Introduction

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Cytochromes P450 (P450s¹) comprise a superfamily of heme-thiolate proteins that catalyze monooxygenation of a large number of lipophilic compounds. They play a central role in metabolizing endogenous products such as cholesterol, steroids, eicosanoids, fatty acids, and/or xenobiotics including drugs, pesticides, environmental pollutants, and chemical carcinogens (Guengerich et al., 1991). P450s are classified into many gene families and subfamilies on the basis of their amino acid sequence and similarities (Nelson et al., 1996). Although first identified in liver, their presence and activity have been extended to extrahepatic tissues including kidney, colon, lung, and brain (Krishma and Klotz, 1994; Strobel et al., 2001). In addition to broad and overlapping substrate specificities, P450s also display complex sex-, tissue-, and species-specific expression (Waxman et al., 1995). There is increasing evidence that P450s show gender-specific expression, which is controlled by hormones and growth factors (Waxman et al., 1995; Bergh and Strobel, 1996; Wang and Strobel, 1997; Pampori and Shapiro, 1999) and could account for gender differences in drug metabolism (Nebert, 1991; Kato and Yamazoe, 1992).

Recently, mouse CYP4a isoforms have been shown to play a role in male-specific hypertension indicating a relationship between blood pressure regulation, sex hormones, and P450 -hydroxylases (Holla et al., 2001). This raises the question whether CYP4Fs, which are leukotriene and prostaglandin -hydroxylases also show a sexual dimorphic expression. CYP4Fs constitute a growing subfamily that to date contains five human (Kikuta et al., 1993, 2000b; Bylund et al., 1999; Cui et al., 2000; Bylund and Oliw, 2001), four rat (Chen and Hardwick, 1993; Kawashima and Strobel, 1995), five mouse (Kikuta et al., 2000a; Cui et al., 2001), one sheep (Bylund et al., 2001), and one fish genes (Sabourault et al., 1999).

Many attempts to describe regulation of individual P450s in animal tissues have been complicated by the lack of sensitivity and specificity of the assays used. Overlapping substrate specificity of these enzymes hampers most enzymatic assays and generation of high affinity antibodies for the individual isoforms is a hard task. Also, high homology at the RNA nucleotide sequence level makes it hard to discriminate among these mRNAs using filter-hybridization techniques. Quantitative real-time reverse transcription-polymerase chain reaction (QRTPCR) provides a rapid and excellent alternative (Gibson et al., 1996; Bustin, 2000) for RNase protection assays that are specific but cumbersome. Real-time RT-PCR quantitation of nucleic acids is based on detection of fluorescence as the product is amplified at the end of each cycle. This permits a quantitative calculation of target mRNA in an unknown sample by comparison with the kinetics of a PCR product of known quantity. Real-time RT-PCR fluorescence is typically monitored during amplification by the hybridization of additional gene-specific oligonucleotide(s) that are fluorescently labeled to allow detection during PCR (Heid et al., 1996). In the TaqMan assay, detection occurs following Taq-driven release of a 5' reporter dye from a 3' quencher molecule on a single hybridized probe during polymerase extension (Livak et al., 1995)

To investigate whether CYP4Fs show a tissue- and/or sex-dependent expression, we developed QRTPCR assays (TaqMan) that allow us to carry out specific quantitation of mRNA expression of all four members of the rat CYP4F subfamily. As there are no CYP4F inducers known, we validated these assays using clofibrate, a peroxisomal proliferator, and a known suppressor of rat CYP4Fs. Additionally, Northern blot hybridization and Western blot analysis were also carried out to determine whether the mRNA expression and protein expression profile matched.

	Materials and Methods

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Animal Treatment. Sprague-Dawley rats of either sex (200g b.wt.) were purchased from Harlan (Indianapolis, IN). Animals were allowed free access to food and water and were subjected to a 12-h light/dark cycle. For validation assays, male rats were intraperitoneally injected with a single dose of 500 mg/kg clofibrate (Sigma-Aldrich, St. Louis, MO) or corn oil and sacrificed the following morning. For the remainder of the study, male (n = 3) and female rats (n = 9) were housed in groups in a native animal house environment for 1 week before experimentation. One group of female rats (n = 6) was ovariectomized on day 1 and allowed to recover until day 5. They were treated with either estrogen (50 µg/kg/animal/day) or sesame oil s.c. for 2 consecutive days. The following day, all rats including controls were sacrificed, tissues excised, immediately frozen in liquid nitrogen, and stored at 80°C until used.

RNA Preparation and Northern Blotting. Frozen tissues were thawed on ice, and total RNA was isolated using RNA Stat reagent (Tel-Test Inc., Friendswood, TX). All samples were DNase treated using RQ1 DNase (Promega Corp., Madison, WI). The quality of the isolated RNA was assessed by electrophoresis on 1% agarose gels based on the integrity of 28S and 18S bands after ethidium bromide staining. Thirty micrograms of total RNA for each of the samples were denatured and electrophoresed on 1% agarose gel containing 2.2 M formaldehyde. RNA was transferred onto a Zeta probe nitrocellulose membrane (Bio-Rad, Hercules, CA) and hybridized with ³²P-labeled DNA probes specific for CYP4F1, CYP4F4, CYP4F5, or CYP4F6. After washing the membranes twice for 15 min with 0.2× SSC and once for 15 min with 0.2× SSC and 0.1% SDS solution, they were exposed onto X-ray films.

Design of Real-Time Quantitative PCR Primers and Probes. PCR primers and fluorescent probe sequences were designed at the 5'-untranslated region (region of maximal specificity) of the CYP4F genes using Primer Express software (Applied Biosystems, Foster City, CA) and custom synthesized by Integrated DNA Technologies, Inc. (Coralville, IA) The sequences of primers and probes for individual CYP4Fs are shown in Table 1. The specificity of P450 primers and probes was confirmed through alignment with the other members of the CYP4F subfamily.

Reverse Transcription and Quantitative Polymerase Chain Reaction. Aliquots (200 ng) of total RNA to be analyzed were reverse-transcribed in triplicate [including an RT () blank to account for amplification of contaminating genomic DNA] for each sample with 1× SSII buffer, 300 nM reverse primer, 500 µM dNTPs, and 10 U/10 µl Superscript II (Invitrogen, Carlsbad, CA) at 50°C for 30 min, followed by 72°C for 5 min. Forty microliters of PCR mix (containing 1× PCR buffer, 300 nM forward primer, 300 nM reverse primer, 4 mM MgCl₂, 2.5 U/50 µl Taq polymerase, and 100 nM fluorogenic probe) was added to 10 µl of the RT reaction. Amplification was performed using an ABI Prism 7700 (Applied Biosystems) at 95°C for 1 min, followed by 40 cycles of 95°C for 12 s and 60°C for 1 min. Increases in fluorescence, which were due to the cleavage of the reporter dye as the PCR proceeded relative to the starting values of delta-normalized reporter fluorescence (Rn), were determined and plotted by the instrument against cycle number. Ct values (the PCR cycle number required for fluorescence intensity to exceed an arbitrary threshold in the exponential phase of the amplification) were then determined for a series of standards. Standard curves were generated by plotting Ct versus the log of the amount of amplicon (custom made from Integrated DNA Technologies, Inc.) for specific CYP4F (500 ag-5 pg), and were used to compare the relative amount of a particular CYP4F mRNA in the samples (Fig. 1). Calculations for Ct and Rn, and standard curve preparation were performed by the software provided with the ABI 7700 system (Heid et al., 1996). Data were analyzed and absolute values of mRNAs were generated by normalizing the copy number values of the gene of interest to the copy number values of r-cyclophilin (internal standard).

View larger version (83K): [in this window] [in a new window]   Fig. 1.   Quantitative real-time PCR standard curve for CYP4F6 gene. Standard curves were generated by plotting Ct versus the log of the amount of amplicon for specific CYP4F (500 ag-5 pg), and were used to compare the relative amount of a particular CYP4F mRNA in the unknown samples. Here, standard curve is shown for CYP4F6 only.

Microsome Preparation and Western Blotting. Microsomes from different tissues were prepared as previously described by Saito and Strobel (1981). Briefly, tissues were homogenized (20 strokes) in 6 volumes of potassium phosphate buffer (pH 7.4) containing 20 mM KPi, 0.25 M sucrose, 1 mM EDTA, and a cocktail of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin and 0.7 µg/ml pepstatin). Each homogenate was centrifuged at 8,500 rpm for 20 min and supernatant was collected. The supernatant was further centrifuged at 100,000g for 45 min, the pellet washed in fresh buffer, and again centrifuged at 100,000g for 45 min. The washed pellets were resuspended and stored in 80°C until analyzed. The total microsomal proteins in each sample were determined by bicinchonimic acid assay using bovine serum albumin as the standard. Samples were boiled in Laemmli buffer and resolved on 4 to 15% Tris-glycine SDS-polyacrylamide gel electrophoresis gels. Proteins were transferred to nitrocellulose membranes using a semidry transfer apparatus. Membranes were blocked overnight with 5% dried nonfat milk followed by a 2-h incubation in 1:250 dilution of polyclonal primary antibody against CYP4F5 (is specific for CYP4Fs but does not distinguish among the isoforms). Membranes were then washed and incubated at room temperature with horseradish peroxidase-conjugated secondary antibody (1:1,000 dilution) for 1 h. Immunoreactivity was detected using a horseradish peroxidase chemiluminescence system (Pierce, Rockford, IL). These experiments were repeated three times each.

Statistical Analysis. Data are presented as mean ± S.E. Statistical significance for effects of clofibrate on CYP4Fs and differences among CYP4F expression between male and female groups was determined using a two-tailed unpaired Student's t test. Multiple groups in the ovariectomy study were compared using one-way analysis of variance. Statistical differences were considered significant if P < 0.05.

	Results

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Effects of Clofibrate on CYP4F Isoforms in Rat Liver

The data in Fig. 2a show that a single dose of clofibrate treatment decreased CYP4F1 expression by 65% (P < 0.05) compared with control liver expression. CYP4F5 also showed significantly reduced expression, although to a lesser extent (by 32% from untreated levels, P < 0.05). Among all the CYP4Fs, CYP4F6 showed a maximal decrease in mRNA expression (by 74% from untreated levels, P < 0.01). CYP4F4 mRNA expression remained unchanged after clofibrate treatment. This is noteworthy, since in the report by Kawashima et al. (1997) it was apparent from the Northern blots that CYP4F4 expression did not change significantly from control group whereas 4F5 and 4F6 showed a significantly reduced expression. Our results for CYP4F1 are also consistent with Hardwick's group where they reported reduced CYP4F1 expression after clofibrate treatment (Chen and Hardwick, 1993). Western blot analysis using a polyclonal antibody also showed a marked decrease in CYP4F expression after clofibrate treatment (Fig. 2b).

View larger version (58K): [in this window] [in a new window]   Fig. 2.   Effects of clofibrate on CYP4Fs expression in rat liver. Rats were treated as described under Materials and Methods. A, quantitation of CYP4Fs mRNA expression. Values are expressed as mean ± S.E for the triplicates of each sample. **, P < 0.01; *, P < 0.05 compared with control. B, Western blot analysis of CYP4Fs after clofibrate treatment. Fifty micrograms of total microsomal proteins were loaded for each sample and blotted with CYP4F5 antibody.

Tissue Distribution of Rat CYP4F Isoforms

Liver. In liver, CYP4F1 showed the maximum whereas CYP4F5 showed the least expression among all CYP4Fs. CYP4F4 and CYP4F6 showed moderate mRNA expression compared with CYP4F1. The CYP4F distribution in liver can be denoted as CYP4F1 > CYP4F4 > CYP4F6 > CYP4F5 (Table 2).

Lungs. On the contrary, in lungs no CYP4F1 expression was detected in either sex, whereas CYP4F6 showed the maximum expression. Compared with CYP4F6 expression, CYP4F4 and CYP4F5 showed lower expression. The comparative CYP4F expression in the lungs was found to be CYP4F6 > CYP4F5 > CYP4F4.

Brain. In brain, CYP4F1 and CYP4F6 showed maximal and comparable mRNA expression. CYP4F4 and CYP4F5 showed lower than CYP4F1 but moderate expression. Thus in brain, all CYP4Fs are represented equally well with a distribution profile as CYP4F1 ~ CYP4F6 > CYP4F4 > CYP4F5.

Kidneys. In kidneys, CYP4F1 showed the highest whereas CYP4F4 showed the least expression. The comparative CYP4F expression in kidneys was observed as CYP4F1 > CYP4F5 > CYP4F6 > CYP4F4. Northern blot hybridization using individual CYP4F probes (data shown for CYP4F5 only) showed results similar to real-time quantitative PCR (Fig. 3). CYP4F5 expression was observed to be much lower in liver and lungs than in kidneys or brain.

View larger version (81K): [in this window] [in a new window]   Fig. 3.   Northern blot hybridization showing CYP4F5 mRNA expression profile in various tissues of male and female rats. Thirty micrograms of total RNA were resolved on a 1% denaturing agarose gel, transferred to a nitrocellulose membrane, and blotted with 462-base pair 32P-labeled CYP4F5-specific DNA probe generated by using gene-specific primers.

Quantitative Comparison of CYP4Fs mRNA Expression in Male versus Female Rats

QRTPCR analysis of rat CYP4Fs expression showed clearly a higher expression in females compared with males (Fig. 4). CYP4F1 showed a 4.2-fold higher expression in female liver compared with males (P < 0.001). Also, CYP4F1 exhibited 2.4-fold more expression in brain (P < 0.01). No CYP4F1 was detected in lungs of either sex (Fig. 4A). CYP4F4 showed 58% higher expression (P < 0.05) in female liver and 56% more expression in female kidneys (P < 0.05) when compared with male rats (Fig. 4B).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Sex-dependent mRNA expression of individual CYP4Fs in rats. Quantitative analysis of CYP4Fs mRNA expression was done in male versus female rats. A, CYP4F1; B, CYP4F4; C, CYP4F5; and D, CYP4F6. Values are expressed as mean ± S.E. (n = 3). ***, P < 0.005; **, P < 0.01; *, P < 0.05 compared with male expression.

For CYP4F5, female rats showed 1.3-fold more expression in liver (P < 0.001) and 60% more expression in kidneys (P < 0.05) compared with their male counterparts (Fig. 4C).

Interestingly, CYP4F6 did not show any difference in expression in males versus females in liver but it showed 42% higher expression in female lungs (P < 0.05), 93% higher expression in female brain (P < 0.01), and 49% higher expression in female kidneys (P < 0.05) when compared with male rats (Fig. 4D).

Western Blot Analysis of CYP4F Protein Expression in Male versus Female Rats

From the Western blot, it is clearly evident that females show a considerably higher expression of CYP4Fs than males in liver, kidneys, and lungs. In brain, we do not observe any appreciable difference in CYP4F protein levels between the two sexes (Fig. 5).

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Western blot analysis of CYP4F expression in various tissues in male and female rats. Fifty micrograms of total microsomal proteins for each sample were run electrophoretically then transferred onto nitrocellulose membrane. Membranes were blotted and analyzed using CYP4F5 antibody as described.

Effects of Ovariectomy and Estrogen Treatment on CYP4F Expression

After ovariectomy, we observed 66 and 80% decrease of CYP4F1 expression from control females in liver and kidneys that was restored up to 75 and 63% upon estrogen treatment, respectively. Similarly after ovariectomy, CYP4F4 and CYP4F6 showed up to 45 and 42% loss of message in liver and up to 95 and 78% loss of message in kidneys, respectively. With exogenous estrogen treatment, this loss of expression of CYP4F4 and CYP4F6 due to ovariectomy was recovered up to 98 and 95% in liver and 24 and 60% in kidneys, respectively (Table 3). We did not observe any appreciable changes in CYP4F5 expression after ovariectomy or estrogen treatment in any of the tissues.

	Discussion

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To investigate whether CYP4Fs in rats show a tissue-dependent and gender-biased gene expression, we carried out specific quantitation of mRNA expressed for the four isoforms of CYP4F subfamily using fluorescence-based real-time PCR assays. Suppression of CYP4F1, CYP4F5, and CYP4F6 message in rat liver by administration of clofibrate is in complete accord with the reports published earlier (Chen and Hardwick, 1993; Kawashima et al., 1997). We do not detect any significant changes in CYP4F4 message after clofibrate treatment. This indicates that although being in the same P450 subfamily, all the members do not necessarily exhibit the same characteristic behavior in terms of their gene regulation.

Clofibrate, a peroxisomal proliferator, acts as a ligand for peroxisome proliferator-activated receptor  (PPAR), which then heterodimerizes with retinoid X receptor, binds to the peroxisome proliferator-responsive element in the 5'-flanking region of target genes such as CYP4A gene and activates its transcription (Muerhoff et al., 1992). As has been seen previously, we observe in this study that rat CYP4Fs show a response to clofibrate completely opposite that of CYP4A, i.e., repression of gene expression. Western blot data also demonstrate a clear decrease in CYP4F protein level after clofibrate treatment. Recently an isoform-specific response has been reported to clofibrate by CYP4Fs in mouse kidneys (Cui et al., 2001). Vu-Dac et al. (1998) have argued previously that fibrate administration can cause both positive (through peroxisome proliferator-responsive elements) and negative (through RevREs) regulation in related genes. Thus the molecular mechanisms underlying this phenomenon seem to be due to the sequence differences in the promoter region of these genes. Whether or not lack of suppressive response by CYP4F4 to clofibrate treatment reflects the same mechanism needs further characterization of the regulatory elements in its promoter region and identification of the transactivating factors that control its gene transcription. However, this difference in expression raises an issue about the functionality of CYP4F4 in the rat in terms of its catalytic activity when compared with other CYP4Fs.

Furthermore, the results from real-time PCR clearly show an isoform-specific CYP4F expression in various tissues. In this study among all CYP4Fs, we observe the highest expression for 4F1 in the liver, kidneys, and brain but no CYP4F1 is detected in the lungs of either sex. In the lungs, CYP4F6 exhibits the maximal gene expression. CYP4F6, which shows comparable expression to CYP4F1 in the brain, is also expressed abundantly in liver and kidneys. Although, CYP4F4 is not expressed in lungs and kidneys as much as it is expressed in liver, it is interesting to note that it can be induced up to 10-fold in lungs at 24 h after traumatic brain injury when compared with its control level (data not shown). It is evident from this study that rat CYP4Fs are regulated differentially in different tissues. This tissue distribution can be partially explained by tissue-specific distribution of nuclear receptors that are involved in their gene regulation i.e., PPAR is expressed in the order liver > kidney > heart compared with other extrahepatic tissues (Waxman, 1999).

Maximum expression of P450s is detected in the liver, as it is the main site for endogenous substrates and drug metabolism. It is, however, important to study P450s in extrahepatic tissues, since they are known to play important roles in organ function as well as pathophysiological states associated with it. Kidney CYP4Fs are important since P450-mediated arachidonic acid metabolites are implicated in the genesis of renal hypertension. Although the specific content of P450 in the brain is much lower than that in the liver and kidney, P450 plays a unique and important role in the brain (Strobel et al., 2001). To date, a role for P450s has been demonstrated in several brain functions including neurotransmission, neurosteroid metabolism, and neurotoxicity (Corpechot et al., 1983; Delaforge et al., 1995). The presence of CYP4Fs in brain may have important consequences regarding pharmacokinetics or neurotoxicity of xenobiotics as well as metabolism of neuroactive drugs for mental disorders. Previous studies in our laboratory have demonstrated that CYP4F4 and CYP4F5 can catalyze the in vitro metabolism of psychoactive drugs such as haloperidol, chlorpromazine, and imipramine (Kawashima et al., 1996; Boehme and Strobel, 1998).

Not only do we see a tissue-dependent expression of the CYP4F subfamily, we report for the first time that CYP4Fs are enriched in female rats. Interestingly, different female tissues show different preferences in terms of CYP4F expression when compared with their male counterparts. For example in liver, CYP4F1, CYP4F4, and CYP4F5 show significantly higher expression in females compared with males but CYP4F6 shows no difference between the sexes. In brain, lungs, and kidneys, CYP4F6 shows much higher expression in females than in males. CYP4F4 and CYP4F5 show a much higher expression in female liver and kidneys but not in brain or lungs. At this point in time we cannot speculate as to the significance of tissue-specific gender differences shown by CYP4Fs. A detailed study of each isoform in terms of its cis regulatory elements as well as its endogenous function, i.e., catalytic activity, is required to explain this tissue-dependent sex-specific behavior. Furthermore after ovariectomy, we observe a considerable decrease in CYP4F1, CYP4F4, and CYP4F6 message in liver and kidneys only. This loss of expression is partially recovered with estrogen treatment in both tissues. There is no significant change seen in CYP4F5 expression after ovariectomy or estrogen treatment. These results suggest a possible role for estrogen in CYP4F gene regulation. Additionally, the expanse of E₂ (estradiol) actions and specificity with which the multicellular pathways of estrogen receptors are activated in different target tissues could also affect the tissue specificities (Hall et al., 2001). To address whether these changes are direct or indirect effects of estrogen and/or its receptors, awaits experiments defining estrogen effects on isolated rat hepatocytes to study CYP4F expression or perhaps employing a mouse model system to define CYP4F expression in estrogen receptor null mice.

Sex-dependent expression of cytochromes P450 and their influence on drug metabolism and drug toxicity have been previously reported. Animal experiments, principally in rats, have revealed that expression of the number of P450 enzymes is sex-dependent (Waxman et al., 1995; Wang and Strobel, 1997). At the molecular level, it is reported that Stat5b is necessary to maintain sexual dimorphic response of P450 gene expression. It is shown that Stat5a-5b heterodimers regulate the expression of some female-specific gene expression (Davey et al., 1999). Kato and Yamazoe (1993) defined a P450 isoform as "sex-specific" if it had 10-fold or higher expression level in one sex compared with another but if the difference in expression level falls below 10-fold then it should be called "enriched" rather than sex-specific. Particular attention has been paid to members of CYP2 and CYP3 families and to their involvement in hepatic steroid hormone hydroxylation, which differs for each sex and is also subjected to postnatal developmental control (Waxman et al., 1995). Recently the CYP4A family, which is mainly involved in -hydroxylation of fatty acids, has been shown to be induced by lipopolysaccharide or the particulate irritant BaSO₄ in male but not in female rats (Mitchell et al., 2001). Also, CYP4a isoforms play a role in gender-specific hypertension in mice indicating an interrelationship between blood pressure regulation, sex hormones, and P450 -hydroxylases (Holla et al., 2001). Our findings with CYP4Fs showing higher expression in female than in male rat, in contradistinction to the male enriched expression of CYP4As, may be important because as a consequence of these sex differences in P450 gene expression, males and females may differ substantially in rates of drug metabolism.

The existence of sex differences in drug metabolism is not unique to rats. Such differences have been shown in humans (Hunt et al., 1992). Since we have shown that rat CYP4Fs are present more in females than in males and they can metabolize tricyclic antidepressants to the inactive 10-hydroxy forms (Kawashima et al., 1996), this might explain why men seem to respond consistently better to tricyclic antidepressants than women in both functional and neurologic disease.