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Original Studies |
Department of Medical Nutrition (H.H., T.R., M.M., M.W., J.-Å.G.), Center for Nutrition and Toxicology (H.H., M.W.), and Department of Surgery (ER), Karolinska Institute at Huddinge University Hospital, Novum, S-141 86 Huddinge, Sweden
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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-hydroxylation of fatty acids (families 5, 7, 8, 11,
17, 19, 21, 24, 27, 51, and 4) and those that facilitate elimination of
xenobiotics and steroids from the body (families 1, 2, and 3) (1).
These are well characterized functions in tissues where the levels are
high, such as the liver, adrenals, and gonads (2, 3). In other tissues
where the P450 content is lower, tissue or cell-specific functions have
been demonstrated for P450 isozymes. In the kidney, blood flow is
regulated by P450-catalyzed metabolites of arachidonic acid (4, 5). In
the brain and prostate, where the P450 content is even lower,
metabolism of GABAA receptor-active steroids and
elimination of dihydrotesterone are important tissue-specific P450
functions (6, 7). A list of the human P450 isoforms investigated in
this study is shown in Table 1
. The P450
content of breast tissue in rats is approximately 0.1% of that in the
liver, and a number of hepatic forms have been detected (8). Several
specific forms of P450 are regulated in the breast, as a function of
age and hormone status of the rats. What remains to be determined is
whether this small amount of P450 is highly localized in a
subpopulation of cells in the rat breast and whether these enzymes play
some physiological, pharmacological, or toxicological role in the
breast. Several forms of P450, such as CYP1A1 and CYP1B1, which are
expressed in the breast, can metabolize estradiol and may influence the
hormone sensitivity of the cells which harbor them (9). In the rat,
breast P450 is inducible also by aryl hydrocarbon receptor ligands and
by pregnenolone-16
-carbonitrile (8, 10). The influence of dietary
and xenobiotic P450 inducers on the sensitivity of the breast to
hormones and hormone antagonists remains to be investigated. Little is
known about the levels of P450 and the isozyme profile in human breast.
RT-PCR has been used to detect the messenger RNA (mRNA) of CYP1A1,
CYP1B1, CYP2C, CYP3A, and CYP2D6 in human breast tumors and in normal
human breast (11). So far there is no information as to whether the
transcripts are translated in the breast. Previous studies have
attempted to characterize the breast P450 profile by Western blotting
of breast microsomes (12, 13, 14). Because of the low level of these
enzymes, large amounts of microsomes have to be loaded on the gels, and
this results in a poor signal-to-noise ratio. We have overcome this
problem by isolating P450 from a total membrane fraction. The present
study was designed to measure the P450 content of the human breast and
to characterize the P450 profile at the RNA and protein levels and,
when possible, to measure catalytic activity.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Breast tissue (110–150 g) was obtained at reduction mammaplasty in healthy and normal-weight women with macromastia. All patients completed a questionnaire concerning factors that may influence the expression of CYP, such as medication, smoking, alcohol consumption, parity, and diet. The liver biopsy specimen (1 g) was taken at cholecystectomy. Informed consent was obtained from all subjects, and the study was approved by the Ethics Committee at Huddinge University Hospital.
Chemicals
Thermus aquaticus (Taq) polymerase, avian myeloblastosis virus RT, ribonuclease inhibitor, oligo(dT), and deoxynucleotide triphosphates were purchased from Promega (Madison, WI). Horseradish peroxidase conjugated streptavidin (HRP-streptavidin) was obtained from Chemicon (Stockholm, Sweden), and an enhanced chemiluminescence detection kit was purchased from Amersham (Little Chalfont, England). Nitrocellulose membranes were obtained from Schleicher & Schuell (Dassel, Germany). Coumarin was obtained from ICN Pharmaceuticals (Costa Mesa, CA), and 7-hydroxycoumarin was obtained from EGA Chemie (Steinheim, Germany). All other chemicals were of analytical grade obtained from either Kebo Lab AB (Stockholm, Sweden), Sigma Chemical Company (St. Louis, MO), or Merck AG (Darmstadt, Germany).
Tissues
Histopathology of the breast samples showed normal breast, rich in fat. The samples were immediately frozen in liquid nitrogen and stored at -80 C. Samples from mammary ductal carcinomas were a kind gift from Dr. Martin Bäckdahl (Department of Surgery, Karolinska Institute). A piece (1 g) of epithelial rich tissue was dissected from one additional mammaplastie sample.
Preparation of total membrane fractions
The frozen tissue was crushed with a mortar and pestle in liquid nitrogen and transferred to homogenization buffer, composed of 100 mmol/L Tris-HCl (pH 7.4), 20% glycerol, 150 mmol/L KCl, 0.2 µmol/L dithiothreitol, and 1 mmol/L EDTA. Phenylmethylsulfonyl fluoride (0.2 mmol/L) was added before homogenization with a Polytron homogenizer (PT 3000; Kinematica, Lucerne, Switzerland). The homogenate was filtered through a piece of gauze. Total membrane fractions were obtained by centrifugation of the homogenate at 105,000 g for 1 h at 4 C.
Preparation of microsomes
The tissues were homogenized as described, and microsomes were obtained by differential centrifugation at 9,000 g for 30 min, followed by a centrifugation at 105,000 g for 1 h at 4 C. Microsomes were resuspended in 50 mmol/L sodium phosphate buffer (pH 7.4) containing 1 mmol/L EDTA and 20% glycerol. The protein content of the microsomes was measured according to Lowry et al. (15) using BSA as reference.
Purification of P450 by hydrophobic chromatography
P450 was partially purified as described previously (8, 16, 17, 18). In short, the total membrane fraction was resuspended in solubilization buffer, composed of 50 mmol/L potassium phosphate buffer (pH 7.5), 20% glycerol, 0.5% (w/v) sodium cholate, 0.2% Emulgen 911, and 0.2 mmol/L EDTA. Phenylmethylsulfonyl fluoride (0.2 mmol/L) was added before the solubilization. After 16 h, insoluble material was sedimented by centrifugation at 105,000 g for 1 h. The solubilized material was diluted 4-fold with 50 mmol/L potassium phosphate buffer containing 20% glycerol, and chromatographed on a column (5 x 2.5 cm) of p-chloroamphetamine coupled Sepharose. The column was washed with 100 mL (1:4 diluted) solubilization buffer, and P450 was eluted with solubilization buffer. P450 was quantitated according to Omura et al. (19) with a Hitachi U-3200 spectrophotometer, and the fractions containing P450 were pooled and, if necessary, concentrated by dialysis against Aquacide II (Calbiochem-Novabiochem Corporation, La Jolla, CA). The purified P450 fractions were used for Western blot analysis.
Western blotting
Proteins in the isolated P450 fraction were precipitated with chloroform/methanol and separated by gel electrophoresis according to Laemmli, with a 9% separating gel (20). Five picomoles of breast P450 was loaded per 0.3-cm well. Transfer of the proteins to a nitrocellulose filter was performed as described by Towbin et al. (21). The filter was blocked in TBS [20 mmol/L Tris-HCl (pH 7.5) and 150 mmol/L NaCl] containing 0.2% Nonidet P-40 and 10% fat-free milk, and was rinsed in TBS and incubated with primary antibodies, and a secondary IgG coupled to horseradish peroxidase. The protein antibody complex was visualized by enhanced chemiluminescence.
Antibodies
Generous gifts of antibodies were obtained as follows: rabbit IgG’s raised against peptides from human CYP1A1 and CYP1A2, from Dr. R.J. Edwards (Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, England) (22, 23); rabbit IgG against rat CYP2A1, also recognizing human CYP2A6, from Dr. F.J. Gonzalez (National Cancer Institute, National Institutes of Health, Bethesda, NMD); sheep IgG against rat CYP4A isozymes, from Dr. G. Gibson, (University of Surrey, Guildford, UK) (24); mouse IgG against human CYP19 (aromatase), from Dr. E. R. Simpson (University of Texas, Dallas, TX) (25); and mouse IgG specific to human CYP2D6, from Dr U. Meyer (Biocenter of the University of Basle, Basle, Switzerland). Rabbit IgG against rat CYP2E1 was obtained from Oxygene Dallas (Dallas, TX) (26). Rabbit IgG’s against rat CYP2B1/2 and CYP3A isozymes were obtained from Human Biologics (Phoenix, Arizona). Microsomes from cell lines expressing human CYP1A1, CYP1A2, CYP2D6, CYP2B6, CYP2A6, CYP3A4, CYP2C8, and CYP2C9 were obtained from Gentest Corporation (Woburn, MA) and used as positive controls and also to test crossreactivity of the antibodies.
Coumarin-7-hydroxylation
Coumarin-7-hydroxylation was performed essentially as described by Aitio et al. (27). The reaction mixture contained 1 mg microsomal protein, 10 nmol/L coumarin, and an nicotinamide adenine dinucleotide phosphate (reduced form)-generating system (5 mmol/L NADP, 0.6 U isocitrate dehydrogenase, 5 mmol/L isocitrate, and 15 mmol/L MgCl2) in a total vol of 1 mL 0.05 mmol/L Tris-HCl buffer, pH 7.5. The incubation was performed in 1.5-mL Eppendorf tubes. Incubations were started after 3-min preincubation at 37 C, by the addition of the nicotinamide adenine dinucleotide phosphate (reduced form)-generating system. After 20 min, the reactions were quenched by addition of 25 µL perchloric acid, and the protein was removed by centrifugation at 1,500 g. The supernatant was extracted with 3 mL chloroform, by vortexing for 30 sec. The layers were separated by centrifugation at 1,500 g for 10 min; 2.5 mL of 30 mmol/L sodium borate, pH 9.3, was added to the organic phase; and the tube was vortexed before centrifugation for 5 min at 1,500 g. The concentration of 7-hydroxycoumarin was measured fluorometrically using a Shimadzu RF 510 spectrofluorometer set at an excitation wavelength of 390 nm and an emission wavelength of 440 nm.
Isolation of total RNA
Total RNA was isolated by the guanidinium thiocyanate single-step method described by Chromczynski et al. (28).
Reverse transcription
Five micrograms of RNA was denatured at 94 C for 2 min. The RNA was added to PCR buffer, consisting of 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 7.5 mmol/L MgCl2, and 0.1% Triton X-100, together with 1.0 mmol/L of each deoxynucleotide triphosphate, 2.2 µmol/L oligo(dT), 50 U ribonuclease inhibitor, and 20 U avian myeloblastosis virus RT in a final vol of 50 µL. The reaction was allowed to proceed for 1 h at 42 C, after which the enzyme was inactivated at 95 C for 10 min. The reverse-transcribed RNA was stored at -20 C. Two negative controls were included (one without the RNA template and one without the RT).
Oligonucleotide primers and probes
Oligonucleotides were synthesized on an Applied Biosystems 380B
DNA synthesizer, and the internal probes were end-labeled with biotin
and purified by high-performance liquid chromatography (Cyber Gene AB,
Huddinge, Sweden). Table 2 shows primers
and probes used for PCR and Southern blot analysis. Conserved
oligonucleotide primers were designed for the P450 subfamilies 1A, 2C,
and 3A, and the internal probe of 3A was conserved for CYP3A3, CYP3A4,
CYP3A5, and CYP3A7. Specific oligonucleotide primers were designed for
CYP1B1, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP4A11, CYP19, and ß-actin;
and specific biotinylated probes were designed for CYP1A1, CYP1A2,
CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP18, CYP9/19, CYP2D6, CYP2E1,
CYP4A11, CYP19, and ß-actin. In addition, conserved primers and
probes for CYP2C8, CYP2C9, CYP2C10, CYP2C17, CYP2C18, and CYP2C19 were
used according to Huang et al. (11). Leptin primers and
probe were synthesized according to Isse et al. (29).
Beta-actin was used as a positive control.
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The PCR was run on a Gene Amp PCR system 2400 (Perkin Elmer, Sundbyborg, Sweden). The PCR reaction consisted of 5 µL complementary DNA (cDNA), PCR buffer, 0.2 mmol/L of each deoxynucleotide triphosphate, 1 µmol/L of the specific primer pair or 0.12 µmol/L of the primers amplifying the ß-actin gene, and 1 U Taq-polymerase, in a total vol of 100 µL. The conditions used for amplification were: 30 sec at 94 C, 30 sec at 50 C, and 1 min at 72 C, for 30 cycles. The PCR-mixture, without the polymerase and cDNA, was used as a negative control, together with the negative controls from the reverse transcription reaction. Amplification of ß-actin was performed for each RT-reaction as a positive control. The PCR products were stored at -20 C.
Southern blot analysis
The PCR products obtained were loaded on a 1.5% agarose gel. The DNA was denatured and then neutralized by soaking the gel first in 0.5 mol/L NaOH containing 1.5 mol/L NaCl, for 45 min, and then in 1 mol/L Tris-HCl (pH 7.4), containing 1.5 mol/L NaCl, for 30 min. The DNA was transferred to nitrocellulose by the capillary transfer method using standard saline citrate (SSC) transfer buffer (20 x SSC containing 150 mmol/L NaCl and 15 mmol/L sodium citrate). To fix the DNA, the membrane was baked for 2 h at 80 C. The membrane was prehybridized in 6 x SSC, 1% SDS, 1 mg/mL ficoll, 1 mg/mL polyvinyl pyrrolidone, 1 mg/mL of BSA, 0.2 mg/mL of denatured fragmented salmon sperm DNA, and 5 µmol/L EDTA for 4 h at 65 C. Hybridization was carried out under the same conditions with 100 ng/mL biotinylated probe for 16 h at 42 C. The membrane was washed, for 1–2 h at 42 C and for 30 min at 55 C, in 6 x SSC.
Detection of PCR products
Membranes hybridized with biotinylated probes were incubated for 1 h at 37 C in PBS containing 137 mmol/L NaCl, 2.7 mmol/L KCl, 4.3 mmol/L NaHPO4, 1.4 mmol/L KH2PO4, 0.2% Nonidet P40, and 3% BSA. The membranes were then incubated for 1 h at 37 C in a solution of 1.0 ug/mL of HRP-streptavidin in PBS containing 0.2% Nonidet P40. Unbound HRP streptavidin was removed by washing three times in 10-min changes in PBS containing 0.2% Nonidet P40. The DNA/HRP-streptavidin complex was visualized by enhanced chemiluminescence.
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Results |
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The presence of interfering chromophores made it difficult to
measure P450 in microsomal and total membrane fractions from human
breast tissue. The P450 in these samples could be quantitated only
after chromatography of the solubilized membranes over a hydrophobic
column. This method has been used previously to isolate P450 from the
rat breast and other extrahepatic tissues and has been shown to give
high recovery of P450 from total membrane fractions (8, 16, 17, 18). The
yield of P450 varied between 0.5 and 7.8 pmol per gram of tissue.
Individual values are given in Table 3,
together with information on the patients regarding age, parity,
smoking, and contraceptive use. A representative P450 spectrum of
sample 18 is shown in Fig. 1. No attempt
was made to separate fat from epithelium, but clearly, the tissue was
composed mostly of fat.
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RT-PCR was used to confirm the identity of the investigated P450
isozymes. Table 4 shows a summary of the
15 reduction samples that were analyzed by Western blotting and RT-PCR.
In the case of sample number 16, the amount of P450 was limited; and
only CYP2E1, CYP3A, CYP2B6, CYP2D6, and P450 19 were analyzed by
Western blot. Antibodies recognizing human CYP1A1, human CYP1A2, rat
CYP2A1, rat CYP2B1/2, rat CYP3A, human CYP2D6, rat CYP2E1, rat CYP4A1,
and human CYP2C10 and CYP19 (aromatase) were used for immunoblotting.
The standards used were either expressed P450 isoforms (Gentest
microsomes) (0.5 pmol per lane), human liver microsomes (2.5 pmol per
lane), or in the case of aromatase, rat ovary microsomes (5 pmol per
lane). The molecular mass of all investigated P450 isoforms was
approximately 50 kDa. No suitable antibody was available for CYP1B1.
Specificity of the antibodies was checked with microsomes expressing
human P450 isoforms (Gentest). There was no crossreactivity detected
with the used antibodies on microsomes expressing human CYP1A1, CYP1A2,
CYP2D6, CYP2B6, CYP2A6, CYP3A4, CYP2C8, and CYP2C9.
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. CYP1A2 could not be detected in
any sample by RT-PCR or Western blot analysis.
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and 4) were detected by RT-PCR in all
reduction samples, the carcinoma samples, and the epithelial sample,
when 10% of the PCR reaction was loaded. Sample number 26 was the only
breast where 100% of the PCR reaction was needed for detection of CYP
2E1. For all other P450 forms, 100% of the PCR reaction was used in
the Southern analysis of the reduction samples. CYP2E1 was the most
easily detected P450 on Western blots and was detected in all reduction
samples. From the intensity of the signals in the samples, compared
with the standard, an estimate can be made about the specific isozyme
content of the breast. If samples 23 and 25 are compared with the
CYP2E1 standard (0.5 pmol), it can be estimated that CYP2E1 represents
approximately 10% and 50% of the P450 loaded onto the gels in samples
23 and 25, respectively. The total P450 content in sample 23 was 1.1
pmol/g tissue and 7.8 pmol/g tissue in sample 25. Thus, per gram of
tissue, sample 25 has approximately 30 times more CYP2E1 than sample
23.
CYP19 and CYP4A11 (Figs. 5 and 6) also were easily detected on Western
blots and could be detected in 11 and 12 reduction samples,
respectively. There was a good correlation with mRNA detection. The
mRNAs of CYP19 and CYP4A11 were detected in 14 reduction samples, CYP19
in all tumor samples, and CYP4A11 in 2 tumor samples. The mRNA of CYP19
was detected in the epithelial sample, whereas CYP4A11 was the only
isoform that was not detected in the epithelial sample. P450s that were
less widespread in the reduction samples were CYP2A6 (Fig. 7), CYP3A (Fig. 8), CYP2B6 (Fig. 9), and CYP1A1 (Fig. 10). These forms were also variably
expressed in the tumor samples, but they were detected in the
epithelial sample. CYP2A6 was detected on Western blots in 13 reduction
samples. In 3 of these patients, there was a band for CYP2A6 on Western
analysis but no detectable mRNA. The antibody is known to recognize
several CYP2A forms in the rat, and it is possible that it recognizes
some unknown P450 protein also in the human. CYP3A was detected by
RT-PCR in 11 reduction samples with the use of conserved primers and
probe. Although the CYP3A antibody recognized expressed human CYP3A4,
no bands of the correct size were detected in any breast sample (data
not shown). It is not known whether this antibody recognizes other
human CYP3A isoforms. The mRNA of CYP2B6 was easily detected in the
tumor samples when 10% of the PCR reaction was loaded, whereas the
detection in the reduction samples and the epithelial sample required
100% of the reaction (Fig. 9). CYP2B6 protein was detected only in 1
of the reduction samples. The absence of CYP2B6 protein, despite
detection of mRNA, may indicate posttranscriptional regulation of this
isoform, or it may reflect the difference in sensitivity between RT-PCR
and Western blot analysis.
The mRNAs of CYP1A1 and CYP2D6 were in the order of 10-fold lower
than CYP2E1. In the case of CYP2D6 (Fig. 11), an additional fragment of larger
size was detected in two carcinoma samples, whereas in the epithelial
sample, only the unspliced CYP2D6 fragment could be detected. As shown
in Figs. 10 and 11, detection of CYP1A1 and CYP2D6 protein on Western
blots was difficult. Bands of the correct size were present, but there
was a high background in most samples.
By RT-PCR analysis (Fig. 12), a CYP2C
fragment was detected in all reduction samples, the carcinoma samples
and the epithelial sample using the consensus primers and probe. The
CYP2C mRNA signal could not be fully accounted for by the known CYP2C
isozymes. CYP2C8 and CYP2C9/19 were present in only two and six
reduction samples, respectively. A CYP2C18 probe recognized a band of
the right size in the liver but did not detect any mRNA in the human
breast samples (data not shown). The antibody against human CYP2C10
recognized expressed CYP2C8 and CYP2C9 and produced a strong band in
human liver microsomes. It is not known whether this antibody
recognizes other CYP2C isoforms. No bands of the correct size were
detected in the breast samples (data not shown). This result is
compatible with the RT-PCR result, which also indicates that the CYP2C
in the breast may be a novel member of this subfamily.
Catalytic activities in human breast microsomes
CYP2A6-specific 7-hydroxylation of coumarin was measured in four
human breast samples to determine whether signals detected on Western
blots are indicative of functionally active enzymes. This catalytic
activity is characteristic of CYP2A6 (30, 31). The formation of
7-hydroxycoumarin was linear with time, up to 20 min, and with protein
concentration, up to 1 mg, using mouse and human liver microsomes (as
shown in Fig. 13). The catalytic
activity measured in four human breast samples varied between 1.6 and 4
pmol/mg·min of microsomal protein (Fig. 13). The yield of microsomal
protein was approximately 0.6 mg per gram of tissue. In human liver
microsomes, the catalytic activity was approximately 24 nmol/mg·min.
The value in the breast, approximately 0.01 percent of that in the
human liver, is compatible with the P450 contents of the tissue and the
small contribution of CYP2A6 to the total breast P450 contents.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Aromatase has been localized to breast adipose stroma and has been suggested to play a significant role in postmenopausal breast cancer (32). In the present study, it was not possible to assess the amount of stroma vs. epithelium in the samples. To address the question of whether samples from reduction mammoplasty, which are largely composed of fat, have any relevance to the overall content of P450 in the breast, a comparison was made between P450 profiles in reduction samples, mammary ductal carcinomas, and dissected normal epithelium. Surprisingly, tumors, reduction samples, and normal epithelium had several P450s in common; the exception was CYP4A11, which was not detected in the epithelial enriched sample. The data indicates that CYP4A11 may be localized in the fat. However, firm conclusions can not be made regarding this issue until the P450 profile in fat has been further studied. Leptin, a hormone produced exclusively by adipocytes, shows regional differences in mRNA expression in adipose tissue (33), and little is known about its regulation in human breast fat. In this study, leptin was used to detect the presence of fat in the breast samples. The reason for the absence of leptin in one of the reduction samples, despite the presence of visible fat, is not clear. One interesting possibility, which is being investigated, may be the existence of a defect in the leptin gene in this patient. In future studies, assessment of cellularity may be achieved by measuring the amount of DNA.
Of the P450s identified, there was no obvious correlation to age, smoking, parity, or use of hormones. CYP1B1 was constitutive in all samples, and CYP1A1 was present in only a few. Both enzymes are known to be regulated by the TCDD receptor, but there was no apparent coregulation of these two enzymes in the breast. Those individuals who had high levels of CYP1A1 mRNA did not have a corresponding increase in CYP1B1 mRNA. Because several individuals with CYP1A1 expression were nonsmokers, the presence of CYP1A1 in some breasts is not related to cigarette smoking. It remains to be determined whether it is related to exposure to TCDD receptor ligands in the breast. Both CYP1A1 and CYP1B1 can metabolize estradiol to catecholestrogens (34, 35). High levels of these enzymes in cells may cause rapid metabolism of estrogen and may be one mechanism by which sensitivity to estrogens in individual cells may be regulated. This mechanism has been demonstrated in MCF7 cells in culture (9).
Unlike the situation with CYP1B1 and CYP1A1, CYP2E1 and CYP4A11 both seemed to be constitutive in the breast. In the rat kidney, these two enzymes are coinduced in the diabetic state by acetone and ketone bodies (36) and have, as their substrates, acetone and fatty acids (37, 38). CYP4A is regulated at the transcriptional level by the peroxisome proliferator-activated receptor (39), whereas ethanol and acetone increase the CYP2E1 content of a cell by stabilization of the protein (40). The presence of CYP2E1 as a major isoform in the human breast is of interest because it has been shown that ethanol consumption is related to breast cancer (41). This enzyme is efficient in converting ethanol to acetaldehyde (37), which can easily form Schiff bases with free amino groups in proteins and interfere with their functions (42).
The use of drugs such as tamoxifen in the treatment of breast cancer is limited because of the development of resistance to the therapeutic effects. One possible mechanism for the development of this resistance is induction of P450. Tamoxifen is metabolized by human CYP2B6, CYP2E1, CYP3A, and CYP2D6 (43), and it also induces CYP2B and CYP3A in the rat liver (44). The effects of tamoxifen on the rat breast P450 profile are presently being studied in our laboratory. Further studies are also needed to investigate possible metabolism of tamoxifen in the breast.
Once P450 proteins have been detected in the breast, two further questions arise. Are these enzymes catalytically active, and where are they located? The first question requires a sensitive assay for catalytic activity, and the latter question requires good antibodies that are useful in immunohistochemistry. One fluorescent assay that could be used in the breast is coumarin 7-hydroxylase. This assay is specific for CYP2A6 and, although this enzyme was of low abundance in the breast, its catalytic activity could be reliably measured in breast microsomes. The values of 1.5–4 pmol/mg microsomal protein/min represent approximately 0.01% of the liver level, and this is compatible with the Western blot data. As in the rat breast (8), the levels of P450 in the human breast per gram of tissue were approximately 3 orders of magnitude lower than in the human liver. This low expression of P450 does not necessarily mean that breast P450 is unimportant, particularly if the enzymes are localized in a limited number of cells, as has been demonstrated for CYP2A3 in the rat breast (45).
The possibility that P450 enzymes in the breast are involved in in situ activation of carcinogens is an important issue. Environmental carcinogens have been implicated in the etiology of breast cancer (46). It is currently thought that P450s play a very important role in both the activation and inactivation of procarcinogens in the body. P450 forms detected in the breast can activate polycyclic aromatic hydrocarbons (CYP1A1, CYP1B1) (47, 48), nitrosamines (CYP2A6, CYP2E1, CYP2D6) (49, 50), and food mutagens (CYP2A6) (51) to their ultimate carcinogens and thus could play a role in the initiation of breast cancer.
To evaluate the importance of breast P450 in carcinogenesis and hormone sensitivity, much more data is needed. It is hoped that clinicians with access to breast tissue will begin to accumulate data on the breast P450 profile of their patients with these issues in mind.
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Acknowledgments |
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Footnotes |
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2 To whom correspondence should be addressed.
Received July 16, 1997.
Revised September 25, 1997.
Revised November 25, 1997.
Accepted November 11, 1997.
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References |
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E. A. Triano, L. B. Slusher, T. A. Atkins, J. T. Beneski, S. A. Gestl, R. Zolfaghari, R. Polavarapu, E. Frauenhoffer, and J. Weisz Class I Alcohol Dehydrogenase Is Highly Expressed in Normal Human Mammary Epithelium but not in Invasive Breast Cancer: Implications for Breast Carcinogenesis Cancer Res., June 15, 2003; 63(12): 3092 - 3100. [Abstract] [Full Text] [PDF]
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 H. K. Crewe, L. M. Notley, R. M. Wunsch, M. S. Lennard, and E. M. J. Gillam Metabolism of Tamoxifen by Recombinant Human Cytochrome P450 Enzymes: Formation of the 4-Hydroxy, 4'-Hydroxy and N-Desmethyl Metabolites and Isomerization of trans-4-Hydroxytamoxifen Drug Metab. Dispos., August 1, 2002; 30(8): 869 - 874. [Abstract] [Full Text] [PDF]
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 C. Sridar, U. M. Kent, L. M. Notley, E. M. J. Gillam, and P. F. Hollenberg Effect of Tamoxifen on the Enzymatic Activity of Human Cytochrome CYP2B6 J. Pharmacol. Exp. Ther., June 1, 2002; 301(3): 945 - 952. [Abstract] [Full Text] [PDF]
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