Formation of N-alkylprotoporphyrin IX after interaction of porphyrinogenicxenobiotics with rat liver microsomes

Mechanism-based inactivation of selected cytochrome P450 (CYP) isozymes by xenobiotics can lead to N-alkylation of the heme moiety and the formation o f ferrochelatase-inhibitory N-alkylprotoporphyrin IX (N-alkylPP), resulting in hepatic porphyrin accumulation and porphyria. Therefore, it is importan t to determine which of the CYP isozymes are responsible for N-alkylPP form ation. Our objective was to determine if N-alkylPPs could be: isolated from rat liver microsomes following interaction in vitro with porphyrinogenic x enobiotics, and if so, whether they are produced in amounts sufficient for further studies using microsomes containing cDNA-expressed human hepatic CY P isozymes. The in vitro formation of N-alkylPP was observed following incu bation of 3-[(arylthio)ethyl]sydnone; allylisopropylacetamide; and 3,5-diet hoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine but not 1-aminobenzot riazole, with rat hepatic microsomes. However the overall N-alkylPP yield p er nmol CYP in vitro was much lower than previously observed in vivo. It wa s concluded that microsomal preparations containing cDNA-expressed CYP isoz ymes do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP and human liver microsomes would be a more appropriate source of N-alkylPP because they contain higher levels of total CYP. (C) 2000 Elsevi er Science Inc. All rights reserved.