The human cytochrome P4503A forms show expression patterns subject to developmental influence. CYP3A7 and CYP3A4 are generally classified as the major fetal and adult liver forms, respectively. However, characterization of CYP3A4, -3A5, and -3A7 developmental expression has historically been confounded by the lack of CYP3A isoform-specific antibodies or marker enzyme activities. Therefore, the objective of this study was to characterize the developmental expression of hepatic CYP3A forms from early gestation to 18 years of age using up to 212 fetal and pediatric liver samples. Based on immunoquantitation, CYP3A5 protein expression was found to be highly variable, generally independent of age, and more frequently observed for African-American individuals. For differentiation of CYP3A4 and -3A7 levels, dehydroepiandrosterone metabolite patterns for expressed CYP3A forms were characterized and used for simultaneous quantitation of protein levels within liver microsome samples. The major metabolite formed by CYP3A4, 7␤-hydroxy-dehydroepiandrosterone, was identified based on cochromatography and mass spectra matching with the authentic standard. Kinetic analysis showed a 34-fold greater intrinsic clearance of 7␤-hydroxy-dehydroepiandrosterone by CYP3A4 versus -3A7, whereas CYP3A7 showed the highest 16␣-hydroxy-dehydroepiandrosterone intrinsic clearance. Metabolite profiles for the expressed enzymes were fit to a multiple response model and CYP3A4 and -3A7 levels in fetal and pediatric liver microsome samples were calculated. Fetal liver microsomes showed extremely high CYP3A7 levels (311-158 pmol/mg protein) and significant expression through 6 months postnatal age. Low CYP3A4 expression was noted for fetal liver (Յ10 pmol/mg), with mean levels increasing with postnatal age.

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The CYP2C subfamily is responsible for metabolizing many important drugs and accounts for about 20% of the cytochrome P450 in adult liver. To determine developmental expression patterns, liver microsomal CYP2C9 and -2C19 were measured (n ϭ 237; ages, 8 weeks gestation-18 years) by Western blotting and with diclofenac or mephenytoin, respectively, as probe substrates. CYP2C9-specific content and catalytic activity were consistent with expression at 1 to 2% of mature values (i.e., specific content, 18.3 pmol/mg protein and n ϭ 79; specific activity, 549.5 pmol/mg/min and n ϭ 72) during the first trimester, with progressive increases during the second and third trimesters to levels approximately 30% of mature values. From birth to 5 months, CYP2C9 protein values varied 35-fold and were significantly higher than those observed during the late fetal period, with 51% of samples exhibiting values commensurate with mature levels. Less variable CYP2C9 protein and activity values were observed between 5 months and 18 years. CYP2C19 protein and catalytic activities that were 12 to 15% of mature values (i.e., specific content, 14.6 pmol/mg and n ϭ 20; specific activity, 18.5 pmol/mg/min and n ϭ 19) were observed as early as 8 weeks of gestation and were similar throughout the prenatal period. CYP2C19 expression did not change at birth, increased linearly over the first 5 postnatal months, and varied 21-fold from 5 months to 10 years. Adult CYP2C19 protein and activity values were observed in samples older than 10 years. The ontogeny of CYP2C9 and -2C19 were dissimilar among both fetal and 0-to 5-months postnatal samples, implying different developmental regulatory mechanisms.

The flavin-containing monooxygenases (FMOs) are important for the metabolism of numerous therapeutics and toxicants. Six mammalian FMO genes (FMO1-6) have been identified, each exhibiting developmental and tissue-and species-specific expression patterns. Previous studies demonstrated that human hepatic FMO1 is restricted to the fetus whereas FMO3 is the major adult isoform. These studies failed to describe temporal expression patterns, the precise timing of the FMO1/FMO3 switch, or potential control mechanisms. To address these questions, FMO1 and FMO3 were quantified in microsomal fractions from 240 human liver samples representing ages from 8 wk gestation to 18 y using Western blotting. FMO1 expression was highest in the embryo (8 -15 wk gestation; 7.8 Ϯ 5.3 pmol/mg protein). Low levels of FMO3 expression also were detectable in the embryo, but not in the fetus. FMO1 suppression occurred within 3 d postpartum in a process tightly coupled to birth, but not gestational age. The onset of FMO3 expression was highly variable, with most individuals failing to express this isoform during the neonatal period. FMO3 was detectable in most individuals by 1-2 y of age and was expressed at intermediate levels until 11 y (12.7 Ϯ 8.0 pmol/mg protein). These data suggest that birth is necessary, but not sufficient for the onset of FMO3 expression. A gender-independent increase in FMO3 expression was observed from 11 to 18 y of age (26.9 Ϯ 8.6 pmol/mg protein). Finally, 2-to 20-fold interindividual variation in FMO1 and FMO3 protein levels were observed, depending on the age bracket. The FMOs (EC 1.14.13.8) are important for the NADPHdependent oxidative metabolism of a wide variety of compounds containing nucleophilic nitrogen-, sulfur-, selenium-, and phosphorous-heteroatoms (1-4). Examples of known substrates of relevance to pediatric therapeutics include the antipsychotic chlorpromazine (5), the antihistaminics promethazine (6) and brompheniramine (7), the H 2 -receptor antagonists cimetidine (8) and ranitidine (9), and the gastroprokinetic agent itopride (10). However, given the prevalence of nitrogen-and sulfur-heteroatoms in medicinals, this short list is likely a gross underestimate of FMO's contribution to pediatric drug disposition. Environmental agents of particular concern include several thioether-containing organophosphorous pesticides (11), the carcinogen 2-aminofluorene (12), and the neurotoxicants nicotine (13) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (14). Finally, a few dietary and/or endogenous FMO substrates have been identified, including trimethylamine, a break-down product of dietary choline (15), cysteamine (16), methionine, and several cysteine-Sconjugates (3).Unlike the numerous cytochrome P450-dependent monooxygenases, there are only six mammalian FMO enzymes, each encoded by a distinct gene located on the long arm of human chromosome 1 (17, 18). As such, the FMO are considered more versatile with regard to substrate specificity than the cytochrome P450-dependent monooxygenases, a feat...