CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

CYP1B1 is an important detox enzyme that metabolizes many compounds and is important for the development of the eye. If activity is too high or too low, CYP1B1 can be at the root of a variety of problems. Read on to learn more.

What is CYP1B1?

The enzyme CYP1B1 is one of the cytochrome P450 monooxygenases (CYPs). These are enzymes that eliminate most of the drugs and toxins from the human body [1].

Read more about CYPs here.

CYP1B1 Function

This enzyme metabolizes:

  • steroid hormones [2], including estrogens [3, 4]. CYP1B1 is also responsible for the final steps in the production of cortisol and aldosterone [5].
  • fatty acids and fat-soluble vitamins [2, 6].
  • melatonin [2].
  • retinol and dietary plant flavonoids [7].
  • polycyclic aromatic hydrocarbons (PAHs), biphenyls, N-heterocyclic amines, arylamines, amino-azo dyes, and other cancer-causing and toxic environmental chemicals [8, 2].
  • CYP1B1 metabolizes few, if any, clinical drugs [8].

CYP1B1 Location

This enzyme is found in the liver, but also in various other tissues including fat, skin, breast gland, prostate, heart, blood vessels, kidney, thymus/marrow and immune cells, breast, uterus, brain, and eyes [6, 2, 7, 8, 9].

Beneficial Functions

An important role of CYP1B1 is that it helps reduce oxidative stress [6, 10]. CYP1B1 deficiency results in increased oxidative stress in mice [11].

Also, this enzyme is important for eye development [9, 12]. Many mutations in CYP1B1 cause primary congenital glaucoma [12, 13].

This enzyme can protect from cancer. CYP1B1 deficient mice have more lung tumors [14].

Potentially Detrimental Functions

This enzyme is produced by fat tissue, and it helps increase fat uptake [6].

In mice, CYP1B1 deficiency increases AMPK, reduces obesity, reduces blood pressure, and improves glucose tolerance [6, 15].

Furthermore, this enzyme is capable of activating a wide range of cancer-causing compounds. Increased CYP1B1 activity has been associated with a range of cancers [16, 10].

CYP1B1 activity was shown to promote the growth and metastasis of non-small lung cancer cells [17] and may promote the growth of renal cell carcinoma (kidney cancer) cells [18].

CYP1B1 Gene Polymorphisms

More than 150 SNPs have been reported for this gene [13, 11].

Many mutations of this gene are linked to primary congenital glaucoma [12, 6].


rs10012 alters androgen (testosterone) concentrations (1499 cases and 1373 controls) [19], with the C variant being more active [20].

This may explain the association of the C variant with increased disease aggressiveness in prostate cancer (1387 subjects) [21].

The C variant was also associated with an increased risk of endometrial cancer (200 subjects) [22].

On the other hand, the G variant was associated with urinary bladder cancer (492 subjects) [23].


This variant is also known as Ala119Ser.

At rs1056827, the A allele is the more active variant of the enzyme [20].

This variant (A) correlates with urinary bladder cancer (492 subjects) [23], higher risk of prostate cancer (meta-analysis, 34 studies, 17,796 cases, and 19,891 controls) [24] and breast cancer (same meta-analysis, 17,796 cases, and 19,891 controls) [24].


rs1056836 is also known as Leu432Val.

People with the G variant at rs1056836 tend to have higher enzyme activity (about 3 fold) [25, 26].

G is associated with increased susceptibility to hepatocellular carcinoma (983 subjects) [27], multiple myeloma (1061 subjects) [28], lung cancer (meta-analysis, 22 studies, 2881 cases, and 3653 controls) [29], and endometrial cancer, but decreased susceptibility to ovarian cancer (meta-analysis of 115 studies: 54,124 cases and 62,932 controls) [24] and prostate cancer (1387 subjects) [21].

The CC genotype (two C alleles) is associated with bloating, facial hair, palpitations, and involuntary urination in premenopausal women. CC is also linked to nausea, bloated stomach, facial hair, and vaginal dryness in peri- and postmenopausal women. Carriers of CC or CG were approximately five times more ly to suffer from vaginal dryness than women with the GG genotype (299 women) [30].

G was associated with shorter average telomere length in postmenopausal women taking hormonal therapy (259 subjects) [26]. Short telomere length is associated with premature aging and age-related disease.


Rs1800440 is also known as Asn453Ser. At this SNP, the C allele was associated with a decreased risk of endometrial cancer (meta-analysis, 48 studies, 30,532 cases, and 39,193 controls) [24].


The T allele at rs150799650 was associated with bladder cancer (in 492 subjects) [23].

  • rs1056837
  • rs2617266
  • rs9282671
  • rs9341266

Factors Affecting CYP1B1 Activity

Researchers have observed many factors that increased or decreased CYP1B1 activity in cells or animals; however, it is unclear how relevant these studies may be to CYP1B1 activity in the human body.

Because of the degree of uncertainty and lack of clinical studies, we strongly recommend talking to your doctor before making any significant changes to your diet, lifestyle, or supplement regimen.

Factors that Decrease CYP1B1:

  • St. John’s wort [34]
  • Apigenin [34]
  • Ginseng [35]
  • Lycopene, a red pigment found in tomatoes, carrots, and watermelon [36]
  • Chrysoeriol, present in rooibos tea and celery [37]
  • Naringenin, found in grapefruit juice [38]
  • Zyflamend, a polyherbal formulation produced from the extracts of ten common herbs (rosemary, turmeric, ginger, holy basil, green tea, hu zhang, Chinese goldthread, barberry, oregano, and Baikal skullcap) [39]
  • Quercetin [40]


Cytochrome P450 1B1

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

A cytochrome P450 monooxygenase involved in the metabolism of various endogenous substrates, including fatty acids, steroid hormones and vitamins (PubMed:20972997, PubMed:11555828, PubMed:12865317, PubMed:10681376, PubMed:15258110).

Mechanistically, uses molecular oxygen inserting one oxygen atom into a substrate, and reducing the second into a water molecule, with two electrons provided by NADPH via cytochrome P450 reductase (NADPH–hemoprotein reductase) (PubMed:20972997, PubMed:11555828, PubMed:12865317, PubMed:10681376, PubMed:15258110).

Exhibits catalytic activity for the formation of hydroxyestrogens from estrone (E1) and 17beta-estradiol (E2), namely 2- and 4-hydroxy E1 and E2. Displays a predominant hydroxylase activity toward E2 at the C-4 position (PubMed:11555828, PubMed:12865317). Metabolizes testosterone and progesterone to B or D ring hydroxylated metabolites (PubMed:10426814).

May act as a major enzyme for all-trans retinoic acid biosynthesis in extrahepatic tissues. Catalyzes two successive oxidative transformation of all-trans retinol to all-trans retinal and then to the active form all-trans retinoic acid (PubMed:10681376, PubMed:15258110). Catalyzes the epoxidation of double bonds of certain PUFA.

Converts arachidonic acid toward epoxyeicosatrienoic acid (EpETrE) regioisomers, 8,9-, 11,12-, and 14,15- EpETrE, that function as lipid mediators in the vascular system (PubMed:20972997). Additionally, displays dehydratase activity toward oxygenated eicosanoids hydroperoxyeicosatetraenoates (HpETEs).

This activity is independent of cytochrome P450 reductase, NADPH, and O2 (PubMed:21068195). Also involved in the oxidative metabolism of xenobiotics, particularly converting polycyclic aromatic hydrocarbons and heterocyclic aryl amines procarcinogens to DNA-damaging products (PubMed:10426814). Plays an important role in retinal vascular development.

Under hyperoxic O2 conditions, promotes retinal angiogenesis and capillary morphogenesis, ly by metabolizing the oxygenated products generated during the oxidative stress. Also, contributes to oxidative homeostasis and ultrastructural organization and function of trabecular meshwork tissue through modulation of POSTN expression (By similarity).

Manually curated information which has been propagated from a related experimentally characterized protein.


Manual assertion inferred from sequence similarity toi

  • UniProtKB:Q64429 (CP1B1_MOUSE)

Manually curated information for which there is published experimental evidence.


Manual assertion experiment ini

  • an organic molecule + O2 + reduced [NADPH—hemoprotein reductase] = an alcohol + H+ + H2O + oxidized [NADPH—hemoprotein reductase]Manual assertion experiment ini EC: assertion experiment iniThis reaction proceeds in the forward direction.
  • 17β-estradiol + O2 + reduced [NADPH—hemoprotein reductase] = 2-hydroxy-17β-estradiol + H+ + H2O + oxidized [NADPH—hemoprotein reductase]Manual assertion experiment iniThis reaction proceeds in the forwardManually curated information which has been inferred by a curator his/her scientific knowledge or on the scientific content of an article.More… Manual assertion inferred by curator fromi direction.


CYP1B1 enhances the resistance of epithelial ovarian cancer cells to paclitaxel in vivo and in vitro

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

Ovarian cancer (OC) is the most common cause ofmortality among gynecological malignancies. Epithelial OC accountsfor approximately 85–90% of primary OC cases (1).

Despite the development in surgicaltechnologies and adjuvant chemotherapy using platinum-based drugsin combination with taxanes, the 5-year survival rate for patientswith the disease still remains at 30%.

The poor prognosis is mainlydue to the late presentation of symptoms or only apparent syndromeduring the metastasis of the disease. In addition, OC has anunpredictable response to chemotherapy as an outcome of intrinsicor acquired drug resistance (2).

Therefore, there is an urgent need for the identification of novelprognostic risk factors and for the development of noveltherapeutic strategies for malignant OC. In addition, the furtherelucidation of the mechanisms underlying the chemotherapeuticresistance is required.

There are three major milestones in OC chemotherapy:drug development, including alkylating agents in the 1970s,cisplatin drugs in the 1980s and paclitaxel (PTX, taxol) in the1990s. Paclitaxel is a natural product with anticancer activityobtained through a semi-synthetic process from Taxusbaccata.

Due to no cross-resistance with platinum-based drugs,it has become the first-line drug and by far the most effectivechemotherapeutic agent for the treatment of OC.

However, theapplication of PTX has been progressively limited in recent yearsmainly due to the occurrence of drug resistance and adversecomplications which also diminish its therapeutic effects in OC(3–5).

The cytochrome P450 (CYP) enzymes are a family ofimportant hemoprotein monooxygenases that catalyze the oxidation ofa wide range of endogenous and exogenous xenobiotics, such asanticancer drugs, which results in drug degradation andinactivation (6,7).

A number of cytochrome P450 familymembers can interfere with the metabolism of a range of anticancerdrugs, such as PTX, docetaxel (DTX) and cyclophosphamide, whichhave been used in the chemotherapy of various cancers, including OC(8,9).

These enzymes have cell-ortissue-specific expression, while some of them, particularlyCYP1B1, are overexpressed in a wide range of cancers (10–15). The overexpression of CYP1B1proteins in cancer cells may affect their sensitivity in reactingto anticancer drugs.

In vitro studies have indicated thatCYP1B1 increases the drug resistance of cells exposed to DTX andantagonizes the anticancer effects of DTX (16). However, to the best of ourknowledge, reports on whether CYP1B1 mediates resistance to PTX inOC chemotherapy are limited.

In the present study, we investigated the expressionprofile of CYP1B1 in samples from patients with OC and confirmedits high expression in malignant cases compared to benign cases andnormal ovarian tissue. In PTX-sensitive and -resistant cell lines,we identified the link between PTX-induced CYP1B1 expression andresistance to PTX.

A specific inhibitor of CYP1B1, α-naphthoflavone(ANF), reversed the resistance to PTX and recovered the sensitivityof OC cells in response to PTX in vitro and in vivo.These findings are of great importance in terms of identifyingnovel diagnostic markers and prognostic factors for OC.

Furthermore, our data provide insight into the development ofpotential therapeutic strategies for the treatment ofpaclitaxel-resistant OC patients.

Patient samples and exclusioncriteria

All 53 paraffin-embedded tissue blocks from patientswith OC were provided by the Department of Pathology, TianjinMedical University General Hospital, Tianjin, China. None of thepatients had been previously treated or had received chemotherapyprior to surgery.

The general information of the patients, the FIGOstage and pathological stage, as well as the histopathologicalcharacteristics of the patients are presented in Table I. Other tissue samples werecomprised of 14 metastatic samples from the 53 patients with OC, 30benign ovarian tumor samples and 19 normal ovarian tissue samples.

The control group and the OC group were designed and selected withstrict matching criteria so that there were no differences in age,family history or body mass index of the patients.

Risk factors of CYP1B1 expression inpatients with epithelial ovarian cancer.

Risk factors of CYP1B1 expression inpatients with epithelial ovarian cancer.

nCYP1B1 expressionPositive (%)χ2P-value
Malignantepithelial ovarian cancer5349 (92.45)
Clinical stage
 I + II1715 (88.24)
 III + IV3634 (94.44)4.400.05
Benign epithelialovarian tumor304 (13.33)
Normal ovary190 (0.0)51.9650%), moderate (5–50%) or negative (


CYP1B1 expression is induced by docetaxel: effect on cell viability and drug resistance

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

The cytochrome P450 CYP1B1 is consistently overexpressed in tumour cells as compared to their normal counterparts, but its precise role in drug resistance is yet to be defined. It has been reported that transfection of CYP1B1 results in increased resistance to docetaxel in V79 cells (McFadyen et al, 2001).

In this study, we analysed changes in expression of CYP1B1 mRNA associated with pulse selection of MCF-7 cells with docetaxel.

Docetaxel-selected MCF-7 cells (MCF-7 Txt), which showed increased resistance to this drug as compared to parental MCF-7 cells, showed a noteworthy increase in CYP1B1 mRNA expression, paralleled by increased ethoxyresorufin-O-deethylase (EROD) activity levels.

This effect was not observed in cisplatin- or adriamycin-selected MCF-7 cells, or in docetaxel-selected colon, lung or pancreatic carcinoma cells. Short-term treatment with docetaxel induced CYP1B1 mRNA expression in MDA 453 and BT-20 breast carcinoma cells, but not in MCF-7 cells.

Transfection of MCF-7 Txt cells with CYP1B1 siRNA did not significantly affect docetaxel-induced toxicity, but it decreased cell survival in the absence of drug. Preincubation of docetaxel with recombinant CYP1B1 did not affect drug toxicity in A549 cells. These results suggest that CYP1B1 does not directly inactivate docetaxel, but rather might promote cell survival in MCF-7 Txt cells, providing an explanation for its association with drug resistance.

Breast cancer affects a significant number of women worldwide and is a leading cause of cancer deaths (Chintamani et al, 2004). Current therapies for breast cancer include locoregional (surgical) treatment, anti-hormone therapy, radiotherapy and chemotherapy (O'Driscoll and Clynes, 2006).

Chemotherapeutic treatment of breast cancer, although of great importance in the management of the disease, has limited benefits for many patients due to intrinsic or acquired resistance to the effects of anticancer drugs.

Moreover, chemoresistance is often associated with a more aggressive phenotype with an increased tendency to invade and metastasise (Campbell et al, 2001).

Cytochromes P450 are a family of enzymes implicated in the biotransformation of both xenobiotics and endogenous compounds. Their primary functions are the synthesis of steroids and bile acids and the detoxification of many foreign substances, such as drugs and environmental agents.

CYP1B1 is the only member of the CYP1B subfamily; it catalyses the hydroxylation of 17-β-oestradiol at the C4 position (Jefcoate et al, 2000) and is involved in testosterone biotransformation as well (Shimada et al, 1999). It is also known to metabolise xenobiotics such as ethoxyresorufin, theophylline and caffeine (Shimada et al, 1997).

CYP1B1 is constitutively expressed at the mRNA level in mammal steroidogenic tissues, such as rat granulosa cells (Dasmahapatra et al, 2002) and its expression can be induced in these and other tissues by peptide hormones, cAMP and ligands of the aryl hydrocarbon receptor (AhR).

Cross-talk has been described between the oestrogen receptor (ER)α and the CYP1B1 expression pathways (Spink et al, 1998). Regulation of expression appears to be tissue-specific, since treatment with the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1B1 mRNA in MCF-7 cells but not in HepG2 cells (Spink et al, 1994).

More recently, regulation of CYP1B1 expression by microRNAs has been described (Tsuchiya et al, 2006).

Conflicting data exist in the literature regarding CYP1B1 expression.

A number of studies failed to detect presence or activity of functional CYP1B1 protein in normal (nontumour) tissue (Murray et al, 1997; McFadyen et al, 1999); however, expression of CYP1B1 in normal tissue has been reported, although in much lower levels than in tumour tissue, at both mRNA and protein levels (Muskhelishvili et al, 2001; Gibson et al, 2003). In spite of these inconsistencies, most studies agree that CYP1B1 protein is commonly overexpressed in malignant as compared to normal tissue.

The significance of CYP1B1 overexpression in cancer is not yet fully understood, but the fact that it can inactivate flutamide, an antiandrogen used in the treatment of prostate cancer, with high efficiency suggests that this enzyme might play an important role in the development of resistance to some forms of chemotherapy (Rochat et al, 2001). Also, a significant decrease in the sensitivity of CYP1B1-transfected V79 Chinese hamster ovary cells to docetaxel as compared to the parental cell line has been described (McFadyen et al, 2001); cytotoxicity was restored by pretreating the cells with the CYP1 inhibitor α-naphtoflavone. Although transfection of CYP1B1 has been reported to confer resistance to docetaxel, no metabolites could be detected after incubation of this drug with recombinant CYP1B1 in a number of different conditions (Bournique and Lemarie, 2002). Thus, even though expression of CYP1B1 appears to be associated with drug resistance, its precise role remains obscure.

In this study, we report induction of CYP1B1 mRNA expression by docetaxel in breast cell lines, both after short-term treatment and pulse-selection. The docetaxel-mediated increase in CYP1B1 expression was cell type-specific, as lung, pancreas and colon cell lines pulse-selected with docetaxel did not show such induction.

CYP1B1 small interfering RNA (siRNA) transfection resulted in decreased cell survival, but did not cause major changes in docetaxel toxicity. Overall, expression of CYP1B1 appears to promote cell survival in MCF-7 Txt cells; this effect might significantly contribute to the increase in drug resistance observed in these cells.

Cell culture media were obtained from Gibco BRL (Paisley, UK); serum was from Sigma (Poole, UK). Docetaxel was obtained from Sanofi Aventis Pharmaceuticals (Surrey, UK), cisplatin was from Mayne Pharma Plc.

(Warwickshire, UK) and paclitaxel was from Bristol-Myers Squibb (Dublin, Ireland). 5-fluorouracil was obtained from Faulding Pharmaceuticals (Warwickshire, UK) and adriamycin was from Ebewe (Unterach, Austria).

Reverse transcription polymerase chain reaction (RT–PCR) reagents and all other chemicals were purchased from Sigma.

Cell lines

MCF-7, BT-20, MDA 231 and MDA 453 breast carcinoma and A549 lung adenocarcinoma cells were obtained from the ATCC (American Type Culture Collection). BT-20 cells, MCF-7 cells and their pulse-selected variants were maintained in MEM supplemented with 10% fetal calf serum (FCS), 1 mM sodium pyruvate and 1 mM non-essential amino acids.

MDA 231 and MDA 453 cells were maintained in RPMI supplemented with 10% FCS. A549 cells were maintained in a 1 : 1 mixture of DMEM and Ham's F12 medium supplemented with 5% FCS. Cell lines were cultured in a humidified atmosphere of 95% air/5% CO2.

All docetaxel-selected variants were referred to as Txt, for example, MCF-7 Txt; adriamycin-selected variants were referred to as Adr and cisplatin as CisPt.

Pulse selection of cells

MCF-7 cells were pulse-selected with adriamycin (4 pulses, once a week for 4 h, with 250 ng ml−1 adriamycin), cisplatin (9 pulses, once a week for 4 h, with 800, 100, 300, 300, 300, 300, 300, 350, 350 ng ml−1 cisplatin, respectively for each week) and docetaxel (6 pulses, once a week for 4 h, except for the first one, which was for a half hour, with 50, 10, 10, 13, 13, 13 ng ml−1 docetaxel, respectively) to generate the MCF-7 Adr, MCF-7 CisPt and MCF-7 Txt variants, correspondingly. Drug concentrations and pulse schedule were decided changes in cell morphology and resistance; a dynamic pulse schedule was used here according to our previous experience with this cell line.

In vitro toxicity testing

Cytotoxicity testing of drugs was measured by the acid phosphatase assay as previously described (Martin and Clynes, 1991). Briefly, cells were seeded at 1 × 103 cells per well in a 96-well plate and left to attach overnight in a 5% CO2 incubator at 37°C.

The appropriate concentrations of drug were prepared freshly at 2 × their final concentration and added to the plate on the following day. The assay was terminated after a further 7-day incubation. The concentration of drug causing a 50% kill (IC50 of the drug) was determined using CalcuSyn (Biosoft, Cambridge, UK).

Fold resistance was calculated as the ratio between IC50 values in parent and pulse-selected cells. All assays were performed at least in triplicate.


Adherent cells were grown in 75 cm2 flasks until approximately 80% confluent; mRNA was then extracted using TRI reagent (Sigma) according to the manufacturer's instructions.

RNA was quantified spectrophotometrically at 260 and 280 nm using the Nanodrop (Nanodrop Technologies, Wilmington, DE, USA). One microgram of mRNA was then subjected to reverse transcription and the resulting cDNA amplified by PCR.

The sequences of primers used for CYP1B1 amplification were IndexTermGTATATTGTTGAAGAGACAG for the forward primer and IndexTermAAAGAGGTACAACATCACCT for the reverse primer (Baron et al, 1998); β-actin was used as control.

The mixture was amplified for 35 cycles using the following conditions: 94°C for 3 min, 49°C for 30 s, 72°C for 1 min. Polymerase chain reaction products were then separated on a 2% agarose gel and visualised by ethidium bromide staining on a UV transilluminator.

Determination of EROD activity

Ethoxyresorufin-O-deethylase (EROD) activity of cells in culture was determined as previously described (Bandiera et al, 2005). Briefly, cells were incubated with a 400 nM solution of 7-ethoxyresorufin in PBS pH 7.4 at 37°C/5% CO2 for 60 min.

After incubation, 200 μl of the cell supernatant was transferred to an opaque 96-well plate and fluorescence were determined at 530 nm excitation and 590 nm emission in a fluorescence plate reader (Synergy HT; Bio-Tek, Winooski, Vermont, USA).

The sample measurements were compared to a resorufin (Sigma; R3257) standard curve.

Ethoxyresorufin-O-deethylase activity was then calculated as the measured amount of resorufin (in picomoles), divided by the total protein content of the sample (in milligrams) and by the total reaction time (in minutes).

RNA interference

RNA interference analysis was performed using siRNAs chemically synthesised and purchased from Ambion Inc., Warrington, UK). Three different siRNAs for CYP1B1 were used (Ambion Ids. 2253, 105952 and 112548). Small interfering RNAs were introduced into the cells via reverse transfection with the transfecting agent siPORT™ NeoFX™ (Ambion Inc.).

Conditions for transfection were optimised using kinesin siRNA (Ambion Inc.). Solutions of negative control (scrambled), kinesin and CYP1B1 siRNAs at a final concentration of 30 nM were prepared in optiMEM (Gibco™). NeoFX solutions were also prepared in optiMEM and incubated at room temperature for 10 min.

After incubation, either scrambled, kinesin or CYP1B1 siRNA solution was added to each NeoFX concentration; these solutions were mixed thoroughly and incubated for a further 10 min at room temperature. Replicates of 10 μl of the siRNA/NeoFX solutions were added to a 96-well plate. A total of 7.

5 × 103 cells in 100 μl were then added to each well; the plates were mixed gently and incubated at 37°C for 24 h. After this period, the transfection mixture was removed from the cells and the plates were fed with fresh medium.

Forty-eight hours after transfection, concentrations of the chemotherapeutic agent (at 2 × the final concentration) were added to the plates in replicates of four. The plates were assayed for changes in proliferation after a further 72 h using the acid phosphatase assay.

Drug metabolism by microsomes

Drug metabolism was investigated in the microsomal fraction of recombinant insect cells (microsomes) expressing CYP1B1 and cytochrome P450 NADPH reductase (P450R) (Beckton Dickinson, Oxford, UK) or P450R alone.

Fifty microlitres of a stock solution of the appropriate drug were prewarmed at 37°C in a total incubation mixture volume of 250 μl containing potassium phosphate buffer (PBS, pH 7.4), MgCl2 (25 mM, 25 μl) and NADPH (6.5 mM, 75 μl).

The reaction was initiated by the addition of 100 μl of a 1 mg ml−1 solution of ice-cold microsomes to the incubation mixture, as recommended by the manufacturer. After an incubation period of either 30 or 60 min, the mixture was filter sterilised and used for toxicity assays as described above.

Statistical analysis

Differences between cell lines were assessed using a two-tailed Student's t-test. A P-value


Combined Effect of CYP1B1 Codon 432 Polymorphism and N-Acetyltransferase 2 Slow Acetylator Phenotypes in Relation to Breast Cancer in the Turkish Population

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

  1. 1Institute of Experimental Medical Research, Department of Molecular Medicine, University of Istanbul, Instabul, Turkey
  2. 2Department of Pathology, Bagcilar Education and Research Hospital, Istanbul, Turkey
  3. 3Cerrahpasa Medical School, Department of Pathology, University of Istanbul, Instabul, Turkey
  1. Correspondence to: Professor Dr. Turgay Isbir, Department of Molecular Medicine, Institute of Experimental Medicine (DETAE), Istanbul University, Vakıf Gureba Cad Sehremini, Istanbul, Turkey. Tel/Fax: +90 2126351959, e-mail: tisbir{at}

Background: Breast cancer (BC), is more prevalent in subjects who have had prolonged exposure to heterocyclic amines, aromatic amines and high levels of oestradiol.

Cytochrome P450 1B1 (CYP1B1) and N-acetyltransferase2 (NAT2) have complementary role in metabolism of xenobiotics such as arylamines and heterocyclic amines, CYP1B1 also hyroxylates 17-β oestradiol. CYP1B1*3 polymorphism and seven missense and four silent polymorphisms of NAT2 were investigated.

Patients and Methods: Sixty Turkish female BC patients and 103 healthy controls were phenotyped by polymerase chain reaction (PCR) based restriction fragment length polymorphism (RFLP). Results and Conclusion: The distribution of NAT2 activity in the healthy control group was found to be correlated with that of healthy caucasians.

Patients had slow acetylator phenotypes of NAT2, 1.8 times higher than controls but no statistical differences were found (p=0.07). In addition, the NAT2*5 alelle was more statistically correlated with breast cancer patients rather than the controls (p=0.02). Moreover, NAT2*5B was the most frequent haplotype of the NAT2*5 family (p=0.000).

Breast cancer patients were detected to posses more CYP1B1*3 mutant alleles than the controls (p=0.043). The combined effect of CYP1B1*3 polymorphism and NAT2 slow acetylator genotype contributed to an increased risk for breast cancer in patients in this study (p=0.004).

Differences in the distribution of gene polymorphisms in key discriminant genes involved in oestrogen and xenobiotic metabolism is considered to contribute to variations in breast cancer susceptibility among different racial/ethnic populations (1).

Biotransformation of xenobiotics in the body includes activating hydroxylation reactions by Cytochrome P450 enzymes and detoxification by conjugating enzymes such as catechol-O-methyltransferase (COMT), glutathione-S-transferases (GSTs) and N-acetyltransferases (NATs) (1).

Polymorphisms in these enzymes may cause modifications in catalysing activity and stabilisation of the enzymes, thus increasing susceptibility to cancer due to prolonged exposure to oestradiol and xenobiotics (1).

In this study, genetic polymorphisms of Cytochrome P450 1B1 (CYP1B1*3) and N-Acetyltransferase 2 (NAT2), and their contribution to breast cancer susceptibility was investigated among Turkish Women. Different populations have different polymorphism patterns, and the Turkish population is expected to display the genetic traits of the Caucasian population (1).

CYP1B1 is a haeme-thiolate monooxygenase that is involved in the NADPH-dependent monooxygenation of a considerable variety of carcinogens such as polyaromatic hydrocarbons (PAHs), heterocyclic aromatic amines (HAA), arylamines and nitrosamines formed in cooked food, tobacco products and found in industrial chemicals. CYP1B1 is located on chromosome 2p21 with three exons and two introns, encoding a 543 aminoacid length enzyme. CYP1B1 is mainly expressed in most cell types and secretory cells of breast tissue(2). CYP1B1 also mediates hydroxylation of 17-β oestradiol mainly to 4-hydroxyestradiol, the excess of which reacts further with DNA to form carcinogenic DNA adducts (3). Nearly, 80% of oestradiol is converted to 2-hydroxyestradiol (2-OHE2) by CYP1A1 in the liver and 20% of it to 4-hydroxyestradiol by CYP1B1 in breast tissue and uterus (3, 4). Various studies have established that increased blood oestrogen levels have the potential to induce breast and endometrial cancer, either by binding of oestrogen to its receptors, resulting in cell proliferation or, by conversion of cathechol form of oestrogens to reactive oxygen species (ROS) and related quinone and semiquinone products via redox cycling (5).

N-acetyltransferase-accompanied detoxification is a basic biotransformation pathway for the metabolism of drugs and environmental xenobiotics, specifically hydrazine drugs, homo- and hetero-cyclic amines and arylamines (6).

The NAT2 detoxifying enzyme represents the N- and O-acetylation process in phase 2 metabolisation of biomolecules forming unstable N-acetylated compounds (7-8).

If not sufficiently excreted from the organism, these N-acetoxy metabolites may be converted to aryl nitrenium ions that have the potential to react with DNA (6-9).

NAT2 is located at chromosome 8p21.3-23.1 and polymorphisms in this region cause individual and interethnic variation, thus affecting the acetylation capacity of the individual. Human populations can be divided into subgroups according to their acetylation capacities.

To date 25 polymorphisms have been identified, of which 7 single nucleotide polymorphisms (SNPs) are the most abundant in various human populations, and are all found within the 870 bp coding region of the NAT2. The wild type allele is denoted as NAT2*4. NAT2*12 and NAT2*13 represent other fast phenotypes.

NAT2*5, NAT2*6 and NAT2*7 haplotypes are documented as the slowest acetylating phenotypes (7). Individuals possessing both slow haplotypes are classified as slow acetylators. In this regard, individuals possessing both fast alleles, NAT2*4, NAT2*12 and NAT2*13 are classified as the fastest acetylators.

Individuals possessing one fast and one slow allele are classified as intermediate acetylators (6-8).

This population based case-control study conducted among Turkish women, investigates the genetic frequencies of CYP1B1*3 and NAT2, and their possible individual or cooperative involvement in susceptibility to breast cancer.

Patient selection. A total of 163 unrelated individuals were included in this study: 60 female breast cancer patients, (median age: 53.7±11.31, range 31-72 years) and 103 healthy controls, (median age: 41.87±14.33, range 22-86 years).

Surgical samples from the 60 breast cancer patients were obtained by the Department of Pathology, Istanbul University Cerrahpasa Medicine School, Istanbul. The healthy control group consisted of female healthy blood donors with no prior cancer history or any other vital disease history. Both postmenopausal and premenopausal women were included in the study.

Twenty six (43.3%) patients and 70 (68.0%) controls were premenopausal and 34 (56.7%) patients and 33 (32.2%) controls were postmenopausal.

DNA extraction. Patient DNA was extracted from paraffin-embedded tissue using the method of Wright and Manos (10). Blood samples from the control group were collected in tubes containing EDTA, and DNA was prepared from leukocyte pellets by SDS lysis, ammonium acetate extraction and ethanol precipitation (11).

Genotyping method for CYP1B1*3 and NAT2. For genotyping, DNA was extracted from the blood of the controls and samples embedded in paraffin from non-tumoural neighbouring breast tissue.

The CYP1B1*3 genotype was determined following Polymerase Chain Reaction (PCR) according to the method of Zheng et al. (12). Genotyping for NAT2 polymorphism, including 11 SNPs and 7 missense mutations, was conducted according to the method of Deitz et al. (13).

The NAT2 haplotype nomenclature used in this study was the study of Sabbagh et al. (7, 8).

Statistical analysis. Statistical analyses were performed using SPSS version 7.5 (SPSS Inc. Chicago, USA) including the Chi-square (χ2) test, Fisher's exact test and the Pearson correlation test. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated.

The allelic variant frequencies of CYP1B1*3 polymorphism are shown on Table I. 20.3% of the patients were homozygous for mutant allele (Val allele), while 40.7% of the patients were heterozygous mutant and 39% were homozygous for wild allele (Leu) (p=0.043, χ2: 3.911, OR: 2.58, 95% CI: 0.99-6.75).

The NAT2 gene was analysed for 11 polymorphisms: T111C, G191A, C282T, T341C, A434C, C481T, G590A, C759T, A803G, A845C, and G857A. None of the polymorphisms at nucleotides 111, 191, 434, 759 and 845 were observed in either control or patient samples.. NAT2 allelic frequencies using the nomenclature of Sabbagh et al. (7, 8) are shown on Table II.

N-Acetyltransferase 2 genotyping in patients and controls was identified through the combinations of all polymorphisms.

When these combined genotypes were evaluated with their phenotypic activities, fast acetylators and intermediate acetylators were found to be less frequent in the patient group (Figure 1). Slow acetylator phenotype in the patient group was observed to be 1.

8 times higher than in the control group. Nevertheless, the results did not reach statistical significance (p=0.07, χ2: 3.24, OR: 1.82, 95% CI: 0.94-3.50).


Evaluation of CYP1B1 Expression, Oxidative Stress and Phase 2 Detoxification Enzyme Status in Oral Squamous Cell Carcinoma Patients

CYP1B1 Enzyme: Function & Ways to Increase / Decrease It

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