Research reportRegulation of expression of cytochrome P-450 2D mRNA in rat brain with steroid hormones
Introduction
The cytochrome P-450 (P-450) mixed function oxidase enzyme system has been studied extensively in liver and has also been characterized in various extrahepatic tissues (i.e. brain, lung, kidney, etc.). Certain subfamilies of P-450 have been shown to be involved in the synthesis of steroid hormones, fatty acids, bile acids, and cholesterol. P-450 is also responsible for the oxidative metabolism of various xenobiotics including the activation of precarcinogens or therapeutic agents. P-450 acts to detoxify certain toxic xenobiotics in the body as well. In light of the many important functions of P-450, it is of interest to understand which subfamilies of P-450 are present in the various tissues and what influences the expression of these subfamilies in these tissues.
In rat brain, P-450 1A1 was the first well known isoform identified using immunohistochemistry [13]. P-450 c and d (P-450 1A1 and 1A2) were also later identified in untreated rat brain using immunohistochemistry [15]. Since these demonstrations, many isoforms such as 2B1, 2B2, 2D, and 2E have been identified in rat brain using PCR [11]. Strobel et al. first reported the expression of rat brain cytochromes P-450 is responsive to phenobarbital and tricyclic amines [20]. Expression of P-450 is also affected by hormone status 1, 9, 17, 18and it has been reported that sex steroid hormones have effects on the expression of the cytochrome P-450 2D (CYP2D) isoform in various brain regions [3]. Bergh and Strobel [3]reported that estrogen treatment increased expression somewhat whereas testosterone treatment brought about a profound increase in CYP2D expression in all brain regions examined. Coadministration of estrogen with testosterone resulted in an induction level lower than that seen by administration of testosterone alone in all brain regions examined.
Down-regulation of a metabolic enzyme after an animal is exposed to a dietary, hormonal, or chemical stimulus has many implications when the modality and efficacy of a drug treatment are considered. The changes in sex steroid hormone levels seen in the post-partum or post-menopausal state exemplify the kinds of alterations in regulatory effectors which impact the level of enzyme expression. Lower levels of a particular enzyme could delay bioactivation, in terms of rate and magnitude, or delay clearance of a drug from an organ. CYP2D is a subfamily of the P-450s responsible for metabolizing, among other things, the tricyclic antidepressants, neuroleptics, and anticonvulsants. Because the brain is the target organ for the actions of these drugs, it is of interest to understand how or if various substances alter expression of CYP2D isoforms in brain. Specifically it is important to understand how changes in sex steroid hormones modify the expression of cytochromes P-450 involved in the activation and metabolism of drugs used in the treatment of brain diseases (e.g. epilepsy) and mental diseases (e.g. depression). Recently we have demonstrated that CYP2D18, a member of the CYP2D subfamily, catalyzes the demethylation of imipramine to desipramine, which is therapeutically active in the treatment of depression [14].
This present work examines how testosterone, progesterone, and testosterone and progesterone together affect the level of CYP2D mRNA in rat brains. Because the P-450 levels in rat brain are low in comparison to liver, a sensitive assay that can detect very small changes in mRNA expression must be used. For this reason cPCR was used as it has been in previous work in our laboratory 6, 7, 11as well as others [16].
Section snippets
Animals
Adult female Sprague–Dawley rats were obtained from Harlan Sprague Dawley, Houston, TX, and were divided into two major groups: ovariectomized and non-ovariectomized. Within these two groups were four treatment groups: (1) testosterone propionate at 1 mg/200–249 g rat and 4-pregnone-3,20-dione at 4 mg/200–249 g rat, (2) testosterone propionate alone at 1 mg/200–249 g rat, (3) pregnone-3,20-dione alone at 4 mg/200–249 g rat, and (4) corn oil. The vehicle used was corn oil. There were six rats
Results
The validity of quantitative and semi-quantitative cPCR relies on the fact that the reactions are executed in the linear (exponential) range of amplification. This consideration was determined for both the CYP2D amplification and the cyclophilin control. The cPCR was carried out at a range of cycle numbers to ascertain where the PCR products were increasing in a linear fashion with cycle number (Fig. 1). The reaction for the 2D product can be seen to be in exponential phase between cycles 30 to
Discussion
This is the first study that uses cPCR techniques to show how progesterone, testosterone, and a combination of the two affect the expression of CYP2D in female rat brains. For several years, PCR has been used as an integral part of quantification assays 2, 8, 21. Due to its increased sensitivity, there are several considerations that need to be taken into account when using cPCR to measure mRNA levels either semi-quantitatively or quantitatively. In this present work, both endogenous and
Acknowledgements
The authors would like to thank Dr. Jun Geng for his assistance with competitors and Laura Bankey for her technical assistance. This investigation was supported in part by the National Institute of Mental Health Grant MH 44923 and in part by The Women's Fund for Health Education and Research, Houston, Texas.
References (21)
- et al.
Administration of testosterone alleviates the constitutive sex difference in rat brain cytochrome P-450
Neurosci. Lett.
(1991) - et al.
Detection and quantitation of 5-HT1A and 5-HT2A receptor mRNAs in human hippocampus using a reverse transcriptase-polymerase chain reaction (RT-PCR) technique and their correlation with binding site densities and age
Neurosci. Lett.
(1994) - et al.
Identification of cytochromes P450 1A2, 2A1, 2C7, 2E1 in rat glioma C6 cell line by RT-PCR and specific restriction enzyme digestion
Biochem. Biophys. Res. Commun.
(1993) - et al.
Presence of sex difference of cytochrome P-450 in the rat preoptic area and hypothalamus with reference to coexistence with oxytocin
Brain Res.
(1990) - et al.
Isolation and amino acid sequence of cyclophilin
J. Biol. Chem.
(1986) - et al.
Immunohistochemical localization of cytochrome P-450 in rat brain
Neuroscience
(1987) - et al.
Genomic cloning and protein expression of a novel rat brain cytochrome P-450 CYP2D18* catalyzing imipramine N-demethylation
J. Biol. Chem.
(1996) - et al.
Immunohistochemical localization of cytochrome P-450 in the rat brain
Neurosci. Lett.
(1988) - et al.
Brain cytochromes P-450 are responsive to phenobarbital and tricyclic amines
Pharmacol. Res.
(1989) - et al.
Absolute mRNA quantification using the polymerase chain reaction (PCR). A novel approach by a PCR aided transcript titration assay (PATTY)
Nucleic Acids Res.
(1989)
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