A Study on the Association of Cytochrome-P450 1A1 Polymorphism and Breast Cancer Risk in North...

Abstract Cytochrome P-450 1A1 (CYP1A1) is in-

volved in the 2-hydroxylation of estrogens and mam-

mary carcinogens into 2-hydroxy catechol metabolites.

Many commonly occurring single nucleotide poly-

morphism (SNP) are reported in CYP1A1 in various

populations that include, isoleucine to valine substitu-

tion at 462 codon in heme binding region in exon 7 (A

to G transition at position 2455; M2), threonine to

asparagine substitution at codon 461 (C to A trans-

version at position 2453; M4), T to C transition at 3801

position (M1) and T to C transition at position 3205

(M3) in 3¢ non-coding region. Epidemiological studies

have shown inconsistent patterns between CYP1A1

polymorphism and breast cancer risk among various

populations. Most of the studies have shown significant

association between CYP1A1 genotype polymorphism

and breast cancer risk. The present investigation was

therefore undertaken to investigate the association of

M1, M2, M3 and M4 polymorphisms and their sub-

sequent contribution in premenopausal and postmen-

opausal women with breast cancer risk in north Indian

women. Genomic DNA was isolated from case con-

trols and breast cancer patients, specific segments of

genomic DNA were amplified and restriction fragment

length polymorphism (RFLP) was performed.

CYP1A1 expression and catalytic activity were also

assessed in premenopausal and postmenopausal case

controls and patients. Polymorphism at M1, M2 and

M4 alleles was detected and odds ratio for W/M1

and M1/M1 was calculated as 1.07 (95% CI, 0.59–1.87)

and 0.74 (95% CI, 0.28–1.96) respectively. Odds ratio

for W/M1 and M1/M1 alleles in premenopausal and

postmenopausal women was 1.09 (95% CI, 0.45–2.49)/

0.62 (95% CI, 0.10–2.66) and 1.60 (95% CI, 0.60–4.22)/

1.06 (95% CI, 0.22–7.33) respectively. Odds ratio for

W/M4 and M4/M4 allele was 1.20 (95% CI, 0.65–2.24)/

4.55 (95% CI, 0.44–226.2) and 0.96 (95% CI, 0.36–

2.64)/4.51 (95% CI, 0.23–273.0) respectively in total

and premenopausal women. In postmenopausal wo-

men odds ratio was calculated as 1.16 (95% CI, 0.45–

2.94) for M4/W but it could not be detected for M4/M4

since this genotype was not found in any postmeno-

pausal case controls. Odds ratio for W/M2 genotype

was calculated 0.57 (95% CI, 0.28–1.02), 1.06 (95% CI,

0.40–2.47) and 0.33 (95% CI, 0.12–0.89) respectively

for total, premenopausal and postmenopausal women,

however, in any group the odds ratio for M2/M2 could

not be detected as M2/M2 genotype was not found in

breast cancer patients. Polymorphism at M1 and M4

alleles was not found significantly associated with

breast cancer risk and only wild type genotype was

found in case controls and patients for M3 allele. Lack

of protective association between CYP1A1 M2 geno-

type was also observed, however, in postmenopausal

women a significant protective association with breast

cancer risk was found (odds ratio, 0.33; 95% CI, 0.12–

0.89; P-value 0.03). Similarly, no significant alteration

in CYP1A1 expression and catalytic activity was

observed in wild type and variant genotypes both in

premenopausal and postmenopausal patients as com-

pared with their respective controls. The results

V. Singh Æ A. Sinha Æ A. Kumar Æ N. Mathur ÆM. P. Singh (&)Industrial Toxicology Research Centre (ITRC), MahatmaGandhi Marg, Post Box 80, Lucknow 226 001, UP, Indiae-mail: [email protected]

N. RastogiSanjay Gandhi Post Graduate Institute of Medical Sciences(SGPGI), Lucknow 226 014, UP, India

Breast Cancer Res Treat (2007) 101:73–81

DOI 10.1007/s10549-006-9264-2

123

EPIDEMIOLOGY

A study on the association of cytochrome-P450 1A1polymorphism and breast cancer risk in north Indian women

Virendra Singh Æ Neeraj Rastogi Æ Ashima Sinha ÆAbhai Kumar Æ Neeraj Mathur ÆMahendra Pratap Singh

Received: 24 April 2006 / Accepted: 28 April 2006 / Published online: 29 June 2006� Springer Science+Business Media B.V. 2006

obtained from the present investigation thus suggest

that probably CYP1A1 (M1, M2, M3, and M4) poly-

morphism alone does not play a significant role in the

breast cancer risk in north Indian women.

Keywords Single nucleotide polymorphism ÆCytochrome-P450 1A1 Æ M1 and M3 genotype ÆM2 and M4 genotype Æ North Indian women

Introduction

Cytochrome P-450 1A1 (CYP1A1) is involved in the

metabolism of mammary carcinogens, environmental

estrogens and polycyclic aromatic hydrocarbons [1, 2].

CYP1A1 is also expressed in the breast tissues and

converts estrogens into 2-hydroxy catechol metabolites

that are utilized in methylation reactions for biosyn-

thesis of 2-methoxy intermediates [3, 4]. 2-Hydroxy

catechol metabolites lack significant estrogenic activity

whereas 2-methoxy derivatives possess potent anti-

angiogenic and anti-proliferative activity [5–8].

Hydroxylation reaction generates reactive metabolites

but unlike 4-hydroxylation metabolites, 2-hydroxyl-

ation metabolites lack significant estrogenic properties

and are not associated with estrogen-induced carcino-

genesis in animals [9, 10]. Although estrogen 2-

hydroxylation is not a very prominent pathway for

potentially active estrogens but functional involvement

of CYP1A1 in estrogen and mammary carcinogens

metabolism prompted investigators to look into the

polymorphism in this gene and its association with

breast cancer risk [11].

CYP1A1 gene is located in q arm of 15th chromo-

some and consists of seven exons, six introns and spans

5810 base pairs [12]. CYP1A1 expression occurs pre-

dominantly in some extrahepatic tissues including

breast tissues [13]. Several polymorphic genotypes of

CYP1A1 gene are reported in various ethnic groups

[14–17]. M1 (CYP1A1*2A), a T to C transition at 3801

position in 3¢ non-coding region, M2 (CYP1A1*2C), an

isoleucine to valine substitution at codon 462 in heme

binding region in exon 7, M3 (CYP1A1*3), specific to

African-Americans, comprises of T to C transition at

position 3205 in 3¢ non-coding region and M4

(CYP1A1*4), a threonine to asparagine substitution at

codon 461 in exon 7 are known in many populations

[18–21]. Epidemiological studies regarding the associ-

ation between CYP1A1 polymorphism and breast

cancer risk have shown inconsistent results [22, 23].

Several studies have shown significant association

between CYP1A1 genotype polymorphism and

breast cancer risk whereas others have shown lack of

association [24, 25]. The dual role of CYP1A1 in

mammary carcinogen activation and estrogen

2-hydroxylation could be responsible for inconsistent

findings. Association of CYP1A1 polymorphism and

breast cancer risk depends on the underlying exposures

to polyaromatic hydrocarbons, heterocyclic amines,

endogenous factors and environmental estrogens [1, 2,

14], therefore, could vary from one population to an-

other or one ethnic group to another depending upon

the localities they occupy and the level of environ-

mental exposure. The present investigation was

therefore undertaken to find out the association of

M1, M2, M3, and M4 genotype polymorphism in

CYP1A1 gene with breast cancer risk in north Indian

women.

Materials and methods

Chemicals

Bovine serum albumin (BSA), sucrose, agarose,

bromophenol blue, xylene cyanol, phenol, chloroform,

ethylene-diamine-tetra-acetic acid (EDTA), Tri-BD

reagent, sodium chloride, potassium chloride, disodium

hydrogen phosphate, potassium dihydrogen phosphate,

magnesium chloride, calcium chloride, glucose-6-

phosphate, glucose-6-phosphate dehydrogenase, mag-

nesium sulphate, trisodium citrate, 7-ethoxyresorufin

and glucose were purchased from Sigma-Aldrich,

USA. Bradford reagent was procured from Bio-Rad,

USA and RT-PCR kits were procured from Fermen-

tas, USA. Taq polymerase, and oligo dT, dNTPs, PCR

primers, PCR reaction kits and other chemicals

required for the study were procured locally from

Bangalore Genei or Sisco Research Laboratory (SRL),

India.

Selection of subjects

The Medical ethics committees of Industrial Toxicol-

ogy Research Centre (ITRC), Lucknow and Sanjay

Gandhi Post Graduate Institute of Medical Sciences

(SGPGI), Lucknow approved the study. The blood

samples from case controls and breast cancer patients

were collected at SGPGI by expert clinicians. The

study was designed to recruit and collect the blood

samples from breast cancer patients and controls that

were between 25 years to 65 years of age. All the cases

and controls were residents of Lucknow or its adjacent

cities in north India. Blood samples were collected

from 116 normal healthy female controls and 105

female breast cancer patients. The case control samples

74 Breast Cancer Res Treat (2007) 101:73–81

123

were collected from healthy human volunteers who

were not diagnosed for any disease especially genetic

disorders as well as did not possess symptoms of any

visible disease. The patient samples were collected

from the individuals belonging to same ethnic group

with a well-known family history and clinically defined

symptoms of breast cancer as evidenced by clinical

history. Patients were classified in premenopausal

and postmenopausal groups based on the information

given by the individuals. A written preinformed con-

sent from the individuals was obtained prior to sample

collection.

Blood collection and DNA extraction

Blood samples from control and breast cancer patients

were drawn through veni-puncture and collected in

vials containing 3.8% tri-sodium citrate (9:1 v/v), pH

6.5. The samples were either processed immediately

for genomic DNA extraction or stored at –80�C until

further use. The genomic DNA was extracted from the

whole blood using salting out procedure [26]. The

genomic DNA was precipitated from the aqueous

phase using ethanol and stored at 4�C till further use.

The extracted genomic DNA samples were used to

amplify the desired gene segments using polymerase

chain reaction (PCR).

PCR and SNP genotyping

PCR amplification and restriction endonuclease

digestion of amplicons were performed in controls and

patients according to the procedure described by Li

et al. [27]. The primers synthesized and PCR condi-

tions were used in this study as reported in literature

[27].

Isolation of white blood cells (WBCs) and cell lysis

WBCs were isolated from the whole blood using

standard protocol [28] with slight modifications. In

brief, whole blood was centrifuged at 250 · g for

20 min at 20�C to remove platelets and plasma.

WBCs were isolated from the buffy coat by dextran

sedimentation and further purified with histopaque

density gradient centrifugation at 700 · g for 30 min

at 20�C. WBCs were recovered from histopaque

11191/10771 and washed thrice with Hank’s balanced

salt solution (HBSS, pH 7.4, 138 mM sodium chloride,

2.7 mM potassium chloride, 8.1 mM disodium hydro-

gen phosphate, 1.5 mM potassium dihydrogen phos-

phate) containing 0.6 mM magnesium chloride,

1.0 mM calcium chloride and 10 mM glucose. The

viability of the cells was tested by trypan blue exclu-

sion test and was never less than 95%. WBCs were

obtained as a pellet following centrifugation and

sonicated in 100 mM phosphate-potassium chloride

buffer, pH 7.4.

Protein estimation

The protein content was measured in cell lysate by

Bradford method [29] using BSA as standard. In brief,

100 ll of cell lysate was mixed with 1.25 ml of com-

mercial Bradford reagent and final volume was made

up to 5.0 ml with water. The mixed contents were

incubated at room temperature for 30 min and absor-

bance was recorded at 595 nm.

Isolation of RNA and cDNA biosynthesis

RNA was isolated from whole blood using Tri-BD

reagent. Polyadenylated RNA was reverse transcribed

using oligo dT primer and RT-PCR was performed using

RT-PCR kit according to manufacturer’s protocol.

Reverse transcriptase polymerase chain reaction

(RT-PCR)

Primers for CYP1A1 were designed and PCR ampli-

fication was performed as reported by Fasco et al. [30].

Forward and reverse primers for glyceraldehyde-3-

phosphate dehydrogenase (GAPDH) were designed as

described elsewhere [31] and amplification was per-

formed concurrently with CYP1A1. Relative expres-

sion of CYP1A1 was normalized with GAPDH

expression.

7-Ethoxyresorufin-O-deethylase (EROD) activity

CYP1A1 activity was measured in terms of EROD

catalytic activity using standard protocol [32] with

slight modifications. In brief, 100 mM phosphate buffer

(pH 7.4), 5 mM glucose-6-phosphate, 2 units glucose-6-

phosphate dehydrogenase, 5 mM MgSO4, 1.6 mg/ml

BSA, 1.5 lM 7-ethoxyresorufin, varying concentrations

of WBC lysate proteins in 100 mM phosphate–KCl

buffer (pH 7.4) and 0.6 nM NADPH were added in

test tubes, gently mixed and incubated at 37�C for

15 min in water bath. Reaction was stopped by the

addition of 2.5 ml methanol and keeping reaction

mixture in ice. Reaction mixture was centrifuged at

3000 rpm for 10 min and supernatant was collected.

Fluorescence was measured at 550 nm excitation and

585 nm emission wavelengths.

Breast Cancer Res Treat (2007) 101:73–81 75

123

Statistical analysis

The statistical analysis was performed using Epi Info-5

software. Statistical significance for odd ratio was cal-

culated using Chi-square test. Odds ratio was calcu-

lated separately with 95% confidence interval for

genotype frequencies in all, premenopausal and post-

menopausal breast cancer patients as compared with

respective controls.

Results

The size of PCR amplicon containing M1 and M3 al-

leles was 739 base pairs (bp) and amplicon containing

M2 and M4 alleles was 214 bp. Amplicons for M1 and

M3 alleles were digested with MspI and SphI and M2

and M4 alleles were digested with BsrDI (M2) and

BsaI (M4) restriction enzymes. Digestion products of

408 bp and 362 bp represented wild type (W) and M1

alleles, however, 331 bp and 226 bp digestion products

represented wild type and M3 alleles. In case of PCR

amplicons that were used for identification of M2 and

M4 alleles, restriction fragment length polymorphism

(RFLP) yielded 149 bp and 55 bp in 2.0% agarose gel

in wild type allele. Variant allele (M2 or M4) were

undigested and appeared as a band of 206 bp since the

enzymatic cutting sites were present in the forward and

reverse primers. Agarose gel electrophoresis of PCR

amplicons and RFLP byproducts obtained in north

Indian population are shown in Fig. 1.

M1 genotype and allele frequencies

Allelic and genotypic frequencies for M1 were not

significantly altered when patients were compared with

case controls. Similarly, when genotypic and allelic

frequencies were compared in case controls and

patients on the basis of menopausal state, there was no

significant alteration observed. Women who were

homozygous for variant M1 allele (OR, 0.74; CI, 0.28–

1.96) or heterozygous for variant M1 allele (OR, 1.07;

CI, 0.59–1.87) did not show significant association with

the breast cancer risk. The distribution of CYP1A1 M1

genotype in north Indian population is shown in Ta-

ble 1. The results obtained clearly suggested lack of

association of M1 allele polymorphism with the breast

cancer risk.




case controls (Table 2). Genotypic and allelic fre-

quencies were compared in case controls and patients

on the basis of menopausal state and no significant

alteration was observed in premenopausal women,

however, in postmenopausal women a significant pro-

tective effect was observed (Table 2) since a significant

association of M2 allelic polymorphism with case

controls was observed (P > 0.05). Heterozygosity for

CYP1A1 M2 allele in postmenopausal women showed

significant (OR, 0.33; CI, 0.12–0.89; P-value 0.03)

protective association with the breast cancer risk.


In the present study, we did not observe any M3 vari-

ant allele both in case controls as well as in patients

irrespective of individual’s menopausal states.




case controls (Table 3). Comparative genotypic and

1 2 3 4 5 6 7 8 9 10 11 12 13

739bp

408bp

362bp

331bp

206bp

149bp

1- 100bp ladder 2- PCR product (M1+M3) 3- W/W+W/W (M1+M3) 4- W/M1+W/W for M3 5- M1/M1+W/W for M36- PCR product (M2+M4) 7- W/W (For M2) 8- W/M2 9- M2/M2 10- PCR product (M2+M4) 11- W/W for M412- W/M413-M4/M4

55bp

Fig. 1 Agarose gel electrophoresis of PCR amplicons and RFLPbyproducts. Lanes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13represent 100 bp ladder, PCR product (M1+M3), W/W+W/W

(M1+M3), W/M1+W/W for M3, M1/M1+W/W for M3, PCRproduct (M2+M4), W/W (M2), W/M2, M2/M2, PCR product(M2+M4), W/W for M4, W/M4 and M4/M4 respectively


123

allelic frequencies in case controls and patients on the

basis of menopausal state showed lack of significant

alteration both in premenopausal and postmenopausal

women (Table 3). Both homozygosity (OR, 4.55;

CI, 0.44–226.2) and heterozygosity (OR, 1.2; CI, 0.65–

2.24) for CYP1A1 M4 variant allele were not found

Table 1 Allelic andgenotypic frequencies ofCYP1A1 M1 allele inpremenopausal andpostmenopausal case controlsand breast cancer patients innorth Indian women

Patients Case controls Odds ratio (95% CI) P-value

Total women n = 105 n = 116Allele frequency (total number of alleles)M1 (T) 0.70 (149) 0.69 (161)M1 (C) 0.29 (61) 0.30 (71)

Genotypic frequency (total number of genotypes)M1 (T/T) 0.50 (53) 0.50 (58) 1.0 (Reference) –M1 (T/C) 0.41 (43) 0.38 (45) 1.07 (0.59–1.87) 0.95M1 (C/C) 0.09 (9) 0.11 (13) 0.74 (0.28–1.96) 0.67Total premenopausal women n = 37 n = 80

Allele frequency (total number of alleles)M1 (T) 0.69 (51) 0.65 (105)M1 (C) 0.31 (23) 0.34 (55)

Genotypic frequency (total number of genotypes)M1 (T/T) 0.46 (17) 0.43 (35) 1.0 (Reference) –M1 (T/C) 0.46 (17) 0.43 (35) 1.09 (0.45–2.49) 0.96M1 (C/C) 0.08 (3) 0.12 (10) 0.62 (0.10–2.66) 0.75Total postmenopausal women n = 68 n = 36

Allele frequency (total number of alleles)M1 (T) 0.72 (98) 0.78 (56)M1 (C) 0.27 (38) 0.22 (16)

Genotypic frequency (total number of genotypes)M1 (T/T) 0.53 (36) 0.63 (23) 1.0 (Reference) –M1 (T/C) 0.38 (26) 0.28 (10) 1.60 (0.60–4.22) 0.42M1 (C/C) 0.09 (6) 0.08 (3) 1.06 (0.22–7.33) 1.00

Table 2 Allele and genotypicfrequencies of CYP1A1 M2allele in premenopausal andpostmenopausal case controlsand breast cancer patients innorth Indian women


Total women n = 105 n = 116Allele frequency (total number of alleles)M2 (A) 0.88 (185) 0.78 (181)M2 (G) 0.11 (25) 0.21 (51)

Genotypic frequency (total number of genotypes)M2 (A/A) 0.76 (80) 0.60 (70) 1.0 (Reference) –M2 (A/G) 0.23 (25) 0.35 (41) 0.57 (0.28–1.02) 0.06M2 (G/G) 0 0.04 (5) – –Total premenopausal women n = 37 n = 80

Allele frequency (total number of alleles)M2 (A) 0.82 (61) 0.78 (125)M2 (G) 0.17 (13) 0.21 (35)

Genotypic frequency (total number of genotypes)M2 (A/A) 0.64 (24) 0.61 (49) 1.0 (Reference) –M2 (A/G) 0.35 (13) 0.33 (27) 1.06 (0.40–2.47) 0.83M2 (G/G) 0 0.05 (4) – –Total postmenopausal women n = 68 n = 36

Allele frequency (total number of alleles)M2 (A) 0.91 (124) 0.77 (56)M2 (G) 0.08 (12) 0.22 (16)

Genotypic frequency (total number of genotypes)M2 (A/A) 0.82 (56) 0.58 (21) 1.0 (Reference) –M2 (A/G) 0.17 (12) 0.38 (14) 0.33 (0.12–0.89) 0.03M2 (G/G) 0 0.03 (1) – –


123

significantly associated with the breast cancer risk. The

results clearly suggested lack of association of M4

allelic polymorphism with the breast cancer risk in

north Indian women.

Differential expression of CYP1A1

The involvement of allelic polymorphism in breast

cancer was analyzed by measuring CYP1A1 expres-

sion in all genotypes of case controls and patients.

Differential expression profile in W/W, M1/W,

M1/M1+M2/W, M4/W, M4/M4 and M2/W+M4/W

genotypes clearly suggested lack of significant alter-

ation in the expression of CYP1A1 gene in north

Indian women (Fig. 2A, B). There was no significant

alteration observed in CYP1A1 expression in variant

homozygous/heterozygous genotypes as compared

with wild type genotype (Fig. 2A, B). Although

CYP1A1 expression was not significantly altered due

to allelic polymorphism both in premenopausal and

postmenopausal control women but some increase

was observed in individuals with M2/W+M4/W and

M4/M4 variant genotype. A typical CYP1A1 expres-

sion profile of wild type and variant genotypes among

case controls is shown in Fig. 2A, B and similar

pattern was also found in patients. Similarly, no sig-

nificant change was observed in CYP1A1 mRNA

expression among wild type and variant genotypes

both in premenopausal and postmenopausal breast

cancer patients.

CYP1A1 catalytic activity

The measurement of EROD activity in WBCs was

used for examining CYP1A1 catalytic activity. EROD

activity was compared in wild type and variant groups

in case controls and patients. As observed in expres-

sion profile, CYP1A1 activity was not significantly al-

tered due to allelic polymorphism; however, some

increase in EROD activity in individuals with M2/

W+M4/W and M4/M4 variant genotypes was observed.

EROD activity in WBCs of groups comprising of W/

W, M1/W, M1/M1+M2/W, M4/W, M4/M4 and M2/

W+M4/W genotypes clearly suggested lack of signifi-

cant alteration in EROD activity in north Indian wo-

men (Fig. 2C). Similarly, no significant change was

observed in CYP1A1 activity between wild type and

variant genotypes among premenopausal and post-

menopausal breast cancer patients and respective case

controls.

Discussion

Breast cancer etiologies not only depend on environ-

mental factors but also on several other contributory

Table 3 Allele and genotypicfrequencies of CYP1A1 M4allele in premenopausal andpostmenopausal case controlsand breast cancer patients innorth Indian women


Total women n = 105 n = 116Allele frequency (total number of alleles)M4 (C) 0.80 (168) 0.85 (198)M4 (A) 0.20 (42) 0.15 (34)

Genotypic frequency (total number of genotypes)M4 (C/C) 0.64 (67) 0.72 (83) 1.0 (Reference) –M4 (C/A) 0.32 (34) 0.27 (32) 1.2 (0.65–2.24) 0.65M4 (A/A) 0.04 (4) 0.01 (1) 4.55 (0.44–226.2) 0.19Total premenopausal women n = 37 n = 80

Allele frequency (total number of alleles)M4 (C) 0.82 (61) 0.86 (138)M4 (A) 0.18 (13) 0.14 (22)

Genotypic frequency (total number of genotypes)M4 (C/C) 0.70 (26) 0.74 (59) 1.0 (Reference) –M4 (C/A) 0.24 (9) 0.25 (20) 0.96 (0.36–2.64) 0.85M4 (A/A) 0.05 (2) 0.01 (1) 4.51 (0.23–273.0) 0.23Total postmenopausal women n = 68 N = 36

Allele frequency (total number of alleles)M4 (C) 0.79 (107) 0.83 (60)M4 (A) 0.21 (29) 0.17 (12)

Genotypic frequency (total number of genotypes)M4 (C/C) 0.60 (41) 0.66 (24) 1.0 (Reference) –M4 (C/A) 0.36 (25) 0.33 (12) 1.16 (0.45–2.94) 0.93M4 (A/A) 0.03 (2) 0 – –


123

factors. Dietary habits play a major role in the patho-

genesis of breast cancer. A correlation between me-

thyl-deficient diets and antioxidant vitamins and breast

cancer risk as a function of MspI genotype is reported

in a small number of cases with polymorphisms at both

sites [33]. However, large-scale epidemiological studies

in African-American and Caucasian women to include

genotype information from controls with more detailed

information on risk factors is still needed for reaching

at any conclusion [33]. Allelic polymorphism in

CYP1A1 gene and its association with breast cancer

risk has been highly inconsistent. Several studies re-

ported significant association, however, some studies

did not show any association [23, 25, 34, 35].

Researchers have suggested that association between

M1, M2, M3 and M4 alleles and breast cancer inci-

dence varies due to ethnicity of the population, envi-

ronmental factors and personal habits such as smoking.

The present study was therefore undertaken in a

population based case controls and breast cancer pa-

tients in individuals who were residents of Lucknow

and adjacent cities of north India.

In present study a significant association between

M1, M3 and M4 polymorphism with breast cancer risk

in north Indian women was not found, however, a sig-

nificant protective association of M2 genotype poly-

morphism with breast cancer risk was observed in

postmenopausal women. Although an association

between breast cancer risk and M1 and M2 genotype

polymorphism was reported in south Indian women

[36] but we did not see significant association between

M1 genotype polymorphism and breast cancer risk in

north Indian women. The reason for discrepancy could

be due to ethnic difference between north and south

Indian populations. Several investigators consistently

observed the discrepancy due to ethnic differences. A

moderate to strong association for M1 genotype was

reported in African-American women and a weak

positive association for M2 genotype was reported in

Caucasians [23, 25]. Earlier studies have confirmed

either population specific association or lack of associ-

ation between CYP1A1 variants and breast cancer risk

[34, 35]. CYP1A1*2A (M1) and CYP*2C (M2) alleles

were not found significantly associated with breast

cancer risk in Caucasian population [22, 25, 35, 37],

however, in Chinese population homozygosity for M1

and M2 alleles were found significantly associated with

breast cancer risk, particularly in postmenopausal wo-

men with a long duration of estrogen exposure [14]. A

positive association between CYP1A1 M3 genotype

with the breast carcinoma was reported in African

population [20], however, in our study we could not find

any association with M1 and M3 polymorphism. Lack

of CYP1A1 M3 genotype in any case controls and

patients in north Indian population showed M3 speci-

ficity to African-American population [20].

The expression study of CYP1A1 polymorphic

patients and respective controls were performed and

results obtained clearly showed lack of significant

alteration in the expression of CYP1A1. Furthermore,

EROD activity in WBC also showed similar patterns.

The present study clearly showed that CYP1A1 M1, M2

and M4 polymorphism neither in case controls nor in

premenopausal or postmenopausal patients produced

significant alteration in the expression and activity of

CYP1A1. In the present study, a protective association

between M2 allele polymorphism and postmenopausal

breast cancer risk was observed, however, no significant

change in the expression was found.

It was assumed that the inhibitory or augmentary

effect on CYP1A1 catalytic activity could likely play a

key role in breast cancer risk since relative importance

0

0.1

0.2

0.3

0.4

0.5

0.6

Genotype

CY

P1A

1/G

APD

H

0

2

4

6

8

Genotypepmol

e re

suru

fin/

min

/mg

prot

ein

W/W

M1/W

M1/M1+M2/W

M4/W

M4/M4

M2/W+M4/W

W/W

M1/W

M1/M1+M2/W

M4/W

M4/M4

M2/W+M4/W

1-W/W2-M1/W3-M1/M1+M2/W4-M4/W5-M4/M46-M2/W+M4/W

CYP1A1

GAPDH

1 2 3 4 5 6A

B

C

Fig. 2 Differential expression of CYP1A1 mRNA and ERODactivities in white blood cells. (A) Differential expression ofCYP1A1 in individuals having different genotype. Lanes 1, 2, 3,4, 5 and 6 represent CYP1A1 expression in W/W, M1/W, M1/M1+M2/W, M4/W, M4/M4 and M2/W+M4/W. (B) Bar diagramsshowing differential expression of CYP1A1 gene in individualshaving different genotype. Values are represented in mean ± SE.Bars 1, 2, 3, 4, 5 and 6 represent W/W, M1/W, M1/M1+M2/W,M4/W, M4/M4 and M2/W+M4/W. (C) Bar diagram showingEROD enzymatic activity of CYP1A1 in individuals having W/W, W/M1, M1/M1+M2/W, M4/W, M4/M4 and M2/W+M4/W.Values are represented in mean ± SE. Bars 1, 2, 3, 4, 5 and 6represent W/W, M1/W, M1/M1+M2/W, M4/W, M4/M4 and M2/W+M4/W


123

and expression of CYP1 enzymes in vivo depends on

the specific tissue [38]. Collectively, the data obtained,

favored the idea that probably CYP1A1 alone is not a

significant contributory factor in breast cancer risk

particularly in north India. The contributory risk factor

could be some other toxicant responsive genes in

combination with CYP1A1 that needs to be elucidated.

Since study showed lack of significant association be-

tween CYP1A1 polymorphism and breast cancer risk,

therefore, further investigation in estrogen-metaboliz-

ing genes and estrogen receptor genes are needed for

proper explanation. As reported in Chinese [39], lack

of association could possibly be explained as not only

CYP1A1 polymorphism but estrogen-metabolizing

genes and estrogen receptor genes polymorphism in

combination could play a role in the etiology of breast

cancer risk in north Indian women.

Acknowledgements Authors sincerely thank University GrantCommission (UGC), New Delhi, India for providing researchfellowship to Virendra Singh and Council of Scientific andIndustrial Research (CSIR), New Delhi, India for providing re-search fellowship to Abhai Kumar. Authors also thank Director,ITRC, Lucknow, India for providing necessary facilities requiredfor this study.

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