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cDNA-expressed human cytochrome P450 isozymes. Inactivation by porphyrinogenic xenobiotics
J P McNamee1, M Jurima-Romet, S M Kobus
1Department of Pharmacology and Toxicology, Faculty of Medicine, Queen's University, Kingston, Ontario, Canada.
Abstract:
A number of xenobiotics are known to exert their porphyrinogenic effects in rodents and chick embryos through mechanism-based inactivation of certain cytochrome P450 (P450) isozymes. To facilitate the extrapolation of results from test animals to humans, we have assessed the ability of three prototype porphyrinogenic compounds-namely, 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (DDEP), 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS), and allylisopropylacetamide (AIA)-to cause mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, 2C9-Arg144 (2C9), 2D6-Val374 (2D6), and 3A4 in microsomes from human lymphoblastoid cell lines (Gentest Corp., Woburn, MA). The following catalytic markers of human P450 isozymes were used: ethoxyresorufin O-deethylase (P450s 1A1 and 1A2), diclofenac 4-hydroxylation (P4502C9), dextromethorphan O-demethylase (P4502D6), and testosterone 6 beta-hydroxylation (P4503A4). We found that DDEP and TTMS caused mechanism-based inactivation of cDNA-expressed human P450s 1A1, 1A2, and 3A4, whereas only DDEP was able to cause mechanism-based inactivation of cDNA-expressed human P4502C9; neither xenobiotic caused mechanism-based inactivation of cDNA-expressed human P4502D6. A comparison of the human P450 isozyme data with results previously obtained in rat and chick embryo liver showed a close correspondence between the results obtained with P450s 1A and 3A, but not the P4502C subfamily. Because several rat isozymes (P450s 2A1, 2B1, 2C6, 2C11, and 3A1) undergo inactivation by AIA, it was noteworthy that AIA did not inactivate any of the cDNA-expressed human P450 isozymes. Because mechanism-based inactivation of P450 isozymes is related to the porphyrinogenicity of xenobiotics, our results demonstrate the importance of supplementing studies of mechanism-based inactivation of P450 isozymes in animal models with similar studies on cDNA-expressed human P450 isozymes.
Insights
Three compounds were tested for their ability to inactivate human cytochrome P450 (P450) enzymes. DDEP and TTMS inactivated human P450s 1A1, 1A2, and 3A4, highlighting the need for human P450 studies.
Area of Science:
- Biochemistry
- Toxicology
- Pharmacology
Background:
- Xenobiotics can cause porphyrin toxicity by inactivating cytochrome P450 (P450) enzymes.
- Understanding these mechanisms in animal models is crucial for human risk assessment.
Purpose of the Study:
- To evaluate the mechanism-based inactivation of human P450 isozymes by prototype porphyrinogenic compounds.
- To compare these effects with findings in animal models for improved extrapolation of results.
Main Methods:
- Assessed inactivation of cDNA-expressed human P450s (1A1, 1A2, 2C9, 2D6, 3A4) using specific catalytic markers.
- Tested three compounds: DDEP, TTMS, and AIA in human lymphoblastoid cell microsomes.
Main Results:
- DDEP and TTMS inactivated human P450s 1A1, 1A2, and 3A4.
- DDEP also inactivated human P450 2C9, while neither compound affected P450 2D6.
- AIA did not inactivate any tested human P450 isozymes, unlike in rat models.
Conclusions:
- Results show species-specific differences in P450 inactivation, particularly for the P450 2C subfamily.
- Mechanism-based inactivation studies using human P450 isozymes are essential for accurate xenobiotic risk assessment.
- Findings emphasize the need to supplement animal studies with human P450 data.