Immunoquantification of epoxide hydrolase and cytochrome P‐450 isozymes in fetal and adult human liver microsomes

Abstract

Epoxide hydrolase and three cytochrome P-450 isozymes were immunochemically determined in microsomes from adult and fetal human liver and tentatively correlated with some enzyme activities. The P-450 isozymes 5, 8 and 9 present in adult liver could not be positively correlated with the total cytochrome P-450 concentration spectrophotometrically determined. In fetal liver microsomes, isozyme 8 could not be detected by either electrophoretic or immunochemical procedures. Isozyme 5 was the major isozyme present in the fetal liver and its concentration increased in close relation with the total P-450 level. As shown previously, arylhydrocarbon hydroxylase activity was related to the concentration of isozyme 8 in adult liver. In fetal preparations, the absence of isozyme 8 was associated with a very low arylhydrocarbon hydroxylase activity. Aldrin epoxidase and benzphetamine-N-demethylase activities were correlated with isozyme 5 concentration, but with different slopes for adult and fetal microsomes: adult preparations catalyzed these two reactions more efficiently. Conversely, the dehydroepiandrosterone 16B-hydroxylase, also associated with isozyme 5 concentration, was more active in fetal than in adult microsomes. Moreover, if acetanilide hydroxylase increased with isozyme 5 concentration in adult samples, no correlation occurred between activity and P-450 isozyme level in fetal microsomes. Hydroxylations of lauric acid in positions 11 and 12 and of dehydroepiandrosterone in position 16a increased with total P-450 concentration but not with isozyme concentrations whatever the age considered. Lastly, epoxide hydrolase activity towards benzopyrene 4,5-oxide was closely associated with its immunochemically determined level. These results clearly suggest that multiple mechanisms are involved in the regulation of different drug-metabolizing enzymes in the human fetus.The metabolism of xenobiotics by hepatic enzymes is now well documented. Hydrophobic molecules are transformed by a multistep system which allows their elimination as watersoluble forms: this biotransformation leads first to the formation of a hydroxylated molecule (phase I) which is conjugated with UDP-glucuronic acid, sulfate or glutathione (phase 11). The major component involved in the oxidative process is cytochrome P-450. Multiple cytochromes P-450 have been shown to be present in the liver of laboratory animals and are characterized by an overlapping substrate specificity. Moreover, these isozymes of cytochrome P-450 can be differently affected by the pretreatment with some inducers [l].In man, P-450-related monooxygenase activities have been measured in microsomes from samples obtained from biopsies, from autopsies, or from donors for renal transplantation

Expression of CYP2D6 in developing human liver

“…Another crucial element is the maturity of the enzymatic system implicated in its metabolism. To date, only few data have reported the onset of P-450 isoenzymes in the human liver: these studies concluded on the presence of CYP3A protein in the fetal liver as early as the 14th week of gestation [2,[19][20][21][22][23] and the absence of proteins of the CYP2C subfamily, explaining the very low activity towards benzopyrene and mephenytoin [2, The CYP2D subfamily is a third group of P-450 genes, composed of three genes in the human species, but only one is functional 1241. We have accumulated evidence indicating that, during ontogenesis, the surge in CYP2D6 protein is mainly postnatal since its concentration rises in newborns 24 h after birth.…”

“…Microsomes were prepared as described elsewhere [2]. Total P-450 content was determined according to Greim [13] and protein by the procedure of Lowry et al [14].…”

“…These P-450s exhibit different profiles for their developmental evolution. In fetal liver, only one P-450 has been repeatedly detected: a CYP3A protein is present at a level roughly similar to that of the adult liver when determined by Western blotting [2] and is able to catalyze enzymatic activities like benzphetamine demethylation, aldrin epoxidation and dehydroepiandrosterone 16P-hydroxylation as the adult isoenzyme does. CYP2C8, one of the major P-450 isozymes immunologically detected in adult liver, is totally absent from fetal liver.…”

Biotransformation of caffeine in human liver microsomes from foetuses, neonates, infants and adults.

“…This suggests that caffeine metabolism may depend in the foetus on the major foetal liver cytochrome, CYP3A [14,[20][21][22][23]. The C-8-hydroxylation of caffeine may also be mediated by CYP3A.…”

“…Human livers were obtained within the first hour after death and microsomes were prepared as described previously [14]. No difference in the time between death and tissue freezing was noted between age groups.…”