FUNCTIONAL CYTOCHROME P4503A ISOFORMS IN HUMAN EMBRYONIC-TISSUES - EXPRESSION DURING ORGANOGENESIS

Expression of functional cytochrome P450 (CYP) isoforms in human embry onic tissues was explored during organogenesis (days 50-60 of gestatio n) with substrate probes, inhibitor probes, and immunoprobes and by re verse transcription-polymerase chain reaction (PCR), cloning, and sequ encing. Evidence was obtained for the presence of relatively high leve ls of one or more functional CYP3A isoforms in embryonic livers. This was manifested as relatively extensive hydroxylation of (R)-warfarin a t carbon 10 and as triacetytoleandomycin-inhibited O-debenzylation of benzyloxyresorufin when human embryonic hepatic microsomal fractions w ere used as enzyme sources. Immunoblots with anti-CYP3A4 antibody exhi bited a strong signal in embryonic hepatic tissues but, in contrast, i ndicated very low or negligible CYP3A levels in human embryonic rung, kidney, heart, adrenal, and brain tissues. To explore expression of in dividual members of the CYP3A subfamily in human embryonic hepatic tis sues at this early gestational stage, CYP3A cDNA was generated by reve rse transcription, amplified by PCR, cloned, and sequenced. Oligonucle otide primers used for PCR were designed to flank target sequences uni que to CYP3A but also common to all human CYP3A subfamily members for which GenBank nucleotide sequence information was available (CYP3A3, C YP3A4, CYP3A5, CYP3A5P, and CYP3A7). Sequencing data indicated that pl asmids in 58 of 59 recombinant positive colonies contained an insert w ith a sequence identical to that present in CYP3A7 cDNA and the plasmi d of only one colony contained an insert with a sequence identical to that present in CYP3A5 cDNA. No evidence was found for expression of C YP3A3 or CYP3A4. Thus, during organogenesis, human embryonic hepatic t issues express primarily CYP3A7 and are capable of significant CYP3A7- catalyzed xenobiotic monooxygenation during this very early stage of g estation.