Comparison of the formation of N-alkylprotoporphyrin IX after interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes prepared from baculovirus-infected insect cells and human lymphoblastoid cell lines.
In a previous study using microsomes from human lymphoblastoid cell lines (HLCL) containing single cDNA-expressed human cytochrome P450 (P450) enzymes, human P450 enzymes were identified that are susceptible to mechanism-based inactivation by the porphyrinogenic xenobiotics, 3-[(arylthio)ethyl]sydnone (TTMS), 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethylDDC) and allylisopropylacetamide (AIA). In this study, we tested the hypothesis that N-alkylprotoporphyrin IX (N-alkylPP) formation following interaction of porphyrinogenic xenobiotics with single cDNA-expressed human P450 enzymes in microsomes from HLCL would occur only with P450 enzymes that had undergone mechanism-based inactivation. In a previous study, when 4-ethylDDC and NADPH interacted with human liver microsomes possessing elevated levels of CYP1A2 and 2C9, N-ethylprotoporphyrin IX (N-ethylPP) was not formed despite the fact that it was formed in microsomes from baculovirus-infected insect cell lines (BIICL) containing either CYP1A2 or 2C9. In this study, we tested the hypothesis that 4-ethylDDC underwent biotransformation by CYP3A4 present in human liver microsomes, diverting the xenobiotic from CYP1A2 and 2C9. Fluorometry was used to measure N-alkylPP formation following interaction of porphyrinogenic xenobiotics and NADPH with cDNA-expressed human P450 enzymes in microsomes from HLCL or BIICL. With TTMS and 4-ethylDDC but not with AIA, N-alkylPP formation was observed only with human P450 enzymes CYP2D6, 1A2, 3A4, or 2C9 in microsomes from HLCL, which had undergone mechanism-based inactivation. Microsomes from BIICL containing CYP3A4 were added to a mixture of NADPH, 4-ethylDDC, and microsomes from BIICL containing CYP1A2 and 2C9. The addition of CYP3A4 to CYP1A2 and 2C9 did not decrease N-ethylPP formation, providing no support for the hypothesis.
Gamble
Jeremy T
JT
Department of Pharmacology and Toxicology, Faculty of Health Sciences, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Nakatsu
Kanji
K
Marks
Gerald S
GS
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002461:Cell Line, Transformed; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D008213:Lymphocyte Activation; D008861:Microsomes / Q000378:metabolism*; D009036:Moths / Q000821:virology; D011165:Porphyrinogens / Q000737:chemistry / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism*; D015262:Xenobiotics / Q000737:chemistry / Q000378:metabolism*
D018076:DNA, Complementary; D011165:Porphyrinogens; D011524:Protoporphyrins; D015262:Xenobiotics; D003577:Cytochrome P-450 Enzyme System; C028025:protoporphyrin IX