; Brosen et al., 1996; Madsen et al., 1997; Simon et al., 1997).

In summary, we have demonstrated that human prenatal hepatic tissues exhibited significant, CYP-dependent catalytic activities for xenobiotic metabolism even at very early stages of development (embryonic, gestational days <60) using imipramine as a model substrate. With this model substrate probe and with chemical inhibition, relatively high catalytic activities were detected. Taken together, the results indicated that CYP3A7 was by far the most important CYP isoform for catalysis of imipramine demethylation in human embryonic hepatic tissues. Although CYP1A1/1B1 could also catalyze the reaction, contributions of CYP1A1/1B1 appeared to be minimal, probably due to their extremely low expression in human embryonic hepatic tissues (Juchau et al., 1998).