Abstract
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.
Footnotes
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This work was supported by the Canadian Institutes of Health Research
- Abbreviations used are::
- P450
- cytochrome P450
- TTMS
- 3-[(arylthio)ethyl] sydnone
- 4-ethylDDC
- 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine
- AIA
- allylisopropylacetamide
- N-vinylPP
- N-vinylprotoporphyrin IX
- HLCL
- human lymphoblastoid cell lines
- N-alkylPP
- N-alkylprotoporphyrin IX
- BIICL
- baculovirus-infected insect cell lines
- N-AIAPP
- N-AIAprotoporphyrin IX
- N-ethylPP
- N-ethylprotoporphyrin IX
- LLD
- lower limit of detection
- DCM
- dichloromethane
- Received September 10, 2002.
- Accepted November 7, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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