Abstract
The cytochromes P450 belong to a superfamily of enzymes involved in a diverse array of endobiotic and xenobiotic metabolic pathways. Several members of a novel family of cytochrome P450 (CYP4F), which specifically mediate leukotriene B4ω-hydroxylation, have now been identified in various species including rat, mouse, and human. In rats, the CYP4F family consists of four related genes, CYP4F1, CYP4F4, CYP4F5, and CYP4F6. Here we report development of fluorescent real-time quantitative polymerase chain reaction assays (TaqMan), which allow us to carry out specific quantitation of mRNA expression of all four members of this subfamily. Since no inducers for the CYP4F family are known to date, we validated these assays using clofibrate, a known suppressor of rat CYP4Fs. Additionally, Northern blot hybridization was carried out to validate these assays. Using this approach, we demonstrate quantitatively, for the first time, that each of the rat CYP4Fs is expressed in a tissue- and sex-dependent manner showing a significantly higher expression in females vis-à-vis males. Western blot analysis using a CYP4F polyclonal antibody also shows a considerably higher protein expression in female liver, kidneys, and lungs when compared with male rats. Furthermore, we observe a significant decrease in the CYP4F1, CYP4F4, and CYP4F6 message in kidneys and liver of ovariectomized rats when compared with control females. This loss of expression is partially restored by estrogen treatment in both tissues, suggesting a role of estrogen in regulating CYP4F expression.
Footnotes
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This work was supported by Grant MH58297 from the National Institute of Mental Health, Department of Health and Human Services.
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The data presented here form part of the dissertation of Auinash Kalsotra submitted to the faculty of the University of Texas Graduate School of Biomedical Sciences in partial fulfillment of the requirements for the Doctor in Philosophy degree.
- Abbreviations used are::
- P450
- cytochrome P450
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcription-PCR
- QRTPCR
- quantitative real-time RT-PCR
- SSC
- standard saline citrate
- PPARα
- peroxisome proliferator-activated receptor α
- Received February 28, 2002.
- Accepted June 10, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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