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Cytochrome P450 1B1 mRNA measured in blood mononuclear cells by quantitative reverse transcription-PCR

^a Author for correspondence. Fax 39-0362383464; e-mail brambilla{at}desiolab.unimi.it.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cytochrome P450 (CYP) 1B1 activates polycyclic aromatic hydrocarbons and aryl aromatic hydrocarbons to carcinogens. We describe a competitive reverse transcription-PCR (RT-PCR) assay for the quantification of CYP1B1 mRNA in blood mononuclear cells (BMCs) by simultaneous RT and PCR amplification of cellular RNA with decreasing amounts of an internal standard. The concentration of CYP1B1 mRNA is derived from the ratio between the intensities of the bands corresponding to the amplified products. To reduce the variability of mRNA extraction efficiency, the measured amount of CYP1B1 has been calculated in relation to the ß-actin gene products. We measured CYP1B1 expression in the BMCs of 75 human subjects; no significant differences in the CYP1B1:ß-actin ratio were detected between women (range, 0.47–4.35; median, 2.0) and men (range, 0.72–3.85; median, 2.09). The analytical imprecision (CV) of duplicates was 14% (n = 25 pairs), and the intraindividual CV for two samples, 1 month apart, was 22% (n = 20). No significant differences were detected in smokers (n = 25; range, 0.77–3.55; median, 2.14) compared with nonsmokers (n = 50; range, 0.47–4.35; median, 2.0). The method has a wide range of linearity, good sensitivity and precision, and is suitable for studies of individual susceptibility as indicated by CYP1B1 expression in BMCs.

	Introduction

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Among members of the cytochrome P450 (CYP)¹ subfamilies, CYPs 1A1, 1A2, 2E1, and 3A4 are recognized to be the major forms involved in the activation in human liver and lung microsomes of most of the procarcinogens that we are exposed to every day via smoking, diet, and environment (1). Multiple forms of these CYPs contribute substantially to the metabolic activation of a number of procarcinogenic chemicals, which do not produce their biological effects per se but require metabolic activation to their proximate reactive species before they can interact with cellular macromolecules (2). Recently, a new member of the CYP subfamily 1, CYP1B1, was identified in rodent species and in humans (3)(4)(5)(6). CYP1B1 amino acid sequences among humans, rats, and mice are 80% similar; between subfamilies, CYP1B1 is equally similar, ~40%, to both CYP1A1 and 1A2 (7).

Studies conducted with yeast microsomal CYP1B1 recombinant showed that CYP1B1 is able to catalyze the activation of both polycyclic aromatic hydrocarbons and aryl amines: in fact very strong activation of 5-methylchrysene (one of the components of condensed tobacco smoke and a known carcinogen in experimental animals) to dihydrodiols was reported (8)(9). In mammary tissue, where it is expressed in high concentrations, CYP1B1 can give rise to activation of dihydrodiols (4)(8). Human exposure to compounds that are metabolized to dihydrodiols, particularly fluoranthene and benzo[c]phenanthrene, is extensive. It is possible that the stable dihydrodiol metabolites are formed in the liver and transported to mammary tissue.

Extrahepatic expression of CYP1B1 suggests that this enzyme may have an important role in activation of procarcinogens in target tissues in situ (such as endometrium, mammary tissue, or bronchial epithelial cells) or in tissues in direct contact with target tissues (such as alveolar macrophages) (8). CYP1B1 may be a major determinant of individual susceptibility to mammary cancer or, more broadly, to cancer.

CYP1B1 is also involved in the metabolism of steroid hormones, as suggested by its tissue distribution pattern (7)(10), and is inducible by adrenocorticotropin and peptide hormones (4)(10)(11)(12). In particular, human CYP1B1 is a catalytically efficient 17ß-estradiol 4-hydroxylase that is likely to participate in endocrine regulation and the toxicity of estrogens (13).

Because a method for precise measurement of CYP1B1 would be useful to detect its expression, we have developed a quantitative assay based on the competitive reverse transcription-PCR (competitive RT-PCR) applied to blood mononuclear cells (BMCs). Results suggest that this assay requires a minimal amount of biological sample, is reproducible, and is suitable for quantifying low levels of CYP1B1 expression and for studies of individual susceptibility.

	Materials and Methods

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subjects
Blood samples were obtained after informed consent from 75 healthy subjects, 42 women and 33 men, mean ages, 38 and 43 years, respectively. From 20 subjects, two samples were collected 1 month apart to determine intraindividual variability. To evaluate the effect of smoke on CYP1B1 expression, we compared the data of 25 smokers (9 women and 16 men) and 50 nonsmokers (33 women and 17 men). To assess smoking habits, we considered number of cigarettes (range, 5–35/day) and cotinine concentrations in serum. The study was performed in accordance with ethics standards.

cotinine analysis
Serum cotinine concentrations were determined using an HPLC procedure (14), and results were expressed in µg/L.

preparation of rna
Total RNA was extracted according to the method of Chomczynski and Sacchi (15) from 1 mL of suspension of mononuclear cells (mean, 7 x 10 cells/mL) isolated from blood by step gradient centrifugation on Histopaque.

preparation of internal standard
We inserted a 608-bp amplification product of CYP1B1 cDNA in pGEM; we then deleted a 98-bp portion from the CYP1B1 sequence by oligonucleotide-mediated mutagenesis (16)(17) as shown in Fig. 1 . The deleted vector was transcribed by the Riboprobe in vitro Transcription System (Promega). The 1B1 recombinant competitor (rc) RNA contained the same PCR primers as the cellular message but gave a shorter PCR product than the cellular CYP1B1 mRNA after amplification. The internal standard of ß-actin was prepared using the procedure described previously for CYP1B1.

View larger version (25K): [in this window] [in a new window]   Figure 1. Construction of the mimic CYP1B1 by oligonucleotide-mediated mutagenesis.

quantitative competitive rt-pcr
Competitive RT-PCR was performed as described by Gilland et al. (18) and Wang and Mark (19) and modified by Vanden Heuvel et al. (20). The scheme is summarized in Fig. 2 . From each RNA sample obtained from mononuclear cells, six equal aliquots (100 ng for CYP1B1 and 1 ng for ß-actin) were prepared, and a dilution series of the rcRNA internal standard was added to these aliquots.

View larger version (21K): [in this window] [in a new window]   Figure 2. Quantification of CYP1B1 mRNA using competitive RT-PCR. rcRNA internal standards were synthesized such that the RT-PCR products derived from the rcRNA could be differentiated from those of target RNA. Synthetic internal standards in decreasing amounts from 10 to 0.3 amol were added to 100-ng aliquots of total RNA before RT.

RT of RNA was performed in a final volume of 7.5 µL with reagents supplied with the First-Strand cDNA Synthesis Kit (Pharmacia Biotech). The samples were incubated at 37 °C for 60 min and reverse transcriptase-inactivated by heating at 95 °C for 5 min. PCR reagents were added to these cDNA samples to a final volume of 50 µL.

cyp1b1 pcr
The PCR reaction contained 1.5 mmol/L MgCl₂, 1.25 units of Taq polymerase, and 7.5 pmol of forward and reverse primers (forward primer: 5'GTG ATG CCC TGG CTG CAG 3'; reverse primer: 5' AAT CGA GCT GGA TCA AAG TTC 3'). Primers were chosen to include intervening sequences when DNA was amplified. cDNA yielded a 608-bp amplification product, whereas genomic DNA yielded no amplification product, as expected. The reactions were cycled 35 times through a 1-min denaturing step at 95 °C and a 1-min annealing and elongation step at 64 °C. A final 10-min elongation cycle at 60 °C followed. Aliquots of the PCR reaction were electrophoresed on 2% SeaKem-agarose gels, and PCR fragments were visualized by ethidium bromide staining. The amplified cellular fragment (target) was 608 bp, and the mimic was 510 bp. The 98-bp internal deletion within the CYP1B1 sequence accounted for the difference between the mimic and cellular PCR amplification products.

ß-actin pcr
The PCR reaction contained 2 mmol/L MgCl₂, 1.25 units of Taq polymerase, and 7.5 pmol of forward and reverse primers (forward primer: 5' GTG CGT GAC ATT AAG GAG AAG 3'; reverse primer: 5' GAA GGT AGT TTC GTG GAT GC 3'). The reactions were cycled as described previously for CYP1B1, with temperatures of 95 °C and 60 °C, respectively. A final 10-min elongation cycle at 60 °C followed. Aliquots of the PCR reaction were electrophoresed on 3% SeaKem-agarose gels, and PCR fragments were visualized by ethidium bromide staining. The amplified cellular fragment (target) was 213 bp, and the mimic was 171 bp. The 42-bp internal deletion within the ß-actin sequence accounted for the difference between the mimic and cellular PCR amplification products.

image analysis
The initial concentration of CYP1B1 mRNA in the specimens was obtained by comparison of amplification products at different rcRNA:target RNA ratios and corresponded to the amounts of rcRNA at which target and rcRNA amplification products were equal. The fluorescence value obtained by integrating the intensity over the areas of the two bands corresponding to cellular (target) and competitor (mimic) amplification products was measured by the Image Master system (Pharmacia). The ratio of the fluorescence values of the two bands, with correction factors of 1.19 for CYP1B1 and 1.24 for ß-actin accounting for the difference in the sizes of the mimic and the target, provided the basis of quantification. The target:mimic ratios were plotted on a decimal scale against the mimic amounts in each PCR amplification reaction mixture. The amounts of 1B1 mRNA in the sample was calculated from the plotted curve, where the target:mimic ratio was 1. The same procedure was used to quantify the amount of ß-actin. Because the extraction yield can vary, the amount of CYP1B1 mRNA was related to the amount of ß-actin mRNA quantified on the same RNA preparation, and results were expressed as the ratio of CYP1B1 mRNA (amol x 10) to ß-actin mRNA (amol) in 100 ng of total RNA from BMCs.

	Results

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A constant amount of cellular RNA (100 ng) was probed with decreasing amounts of synthetic 1B1 rcRNA (from 10 to 0.3 amol). An example of CYP1B1 RNA agarose gel electrophoresis is shown in Fig. 3 ; the PCR product corresponding to the target mRNA (608-bp band) can be resolved easily from that of the mimic rcRNA (510-bp band). An example of the curve obtained by plotting the target:mimic ratios against the mimic amounts in each PCR amplification reaction mixture is given in Fig. 4 .

View larger version (83K): [in this window] [in a new window]   Figure 3. Ethidium bromide-stained agarose gel showing quantitation of CYP1B1 mRNA in human mononuclear cells. Dilutions of synthetic internal standards (10–0.3 amol/tube) were added to a constant amount (100 ng/tube) of patient RNA. The 608-bp bands correspond to CYP1B1 mRNA; the 510-bp bands correspond to the synthetic internal standard.

View larger version (12K): [in this window] [in a new window]   Figure 4. Quantitative analysis of CYP1B1 mRNA in human BMCs. The gels were analyzed by computer imaging, and the ratio of target to synthetic internal standard was calculated. Data are plotted on a decimal scale (y axis) vs the amounts of synthetic internal standard in the reaction tubes (x axis).

exponential pcr amplification of cyp1b1 and ß-actin mRNA
To determine the exponential range of PCR amplification for CYP1B1 mRNA and the synthetic internal standard, we co-reverse-transcribed 100 ng of total cellular RNA obtained from mononuclear cells for CYP1B1 and 2.5 amol of synthetic internal standard into first-strand cDNAs. The cDNA products were then amplified for different numbers (25–40) of PCR cycles. PCR products were size-fractionated through a 2% agarose gel, stained with ethidium bromide, and quantified. The results obtained from the exponential range experiment are shown in Fig. 5 . The relative amounts of the two amplified products stayed identical throughout the PCR amplification, even after 35 cycles, when the plateau phase was reached. The same procedure was followed for ß-actin, using 1 ng of total cellular RNA and 500 amol of synthetic internal standard (Fig. 6 ).

View larger version (18K): [in this window] [in a new window]   Figure 5. Exponential range of amplification for 100 ng of BMC total RNA and 2.5 amol of CYP1B1 synthetic internal standard.

View larger version (19K): [in this window] [in a new window]   Figure 6. Exponential range of amplification for 1 ng of BMC total RNA and 500 amol of ß-actin synthetic internal standard.

linearity
We probed a constant amount (2.5 amol) of CYP1B1 internal standard with increasing amounts of total RNA from BMCs in the presence of specific primers designed for RT-PCR. After 35 amplification cycles, the products were assayed to evaluate the linearity between the amount of initial cellular CYP1B1 and the PCR product. A linear response (r = 0.96; Fig. 7 ) was observed in a 0.1–5.4 amol range. The results obtained in the subjects were within the observed interval of linearity.

View larger version (18K): [in this window] [in a new window]   Figure 7. Linearity and sensitivity of competitive RT-PCR. CYP1B1, total RNA (250, 190, 130, 100, 50, 10, or 5 ng) from BMCs was probed with a constant amount (2.5 amol) of synthetic internal standard. ß-actin, total RNA (2.5, 1.9, 1.3, 1.0, 0.5, 0.1, or 0.05 ng) from BMCs was probed with a constant amount (500 amol) of synthetic internal standard. Samples were amplified in a 35-cycle PCR, and the amplification products were resolved on a 2% agarose gel. After ethidium bromide staining, the target:mimic ratios were measured and plotted vs target mRNA amounts.

We determined the sensitivity of the assay by using decreasing amounts (250, 190, 130, 100, 50, 10, and 5 ng) of total cellular RNA; the assay detected as little as 0.11 amol of CYP1B1 mRNA (Fig. 7 ). The same procedure was followed for ß-actin, using 500 amol of synthetic internal standard (Fig. 7 ). A linear response (r = 0.95; Fig. 7 ) was observed in a 23–1153 amol range. The results obtained in the subjects were within the observed interval of linearity.

analytical variability
The CV, calculated from results of duplicate RT-PCRs on the same RNA preparation of 25 subjects, was 16% for CYP1B1 and 16% for ß-actin. These variations originate from cDNA synthesis, PCR amplification, image scanning, and processing steps. For the CYP1B1:ß-actin ratio the CV was 14%.

intraindividual variability
The intraindividual CV for the CYP1B1:ß-actin index, evaluated in 20 subjects by comparison of two consecutive samples (collected 1 month apart), was 22%. Values for the ratio ranged from 0.47 to 3.85 (median, 2.38) and from 0.59 to 4.33 (median, 2.60) for the first and second determinations, respectively.

interindividual variability
Interindividual variability for the CYP1B1:ß-actin index was calculated considering the first result available for each subject; values ranged from 0.47 to 4.35 in women (0.70, 2.00, and 4.27 being the 2.5, 50, and 97.5 percentiles, respectively) and from 0.72 to 3.85 in men (0.76, 2.09, and 3.68 being the 2.5, 50, and 97.5, percentiles, respectively).

effect of smoking
We tested 25 smokers and 50 nonsmokers (serum cotinine, 20–696 µg/L), to evaluate the effect of smoke on the CYP1B1 gene: the results obtained are reported in Fig. 8 and do not show significant differences between smokers and nonsmokers. In smokers, the CYP1B1:ß-actin ratio was 0.99–3.55 (median, 2.27) in women and 0.77–3.39 (median, 2.14) in men. In nonsmokers, the ratio was 0.47–4.35 (median, 1.97) in women and 0.72–3.85 (median, 2.08) in men. We did not observe any correlation between CYP1B1 expression and smoking, as assessed by cotinine concentrations (Fig. 9 )

View larger version (12K): [in this window] [in a new window]   Figure 8. Distribution of the ratio of CYP1B1 (amol x 105) to ß-actin (amol) in two groups of subjects: 25 smokers and 50 nonsmokers. , smokers; , nonsmokers; (—–) median of each group.

View larger version (10K): [in this window] [in a new window]   Figure 9. Correlation between the ratio of CYP1B1 (amol x 105) and ß-actin (amol) and cotinine concentrations (µg/L) in 25 smokers.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
A reliable RT-PCR method for quantitative measurements needs to be sensitive, reproducible, and precise; therefore, several aspects and potential sources of variability must be considered. We have developed an assay for the quantification of CYP1B1 mRNA expression by PCR that requires a small amount of biological sample, avoids use of radioactive labels, and offers higher sensitivity than conventional RNA hybridization methods such as Northern blot.

Because RNA extraction could greatly contribute to analytical variability, we decided to keep RNA extraction under control by relating CYP1B1 to ß-actin mRNA.

The test has been designed to have the internal standard covering both RT and PCR, and it seems to be more accurate than tests already published, which control only the amplification step (21). The synthetic CYP1B1 RNA designed as an internal standard for this assay controls for the efficiency of both the RT reaction and the PCR. This synthetic RNA has the same primer sequences as the target mRNA, so that there are no differences in primer efficiencies, and the difference in size between mimic and target (98 nucleotides) allows the separation of the corresponding amplification products in an agarose gel. In addition, false-positive results attributable to the amplification of DNA potentially present in the sample after the RNA extraction process are avoided with an appropriate choice of primers sequences in two different exons.

The wide linearity range indicates that the assay can be used reliably with samples having a wide range of CYP1B1 mRNA concentrations, with a lower detection limit of 0.11 amol of CYP1B1 mRNA.

The variability of the principal components of the test was studied. The analytical variability of RT-PCR from the same total RNA preparation was good and in agreement with data reported to date for RT-PCR of CYP1A1 and MDR1 expression (13)(22). A substantial overlap in the expression of CYP1B1 mRNA was found in nonsmokers and smokers, indicating that smoking does not seem to be a potent inducer of CYP1B1 transcription, at least in mononuclear cells. This result is in agreement with the observation that CYP1B1 is not higher in the placentas of women who smoke than in women who do not smoke (21). However, an increased expression of CYP1B1 was observed in the bronchial epithelial cells of smokers compared with nonsmokers, indicating that CYP1B1 is induced by smoke and polyaromatic hydrocarbons (23) at a concentration achievable in the lung but not at those obtained in the blood of smokers with serum cotinine concentrations up to about 700 µg/L. These observations suggest that CYP1B1 expression might be tissue-dependent, as has already been shown for other members of the cytochrome P450 subfamilies. The apparent insensitivity of CYP1B1 to smoking will be of help when it is used as a cancer susceptibility marker because lifestyle is not expected to greatly affect CYP1B1 expression in BMCs. Therefore, CYP1B1 mRNA concentrations seem to be indicators of constitutive individual expression. Our data indicate a great CYP1B1 interindividual variability, which may be based on genetic polymorphism and can be exploited in susceptibility studies.

In conclusion, competitive RT-PCR is a reliable and accurate method for evaluating CYP1B1 expression and is a good candidate for assessing the association of CYP1B1 expression and cancer susceptibility because it has low analytical variability, its interindividual variability is greater than its intraindividual variability, and it is relatively insensitive to smoking habits.

	Acknowledgments

 
This work was supported by grant no. 465/96 from Regione Lombardia, Milan, Italy. We thank Antonio Musio for helpful discussion and Pierangela Molteni, Raffaella Sala, and Elisabetta Gonella for expert technical assistance.

	Footnotes

 
University Department of Clinical Pathology, Hospital of Desio, Via Mazzini 1, 20033 Desio, Milan, Italy.

¹ Nonstandard abbreviations: CYP, cytochrome P450; RT, reverse transcription; BMC, blood mononuclear cell; and rc, recombinant competitor.

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