genetic variability in cyp2a6 and the pharmacokinetics of nicotine

REVIEWFor reprint orders, please contact:[email protected]

Genetic variability in CYP2A6 and the pharmacokinetics of nicotine
Jill C Mwenifumbo & Rachel F Tyndale†
†Author for correspondenceUniversity of Toronto, Rm 4326 Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, Ontario, M5S 1A8, CanadaFax: +1 416 978 6395;E-mail: [email protected]

part of

Keywords: cessation, cotinine, CYP2A6, ethnicity, lung cancer, metabolism, nicotine, polymorphism, smoking, trans-3´-hydroxycotinine

10.2217/14622416.8.10.1385 ©

Nicotine is the psychoactive substance responsible for tobacco dependence. It is also a therapeutic used to aid smoking cessation. Cytochrome P450 (CYP)2A6 is the human hepatic enzyme that mediates most of nicotine’s metabolic inactivation to cotinine. Genetic variation in the CYP2A6 gene can increase or decrease enzyme activity through altering the protein’s expression level or its structure and function. This article reviews CYP2A6 genetic variation and its impact on in vivo nicotine kinetics, including a description of the individual variants, different phenotyping approaches for assessing in vivo CYP2A6 activity and other sources of variation in nicotine metabolism such as gender. In addition, the effect of CYP2A6 polymorphisms on smoking behavior and tobacco-related lung cancer risk are briefly described. Furthering knowledge in this area will improve interpretation of studies examining smoking behavior, as well as those using nicotine as a therapeutic agent.

Nicotine is an alkaloid that acts on nicotinicacetylcholine receptors in the central and periph-eral nervous systems. Centrally, nicotine canmodify drug taking behavior, learning, memoryand other neurobiological processes. Peripher-ally, nicotine produces a wide range of physio-logical effects via the autonomous nervoussystem, which regulates cardiovascular, digestiveand endocrine function.

Determining sources of variation in nicotine’smetabolism is important for several reasons.First, nicotine is implicated in the developmentand maintenance of tobacco dependence [1], andsmoking results in exposure to a multitude ofcarcinogens [2]. Second, nicotine-replacementtherapy is widely used to aid smokingcessation [3,4]. Third, preliminary trials areunderway investigating nicotine as a treatmentfor diseases/disorders where it has been foundthat smokers are afforded a measure of protec-tion, such as attention deficit disorder [5,6],Alzheimer’s disease [7,8], Parkinson’s disease [9,10],Tourette’s syndrome [11,12] and ulcerativecolitis [13,14].

The primary aim of this article is to review thecurrent state of knowledge regarding the geneticvariability in cytochrome P450 (CYP)2A6 and itsimpact on nicotine pharmacokinetics. The focusis on describing polymorphisms and several large-scale CYP2A6 genetic/in vivo nicotine kineticassociation studies. In addition, other sources ofvariation in nicotine C-oxidation and the implica-tions of this variability will be discussed. CYP2A6makes a sizable contribution to the range of nico-tine metabolic capacity observed in humans; thus,

brief mention will be made of the relationshipbetween CYP2A6 genotype with both smokingbehaviors and tobacco-related lung cancer.

Nicotine metabolismIn humans, nicotine’s primary route of elimina-tion is through hepatic metabolism; on average,70–80% of absorbed nicotine is metabolicallyinactivated to cotinine [15]. CYP2A6 is the drug-metabolizing enzyme that mediates most of nic-otine’s C-oxidation to cotinine [16,17]. This wasestablished experimentally in a study of humanliver microsomes, where cotinine formation washighly correlated with CYP2A6 protein levels(r = 0.90; p < 0.001) [16], inhibited (>75%) by aCYP2A6 monoclonal antibody [16], and alsoinhibited by the specific CYP2A6 substratecoumarin (>85%) [16,18]. In the liver, cotinine issubsequently hydroxylated to trans-3´-hydroxy-cotinine by CYP2A6 [19]. These in vitro studiesall described a wide range of CYP2A6 proteinlevels and activities among human livers and theresultant extensive variability in nicotine C-oxi-dation and cotinine hydroxylation. Takentogether, these findings suggested a highlypolymorphic CYP2A6 gene [16,17,19].

CYP2A gene clusterThe CYP2A6 gene is located on the long arm ofchromosome 19q13.2 [20]. It is found amidst a500-kb cluster of CYP2 family genes and pseu-dogenes (i.e., CYP2A, 2B, 2F, 2G, 2S and2T) [20]. There are four members in the humanCYP2A subfamily: CYP2A6, CYP2A7, CYP2A13and the split pseudogene CYP2A18PC and

2007 Future Medicine Ltd ISSN 1462-2416 Pharmacogenomics (2007) 8(10), 1385–1402 1385

REVIEW – Mwenifumbo & Tyndale

1386

CYP2A18PN [20]. The CYP2A6 gene locusspans approximately 6 kb and consists of nineexons, which encode 494 amino acids [21].CYP2A6 is highly polymorphic [201]. It can existas a deleted or duplicated gene and can containgene conversions, nucleotide deletions andnucleotide insertions, as well as SNPs. The lastfew years have seen substantial advances in theidentification and characterization of CYP2A6polymorphisms; however, it is apparent thatgaps in this knowledge still exist, especially innon-Caucasian and non-Asian populations.

Established CYP2A6 allelesThe discovery of new CYP2A6 alleles has pro-gressed rapidly and over 30 alleles are currentlynamed. Several alleles (e.g., CYP2A6*13) haveyet to be characterized with respect to their func-tional impact on metabolic activity in vivo. Thecurrent state of knowledge regarding CYP2A6alleles and their functional impact is summarizedin Table 1. Multiple additional SNPs (not yetassigned to alleles) remain to be characterizedwith respect to their haplotype, frequency andfunctional impact on enzyme activity [22–24].

CYP2A6*2, *4, *7, *10 and *17 dramaticallyreduce CYP2A6 activity towards nicotine in vivo.Homozygous or hemizygous individuals withthese alleles have substantially reduced capacityfor nicotine metabolism [25–28]. CYP2A6*5, *6,*11, *19 and *20 are predicted to dramaticallyreduce CYP2A6 activity towards nicotine becausetheir cDNA-expressed proteins have little-to-noenzyme activity [29–33]; however, the in vivoimpact on nicotine metabolism has not been con-firmed in homozygous or hemizygous individuals.CYP2A6*9 and *12 are associated with modestlyreduced nicotine metabolism in vitro [34,35] andin vivo [34,36,37]. CYP2A6*1B is associated withincreased mRNA, protein level and activity invitro [38] and moderately increased nicotinemetabolism in vivo [39]. There is evidence that thetwo types of duplication alleles (CYP2A6*1X2Aand *1X2B) may result in increased nicotinemetabolism [40,41]. The impact of CYP2A6*8 [27,42],*13 [23], *14 [23], *15 [23], *16 [23], *18 [32], *21 [43]

and *22 [24] on nicotine metabolism in vivo hasnot been explicitly demonstrated. Of these alleles,CYP2A6*13, *15 and *22 are predicted to have atleast reduced nicotine metabolism owing to thepresence of specific SNPs. CYP2A6*8, *14, *18and *21 have not been associated with reducednicotine metabolism in vivo [37,43], and expressedCYP2A6.18 does not differ from wild-typewhen nicotine is the substrate [32]. Similarly,

CYP2A6*1D, *1G, *1H and *1J, all of which con-tain SNPs in putative regulatory regions, have notbeen associated with reduced nicotine metabolismin vivo [37], despite their demonstrated reducedtranscription in vitro [44,45].

The frequencies of CYP2A6 alleles vary acrossethnic groups (Table 2). Figure 1 shows the differ-ences in proportions of normal, intermediate,slow and poor nicotine metabolism groups, aspredicted by CYP2A6 genotype, among differentethnicities. The proportion of persons with poornicotine metabolism (<25% activity), predictedfrom the frequencies of CYP2A6 variant alleles,is ranked as follows:

• Japanese: 12%

• Korean: 6%

• Chinese: 4%

• African–American: 2%

• Caucasian: less than 1%.

The interethnic variability in the proportion ofCYP2A6 decrease- or loss-of-function alleles isgenerally consistent with the interethnicdifferences in nicotine metabolism [46–48].

In vivo CYP2A6 activity phenotypingTo phenotype the activity of a genetically variabledrug-metabolizing enzyme in individuals, apharmacokinetic study is typically required [49].This type of study involves the administration ofa single oral dose of drug to a study participantwho is free of the drug. The resulting plasma orurine concentrations of the parent drug and theproximal metabolite produced from the meta-bolic pathway of interest are then assessed at anoptimal time point. This method is used to deter-mine the cotinine:nicotine ratio (COT:NIC), ametabolic ratio that estimates the capacity of coti-nine formation from nicotine. Thus, this kineticparameter is a proxy measure of CYP2A6 activityin vivo. However, a second enzyme may contrib-ute up to 10% of the nicotine C-oxidation path-way [16,50], making this pathway somewhat lessspecific for CYP2A6 phenotyping.

Nicotine kinetic studies differ from otherdrug studies because a sizeable proportion ofthe population uses tobacco and consequentlyhas plasma levels of nicotine and its proximalmetabolites. So, while a good measure in non-smokers, COT:NIC may not be an appropriatemeasure of nicotine C-oxidation activity inactive smokers because cotinine, and to a lesserextent nicotine, may be present prior to pheno-typing. A significant limitation to theCOT:NIC approach to CYP2A6 phenotyping

Pharmacogenomics (2007) 8(10) future science groupfuture science group

Genetic variability in CYP2A6 and the pharmacokinetics of nicotine – REVIEW

Tab

le 1

. CY

P2A

6 al

lele

s, t

hei

r im

pac

t o

n C

YP2

A6

leve

ls a

nd

/or

acti

vity

in v

itro

, an

d a

sso

ciat

ion

s w

ith

in v

ivo

met

abo

lism

.

CY

P2A

6 al

lele

Def

inin

g g

eno

mic

DN

A

chan

ges

Am

ino

aci

dch

ang

eLo

cati

on

Alle

le d

escr

ipti

on

Ref

.

*1A

Refe

renc

e

*1B‡

58 b

p C

YP2

A7

gene

co

nver

sion

(3´-U

TR g

ene

conv

ersi

on)

3´-U

TRIn

vitr

o, C

YP2

A6*

1B r

esul

ts in

hig

her

tran

scrip

tion#

thr

ough

mRN

A

stab

iliza

tion.

In v

ivo,

hav

ing

one

or t

wo

copi

es o

f th

e al

lele

res

ults

in f

aste

r ni

cotin

e cl

eara

nce

[18,

38,3

9,11

7]

*1D

-101

3A>

G

5’-P

RE(-

1005

to

-101

9)In

vitr

o, C

YP2

A6*

1D r

esul

ts in

50%

low

er t

rans

crip

tion#

. How

ever

, in

hum

an

liver

sam

ples

, thi

s al

lele

is n

ot a

ssoc

iate

d w

ith m

RNA

leve

ls. I

n vi

vo, t

he im

pact

is

unc

lear

[37,

44]

*1F

5717

C>

TP4

08P

Exon

8In

viv

o, t

he im

pact

is u

ncle

ar[3

7,11

8]

*1G

5717

C>

T58

25A

>G

P408

PEx

on 8

In

tron

8In

viv

o, t

he im

pact

is u

ncle

ar[3

7,11

8]

*1H

-745

A>

G5’

-CC

AA

T(-

748

to-7

45)

In v

itro,

CY

P2A

6*1H

res

ults

in 2

0% lo

wer

tra

nscr

iptio

n#. I

n vi

vo, t

he im

pact

is

unc

lear

[37,

45]

*1J

-101

3A>

G -

745A

>G

5’-f

lank

ing

In v

ivo,

the

impa

ct is

unc

lear

[37,

45]

*217

99T>

AL1

60H

Exon

3Ex

pres

sed

CY

P2A

6.2

is u

nsta

ble,

fai

ls t

o in

corp

orat

e he

me

and

is

enzy

mat

ical

ly in

activ

e. In

viv

o, t

his

alle

le is

inac

tive

[18,

25]

*3C

YP2

A7

gene

con

vers

ions

in

exo

ns 3

, 6 a

nd 8

CY

P2A

6*3

is a

hyb

rid a

llele

. It i

s th

ough

t to

be in

activ

e, a

lthou

gh it

is v

ery

rare

in

all

popu

latio

ns[2

1,11

9]

*4A

-D¶

5’ s

eque

nce

is o

f C

YP2

A7

orig

in a

nd 3

’ seq

uenc

e is

of

CY

P2A

6 or

igin

Hyb

rid-d

elet

ed a

llele

tho

ught

to

have

occ

urre

d by

hom

olog

ous

uneq

ual

cros

sove

r. In

CY

P2A

6*4A

, the

3’-

UTR

is o

f C

YP2

A6

orig

in. C

YP2

A6*

4B is

a

com

plet

e ge

ne d

elet

ion.

CY

P2A

6*4C

and

CY

P2A

6*4A

are

iden

tical

. C

YP2

A6*

4D s

eque

nce

beyo

nd e

xon

9 is

of

CY

P2A

6 or

igin

. In

vivo

, the

se

alle

les

are

inac

tive

[117

,119

–123

]

*565

82G

>T

and

3´-U

TR g

ene

conv

ersi

on

G47

9VEx

on 9

Expr

esse

d C

YP2

A6.

5 is

uns

tabl

e an

d is

enz

ymat

ical

ly in

activ

e. C

YP2

A6*

5 w

as

disc

over

ed in

a h

emiz

ygou

s in

divi

dual

with

poo

r co

umar

in m

etab

olis

m.

Invi

vo, t

he im

pact

is u

ncle

ar

[29]

*617

03G

>A

R1

28Q

Exon

3Ex

pres

sed

CY

P2A

6.6

has

a di

sord

ered

hol

opro

tein

str

uctu

re a

nd h

as 8

5%

low

er c

oum

arin

7-h

ydro

xyla

tion

activ

ity. I

n vi

vo, t

he im

pact

is u

ncle

ar[3

0,37

]

Nu

cleo

tid

e se

qu

ence

nu

mb

ers

are

+1

wit

h r

efer

ence

to

th

e A

TG s

tart

sit

e o

n t

he

refe

ren

ce g

eno

mic

seq

uen

ce N

G_0

0000

8.7.

SNPs

fo

un

d in

co

din

g r

egio

ns

and

th

at r

esu

lt in

an

am

ino

aci

d c

han

ge

are

sho

wn

in b

old

.‡ T

he

CY

P2A

6*1B

alle

les

are

nu

mb

ered

fro

m 1

–16

to d

isti

ng

uis

h b

etw

een

hap

loty

pe

dif

fere

nce

s in

DN

A v

aria

tio

n lo

cate

d in

th

e 5’

- an

d 3

’-fl

anki

ng

reg

ion

s, in

tro

ns

and

syn

on

ymo

us

SNPs

in e

xon

s.§ L

ette

rs in

dic

ate

a d

iffe

ren

ce in

hap

loty

pes

.¶Le

tter

s in

dic

ate

a d

iffe

ren

ce in

th

e cr

oss

ove

r re

gio

n.

# Tra

nsc

rip

tio

n in

a lu

cife

rase

co

nst

ruct

rep

ort

er s

yste

m.

PRE:

Pro

mo

ter

resp

on

sive

ele

men

t; U

TR: U

ntr

ansl

ated

reg

ion

.

1387future science groupfuture science group www.futuremedicine.com


*765

58T>

C a

nd

3´-U

TR g

ene

conv

ersi

onI4

71T

Exon

9Ex

pres

sed

CY

P2A

6.7

is u

nsta

ble,

doe

s no

t m

etab

oliz

e ni

cotin

e an

d ha

s 40

%

low

er c

oum

arin

7-h

ydro

xyla

tion

activ

ity. I

n vi

vo, t

his

alle

le is

ess

entia

lly

inac

tive

tow

ards

nic

otin

e

[27,

124,

125]

*866

00G

>T

and

3´-U

TR g

ene

conv

ersi

onR4

85L

Exon

9In

viv

o, t

he im

pact

is u

ncle

ar. H

owev

er, i

t is

like

ly t

o be

com

para

ble

with

wild

-typ

e [2

7,42

,125

]

*9A

-B§

-48T

>G

TATA

box

In v

itro,

CY

P2A

6*9

resu

lts in

55%

low

er t

rans

crip

tion#

. In

vivo

, thi

s al

lele

has

re

duce

d ac

tivity

[36,

37,4

4,12

6]

*10

6558

T>C

, 660

0G>

T an

d 3´

-UTR

gen

e co

nver

sion

I471

T, R

485L

Exon

9In

viv

o, t

his

alle

le is

inac

tive.

[27,

42,1

25]

*11

3391

T>C

S2

24P

Exon

5Ex

pres

sed

CY

P2A

6.11

res

ults

in a

60%

dec

reas

e in

act

ivity

tow

ard

tega

fur

activ

atio

n an

d a

40%

dec

reas

e in

cou

mar

in 7

-hyd

roxy

lase

act

ivity

. C

YP2

A6*

11 w

as d

isco

vere

d in

a h

emiz

ygou

s in

divi

dual

with

poo

r te

gafu

r m

etab

olis

m. I

n vi

vo, t

he im

pact

is u

ncle

ar

[31]

*12A

-C§

5’-f

lank

ing

regi

on a

nd

exon

s 1–

2 ar

e of

CY

P2A

7 or

igin

and

exo

ns 3

–9 a

re o

f C

YP2

A6

orig

in

Ten

amin

o ac

id

subs

titut

ion

Expr

esse

d C

YP2

A6.

12 is

uns

tabl

e an

d re

sults

in a

40%

dec

reas

e in

cou

mar

in

7-hy

drox

ylas

e ac

tivity

. CY

P2A

6*12

was

dis

cove

red

in a

hom

ozyg

ous

indi

vidu

al w

ith 5

5% lo

wer

cou

mar

in 7

-hyd

roxy

latin

g ac

tivity

. In

vivo

, thi

s al

lele

has

red

uced

act

ivity

[34,

36]

*13

-48T

>G

and

13G

>A

G5R

TATA

box

Exon

1In

viv

o, t

he im

pact

is u

ncle

ar. H

owev

er, t

his

alle

le is

like

ly t

o re

sult

in r

educ

ed

activ

ity b

ecau

se it

con

tain

s th

e -4

8T>

G S

NP

foun

d in

CY

P2A

6*9

[23,

24,3

7]

*14

86G

>A

S29N

Exon

1In

viv

o, t

he im

pact

is u

ncle

ar. H

owev

er, i

t is

like

ly t

o be

com

para

ble

with

wild

-typ

e [2

3,37

]

*15

-48T

>G

and

213

4A>

GK

194E

TATA

box

Exon

4In

viv

o, t

he im

pact

is u

ncle

ar. H

owev

er, t

his

alle

le is

like

ly t

o re

sult

in r

educ

ed

activ

ity b

ecau

se it

con

tain

s th

e -4

8T>

G S

NP

foun

d in

CY

P2A

6*9

[23,

24,3

7]

*16

2161

C>

AR2

03S

Exon

4In

viv

o, t

he im

pact

is u

ncle

ar[2

3,37

]

*17

5065

G>

AV

365M

Exon

7Ex

pres

sed

CY

P2A

6.17

has

60%

low

er n

icot

ine

C-o

xida

tion

and

40%

low

er

coum

arin

7-h

ydro

xyla

tion

activ

ity. I

n vi

vo, t

his

alle

le is

ess

entia

lly in

activ

e to

war

ds n

icot

ine

[28]

Tab

le 1

. CY

P2A

6 al

lele

s, t

hei

r im

pac

t o

n C

YP2

A6

leve

ls a

nd

/or

acti

vity

in v

itro

, an

d a

sso

ciat

ion

s w

ith

in v

ivo

met

abo

lism

(co

nt.

).

CY

P2A

6 al

lele

Def

inin

g g

eno

mic

DN

A

chan

ges

Am

ino

aci

dch

ang

eLo

cati

on

Alle

le d

escr

ipti

on

Ref

.

Nu

cleo

tid

e se

qu

ence

nu

mb

ers

are

+1

wit

h r

efer

ence

to

th

e A

TG s

tart

sit

e o

n t

he

refe

ren

ce g

eno

mic

seq

uen

ce N

G_0

0000

8.7.

SNPs

fo

un

d in

co

din

g r

egio

ns

and

th

at r

esu

lt in

an

am

ino

aci

d c

han

ge

are

sho

wn

in b

old

.‡ T

he

CY

P2A

6*1B

alle

les

are

nu

mb

ered

fro

m 1

–16

to d

isti

ng

uis

h b

etw

een

hap

loty

pe

dif

fere

nce

s in

DN

A v

aria

tio

n lo

cate

d in

th

e 5’

- an

d 3

’-fl

anki

ng

reg

ion

s, in

tro

ns

and

syn

on

ymo

us

SNPs

in e

xon

s.§ L

ette

rs in

dic

ate

a d

iffe

ren

ce in

hap

loty

pes

.¶Le

tter

s in

dic

ate

a d

iffe

ren

ce in

th

e cr

oss

ove

r re

gio

n.

# Tra

nsc

rip

tio

n in

a lu

cife

rase

co

nst

ruct

rep

ort

er s

yste

m.

PRE:

Pro

mo

ter

resp

on

sive

ele

men

t; U

TR: U

ntr

ansl

ated

reg

ion

.

1388 Pharmacogenomics (2007) 8(10) future science groupfuture science group


*18A

-C‡

5668

A>

TY

392F

Exon

8Ex

pres

sed

CY

P2A

6.18

has

100

% n

icot

ine

C-o

xida

tion

activ

ity a

nd 5

0% lo

wer

co

umar

in 7

-hyd

roxy

latio

n ac

tivity

. In

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is keeping smokers abstinent for the long peri-ods of time required to remove nicotine metab-olites. Likewise, this metabolic ratio is notappropriate to measure nicotine C-oxidationactivity in smokers during ad libitum smoking.Following smoking a cigarette, plasma nicotinelevels rapidly increase and then decrease [51],but the concentration of cotinine remainsfairly stable owing to its long half-life [51].Therefore, depending on the time of the lastcigarette, COT:NIC has the potential to varyextensively within an individual.

Another metabolic ratio typically used tophenotype CYP2A6 activity in vivo is the trans-3´-hydroxycotinine:cotinine ratio (3HC:COT). Evi-dence suggests that CYP2A6 is responsible for100% of the hydroxylation of cotinine to trans-3´-hydroxycotinine [19,52,53]. As this pathway is spe-cific to, and selective for, CYP2A6, the metabolicratio of 3HC:COT can be used to assess its relativeactivity [52,54,55]. Plasma and salivary 3HC:COTare highly correlated with the clearance of orallyadministered nicotine in smokers and nonsmokers[52,56]. 3HC:COT is generally independent of thetime of day of sampling in smokers [57] and is notpredicted to vary with smoking patterns owing tothe long half-lives of cotinine and trans-3´-hydroxycotinine [58]. In 137 light smokers, theplasma 3HC:COT ratio derived from ad libitumsmoking highly correlated with the ratio at 1.5 h(Spearman’s rho = 0.87; p < 0.001) and 4.5 hours(Spearman’s rho = 0.89; p < 0.001) after 4 mg oforal nicotine [59]. The ratios at 1.5 and 4.5 hafter oral nicotine were also highly correlated(Spearman’s rho = 0.96; p < 0.001) [Mwenifumbo JC

and Tyndale RF. Unpublished Observations].The clearance of nicotine to cotinine

(CLNIC→COT) quantifies the rate of cotinineformation and is a proxy measure of CYP2A6activity in vivo. To calculate this kinetic para-meter, nicotine’s metabolic clearance (CLnonrenal)and the fractional conversion of nicotine tocotinine (ƒ) must be determined. ƒ is an esti-mate of the percentage of nicotine that is con-verted to cotinine and approximates theproportion that CYP2A6 contributes to an indi-vidual’s metabolic clearance. ƒ significantly corre-lates with total nicotine clearance [15].CLNIC→COT can be calculated by multiplyingCLnonrenal and ƒ. These kinetic parameters can beassessed in both active smokers and nonsmokers ifisotope-labeled nicotine and cotinine are used [15].However, a limitation is the multiple blood sam-pling time points for the measurement of drugand metabolite concentrations.

Nicotine metabolism in individuals homozygous for the CYP2A6 gene deletion alleleTwin studies demonstrate that variation inCYP2A6 activity, as assessed by CLNIC→COT, isstrongly influenced by genetic factors, with aheritability of approximately 60% in a predomi-nately Caucasian population [60]. However, thevariability in CLNIC→COT accounted for byCYP2A6 variant alleles known at that time wasmodest. Individuals homozygous for CYP2A6*4(the gene deletion) provide a classic demonstra-tion of CYP2A6 genotype’s effect on in vivonicotine-metabolism phenotype. The first studyto investigate this specific relationship assessedlevels of urinary cotinine, after smoking six ciga-rettes in 1 h, in a group of Japanese with thehomozygous deletion (CYP2A6*4/*4) (n = 6)and a wild-type control group (CYP2A6*1/*1)(n = 5). The homozygous deletion group had15% of the urinary cotinine level (after a 24-hcollection) of the wild-type controls [26]. A sub-sequent study examined urinary cotinine afterad libitum smoking in a larger group [61]. Again,the homozygous deletion group (n = 9) had 11%of the urinary cotinine level of the wild-typecontrol (CYP2A6*1) (n = 181) [61]. In a study ofJapanese nonsmokers, participants chewed onepiece of nicotine gum and urinary levels of nico-tine and nine of its metabolites were assessedover a 24-h collection period [53]. The wild-typegroup (n = 3) excreted mainly cotinine, trans-3´-hydroxycotinine and their respective glucuro-nides. In the two homozygous deletion individu-als, unchanged nicotine, nicotine N-glucuronideand nicotine 1´-N-oxide made up most of theexcreted metabolites. Very little (at most 5%),was excreted as cotinine, cotinine N-glucuronideand cotinine 1´-N-oxide [53]. Levels of trans-3´-hydroxycotinine were below the limit of quanti-fication in the two homozygous CYP2A6*4 indi-viduals. Generally, trans-3´-hydroxycotinine isthe most abundant metabolite (30–40%) in theurine of smokers [62,63]. These studies demonstrateddramatically different urinary metabolite profilesafter nicotine gum and cigarette smoking inindividuals homozygous for the CYP2A6 deletion.

When considering systemic exposure, follow-ing the oral administration of nicotine, individu-als homozygous for the CYP2A6 gene deletion(n = 3) had 3.6-fold greater mean nicotine plasmaarea under the concentration–time curve (AUC)and their mean AUC for cotinine was only 9% ofthat of the wild-type group (n = 5) (Figure 2) [27].This demonstrated that individuals without any



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functional CYP2A6, in this case owing to the genedeletion, have dramatically altered systemic expo-sure to nicotine and its proximal metabolite coti-nine. Other CYP2A6 substrates such as coumarinare also affected by the deletion genotype. As seenin Figure 2, the homozygous deletion group doesnot form 7-hydroxycoumarin. Coumarin is anexcellent probe substrate for distinguishingbetween those with any metabolic capacity andthose with no metabolic capacity; however, it doesnot discriminate well along the gradient ofCYP2A6 activity (e.g., normal, intermediate, slowand poor) [64]. These studies, and others [25], dem-onstrate that deficient nicotine C-oxidationin vivo can be attributed to CYP2A6 polymor-phisms and that this enzyme has a large effect onnicotine pharmacokinetics.

CYP2A6 polymorphisms & in vivo nicotine metabolism association studiesThe abovementioned pharmacokinetic studieswere relatively small and used the CYP2A6 genedeletion polymorphism to illustrate the impactof CYP2A6 on substrate metabolism. Recently,there have been four larger studies examiningthe association of multiple CYP2A6 alleles withnicotine metabolism in several ethnic groups, asdescribed below.

Study 1One formal pharmacokinetic study quantitativelyassessed nicotine metabolism and kinetics.Approximately 300 predominately Caucasianparticipants were dosed with intravenous infu-sions of isotope-labeled nicotine and cotinine andgenotyped for several CYP2A6 decrease- or loss-of-function alleles [36]. Based on the impact of individ-ual CYP2A6 genotypes, individuals were catago-rized into predicted normal (CYP2A6*1/*1),intermediate (CYP2A6*1/*9 and *1/*12) andslow (CYP2A6*1/*2, *1/*4, *4/*9, *9/*9, *9/*12and *12/*12) nicotine-metabolism groups. Over-all, the total clearance, nonrenal clearance, clear-ance of nicotine to cotinine, half-life, fractionalconversion of nicotine to cotinine and3HC:COT were significantly different betweenthe three groups. The normal metabolism was thereference group with 100% CLNIC→COT, theintermediate metabolism group had 80% CLN-

IC→COT and the slow metabolism group had 49%CLNIC→COT. The differences in nicotine meta-bolic capacity between the groups was supportedby urine data, where the normal metabolismgroup excreted the least amount of unchangednicotine and the most trans-3´-hydroxycotinine

compared with both the intermediate and theslow groups. These data indicated that, owingto greater CYP2A6 activity, the normal-metabolism group had the largest capacity fornicotine C-oxidation [36].

Study 2The association of multiple CYP2A6 decrease- orloss-of-function polymorphisms with kineticdata, acquired during a nicotine-replacementclinical trial, was tested in a treatment-seekingpopulation of Caucasians (n = 310) [54]. The samegenotype-based nicotine metabolism groups thatare described above were employed. The threegroups had different baseline 3HC:COT fromad libitum smoking and different plasma levels ofnicotine achieved from the transdermal patch inabstinent smokers. Compared with the normal-metabolism group, the slow group had 50%lower CYP2A6 activity at baseline and 44%higher steady-state plasma levels of nicotine [54].These findings demonstrated that CYP2A6 gen-otype altered CYP2A6 activity, as assessed by the3HC:COT ratio, and consequently affectedplasma nicotine levels achieved from thetransdermal patch.

Study 3 This pharmacokinetic study examined the varia-bility in CYP2A6 activity and CYP2A6 allelesamong four ethnic groups [37]. Following amethod originally established by Nakajima et al.,study participants were given nicotine gum, andplasma levels of nicotine and cotinine were meas-ured 2 h later. Plasma COT:NIC was used as aproxy measure of CYP2A6 activity [64]. The com-bined frequency of decrease- or loss-of-functionalleles was 9, 22, 43 and 51% in Caucasians(n = 176), African–Americans (n = 160), Kore-ans (n = 209) and Japanese (n = 92), respectively.Consistent with the other studies, within eachethnic group, individual CYP2A6 variant geno-types tended to have lower metabolic activity, butowing to low numbers and no grouping strategy,many associations could not be tested statisti-cally [37]. The mean COT:NIC ratios were quitesimilar between Caucasians, African–Americansand Koreans despite the differences in the fre-quencies of CYP2A6 polymorphisms. This find-ing is not in concordance with the reported 13%slower metabolic clearance of nicotine in Afri-can–Americans compared with Caucasians [46].Japanese were the only group that had considera-bly lower mean COT:NIC relative to the otherethnic groups [37].



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Study 4Another traditional pharmacokinetic study,using an experimental paradigm similar to thestudy described above, examined the associa-tion between CYP2A6 genotype and CYP2A6activity in a nonsmoking population of Thais(n = 120). The genotypes were categorized intoextensive (CYP2A6*1/*1), intermediate(CYP2A6*1/*4, *1/*7, *1/*9, *1/*10 and *9/*9),poor (CYP2A6*4/*7, *4/*9 and *7/*7) and verypoor (CYP2A6*4/*4) nicotine-metabolismgroups. The groups had mean COT:NIC ratiosof 100, 65, 24 and 0%, respectively [64]. Not-withstanding the minor differences in CYP2A6grouping, this study demonstrated a clearCYP2A6 genotype–phenotype association.

The association of CYP2A6 polymorphismswith in vivo CYP2A6 activity and nicotinemetabolism has been consistently demonstratedusing several different modes of nicotine admin-istration and different metabolic capacity assess-ment parameters, among several ethnic groups.Of note, an additional common observation in

all of these studies is the considerable variabilitythat exists in ‘wild-type’ groups, those withoutidentified variant alleles. This may be due to uni-dentified CYP2A6 polymorphisms or poly-morphic proteins involved in the regulation,transcription or translation of the gene. Inaddition, there are several other known contribu-tors to the variability in nicotine C-oxidation, asdiscussed below.

Sources of variability in nicotine C-oxidationGenderIn a study of nicotine pharmacokinetics in apopulation of black African descent, bothwomen smokers and nonsmokers had approxi-mately 25% higher mean CYP2A6 activity(3HC:COT) compared with their male counter-parts [59]. Likewise, women from predominatelyCaucasian populations have been shown to havehigher apparent nicotine elimination rate con-stants [66], lower steady-state plasma levels of nico-tine per cigarette [67], higher CYP2A6 activity [68]

Figure 1. Interethnic variability in the proportion of persons with normal, intermediate, slow and poor nicotine metabolism, as predicted by CYP2A6 genotype.

Genotype frequencies were calculated based on the combined frequencies of CYP2A6 predicted D alleles (CYP2A6*9, *12, *13 and *15) and L alleles (CYP2A6*2, *4, *5, *6, *7, *10, *11, *17, *19 and *20) from several populations [37,42,77]. Grouping for the predicted nicotine metabolism groups was as follows: intermediate had one D allele, slow metabolism had one L allele or two D alleles, and poor had the combination of one L and one D allele or two L alleles.D: Decrease-of-function; L: Loss-of-function.

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1393www.futuremedicine.com


1394

and higher metabolic clearance of nicotine tocotinine (approximately 17%) compared withmen [68]. Moreover, women taking oral contra-ceptives containing estrogen had significantlyhigher metabolic clearance of nicotine to coti-nine compared with women not taking oral con-traceptives [68]. Together, these studies suggestthat CYP2A6 activity may be influenced byestrogen. Of note, pregnant women also havedramatically altered nicotine metabolism anddisposition kinetics [69].

Smoking statusNicotine clearance has been found to be signif-icantly slower in smokers compared with non-smokers [70]. A study that employed a within-subject crossover design found that nicotine’smetabolic clearance was decreased by approxi-mately 10% during a period of heavy smoking(mean >25 cigarettes/day) compared to after aweek of smoking abstinence [71]. This pheno-menon extends to light smokers (mean <10 cig-arettes/day), where, in a different study, lightsmokers had higher mean systemic exposure toorally administered nicotine, as evidenced bytheir 25% greater estimated nicotine AUCcompared with nonsmokers [59]. In nonhumanprimates, chronic nicotine administrationreduces the levels of hepatic CYP2A enzymeand in vitro nicotine metabolism, possibly via atranscriptional downregulation [72]. It is alsopossible that nicotine may directly inactivateCYP2A6 because it is a mechanism-basedinhibitor of human cDNA-expressed CYP2A6[73,74]. The mechanism is not yet clear, but nic-otine, a metabolite, or another component intobacco could be decreasing the rate ofnicotine’s metabolism.

Interethnic variability in nicotine C-oxidationThe observed difference in nicotine C-oxidationbetween ethnic groups may be due to both non-genetic and genetic factors. Dissimilar diets maybe an influential nongenetic factor, for example,compounds in foods such as broccoli [75] andsoy [76] have been demonstrated to induce andinhibit CYP2A6, respectively. Important geneticfactors, such as CYP2A6 [77] and UGT [78] vari-ant allele frequencies, are disparate between eth-nic groups. The considerable interethnicdifference in CYP2A6 levels and the resultantvariability in nicotine C-oxidation activity werefirst demonstrated between Caucasians (n = 30)and Japanese (n = 30) [48]. On average, higher

protein levels and CYP2A6 metabolic activitieswere seen in Caucasian compared with Japaneseliver microsomes [48]. The findings of this in vitrostudy are supported by the interethnic variabilityin nicotine’s pharmacokinetic profile seen in vivoin humans. Asian–American smokers (n = 37)metabolized nicotine 18% and cotinine 31%slower than Caucasian smokers (n = 54) [47].Consistent with this, the urine recovery of nico-tine was higher and trans-3´-hydroxycotininewas lower in Asian–Americans compared withCaucasians [47]. African–American smokers(n = 51) also metabolized nicotine 13% andcotinine 32% slower than Caucasian smokers(n = 54) [46]. Of note, neither study controlledfor the effects of CYP2A6 genetic polymor-phisms; thus, much of the difference may beattributed to the higher proportion of decrease-and loss-of-function CYP2A6 alleles in Afri-can–Americans and Asian–Americans comparedwith Caucasians. In the case of the Afri-can–American and the Caucasian study, theauthors alluded to this when they concluded thatthe slower nicotine C-oxidation (presumably viaCYP2A6) and slower N-glucuronidation were twosources of the ethnic differentiation in metabolism[46]. Regarding polymorphic UGT activity, geneti-cally variable nicotine and cotinine glucuronidationmay affect the extent, but are unlikely to alter therate, of nicotine C-oxidation. There is evidence ofreduced and bimodal N-glucuronidation amongAfrican–Americans, but not Caucasians [46].

Other cytochrome P450s capable of nicotine C-oxidationAs discussed previously, persons homozygous forthe CYP2A6 gene deletion are capable of formingsmall amounts of cotinine from nicotine (Figure 2).In addition to CYP2A6, the genetically variableCYP2A13 and CYP2B6 can mediate nicotineC-oxidation. CYP2A13 is very efficient in catalyz-ing nicotine C-oxidation and cotinine hydroxyla-tion [79]. However, CYP2A13 mRNA is found atvery low levels in the liver [80,81]. The highest levelsof CYP2A13 mRNA are in the human respiratorytract [81]. Thus, despite its high metabolic activitytoward both nicotine and cotinine, CYP2A13 isnot expected to contribute significantly to thesystemic pharmacokinetic profiles of nicotine orcotinine owing to its low hepatic levels [51,82]. Ofnote, expressed CYP2A13 is the most activehuman CYP in the metabolic activation of thetobacco-smoke N-nitrosamine procarcinogen4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone(NNK) [81].




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Expressed CYP2B6 is also capable of nicotineC-oxidation, but in vitro it has a tenfold higherKm and approximately 10% the catalytic effi-ciency of CYP2A6 [50]. Some human liver micro-somes exhibit two-site enzyme kinetics, and ithas been suggested that at higher nicotineconcentrations, CYP2B6 may be involved incotinine formation [50]. CYP2B6 mRNA and/orprotein are found in the liver, lung andbrain [83–85]. It is possible that CYP2B6 could becontributing to the minor cotinine formation inpersons lacking CYP2A6; however, in vivo evi-dence does not support a large role for CYP2B6.A recent smoking-cessation study in Caucasiansdemonstrated that CYP2B6 increase-of-functionalleles did not alter nicotine plasma levelsobtained from the transdermal patch, evenamong those with genetically reduced CYP2A6metabolism [86].

Implications of variable nicotine metabolismThe amount of nicotine in the body is a functionof the dose, route and rate of elimination. Manyexperimental studies have demonstrated that

smokers titrate their cigarette consumption toachieve a particular level of nicotine. Specifically,changing the dose of nicotine alters smokingbehaviors. For example, when asked to use high-, medium- and low-content nicotine cigarettes,smokers used an average of seven high-nicotinecigarettes (3.2 mg), 11 medium cigarettes (theirusual brand) and 13 low-nicotine cigarettes(<0.3 mg) [87]. In a similar experiment, smokerswere asked to use gum containing high or lowconcentrations of nicotine before smoking.The nicotine preload from the high-nicotinegum resulted in fewer puffs being taken on thesubsequent cigarette compared with usinglow-nicotine gum before smoking [88].

Increasing the rate of nicotine elimination alsoaffects smoking behavior. A small group ofsmokers (n = 11) were given oral ammoniumchloride to acidify their urine [89]. Urinary acidi-fication substantially increased renal clearance,which in turn increased nicotine clearance by41% [89]. The higher nicotine clearance reducedblood levels by 15% and the subsequent dailyintake of nicotine extracted from cigarettes was18% higher [89].

6 gene deletion dramatically reduces in vivo substrate metabolism.

omozygous deletion; n = 3) group has a 3.6-fold higher systemic exposure (plasma AUC360 of nicotine) A6*1/*1 (wildtype; n = 5) group after oral administration of 4 mg nicotine. (B) The homozygous deletion group tinine of the homozygous wild-type group. (C) In the homozygous deletion group no 7-hydroxycoumarin is fter the oral administration of 50 mg of coumarin.

Homozygous deletion (CYP2A6*4/*4, n = 3)

Homozygous wild-type (CYP2A6*1/*1, n = 5)

300 3600 180 240

e (min)

300 36000

60 120 180 240

Time (min)

Pla

sma

cotin

ine

(ng/

ml)

10

20

30

40

50

60

70

300 36000

60 120 180 240

Time (min)

Pla

sma

7-hy

drox

ycou

mar

in (

mM

)

1

2

3

4

5

6

7

8



1396

Conversely, pharmacologically blockingCYP2A6 enzymatic activity (mimickingCYP2A6 decrease- or loss-of-function geneticvariants) is a technique that decreases the rate ofnicotine elimination. Methoxsalen (8-methoxy-sporalen) effectively inhibits CYP2A6-mediatednicotine C-oxidation in vitro [90]. The concur-rent oral administration of the inhibitor meth-oxsalen with nicotine in vivo resulted in nicotineplasma levels that were twofold higher comparedwith oral nicotine alone [91]. Plasma nicotine levelswere also maintained for longer periods of timecompared with oral nicotine alone, suggesting areduction in nicotine’s systemic clearance [91].Overall, methoxsalen increased the extent of sys-temic exposure to nicotine after oral administra-tion [91]. With regards to smoking behavior,study participants who received both oral nico-tine and methoxsalen had 50% lower smoking-related breath carbon monoxide, smoked 24%fewer cigarettes, had 83% longer latency to thenext cigarette and had a 25% decrease in thetotal number of puffs taken compared withplacebo [92]. Taken as a whole, there was a sub-stantial improvement in the smoke exposure costof nicotine acquisition. This supports the theorythat when metabolism of nicotine is reducedsmoking is also reduced in dependent smokers.

The studies described above demonstrate thatwith experimentally introduced variability innicotine pharmacokinetics, whether it beincreased nicotine dose, decreased nicotineplasma levels due to urinary acidification orincreased nicotine plasma levels due to CYP2A6inhibition, smokers try to maintain their plasmanicotine concentration within a narrow range bydemonstrating compensatory smoking behaviorand titrating the number of cigarettes smoked orhow they smoked their cigarettes.

The transdermal patch is an alternative sourceof nicotine used to aid smoking cessation. Duringtreatment patients are assumed to be receivingsimilar doses of nicotine from the patch; however,owing to variability in metabolism, an individual’splasma concentrations of nicotine and cotininecan vary considerably. In a large smoking-cessa-tion study (n ≈ 200), the plasma nicotine andcotinine levels achieved from the patch varied 14-fold and 22-fold, respectively [93]. A recent smok-ing-cessation study demonstrated that CYP2A6activity, as assessed by baseline 3HC:COT fromad libitum smoking, was associated with steady-state nicotine plasma levels obtained from thetransdermal patch in abstinent smokers [55].Slower CYP2A6 activity was a determinant of

higher mean plasma nicotine concentrations andless-severe cravings for cigarettes [55]. SlowerCYP2A6 activity was also a determinant of theeffectiveness of transdermal nicotine at the end oftreatment (8 weeks) and at follow-up (6 months)[55]. The odds of abstinence were lower by almost30% with each increasing quartile of the3HC:COT metabolic ratio [55]. This study is con-sistent with another that suggests that the effec-tiveness of nicotine-replacement therapy can bemaximized with individualized dosing [94].

CYP2A6 genotype, smoking & lung cancerAs mentioned, nicotine is the primary compoundin tobacco that establishes and maintains tobaccodependence [1], and smokers adapt their behaviorto maintain preferred nicotine levels; genetic vari-ation in CYP2A6 affects the pharmacokinetics ofnicotine and smoking behavior (as reviewed inMalaiyandi et al. [95]). Therefore, it follows thatCYP2A6 decrease- or loss-of-function variantshave been associated with an altered risk ofbecoming nicotine dependent [96,97], a decreasedrisk of being a smoker [77,98], lower cigaretteconsumption [40,54,59,77,99–101], reduced inhala-tion [102,103], and a greater likelihood of cessa-tion [104]. However, these findings have not beenuniformly confirmed [105,106].

Individuals with CYP2A6 decrease- or loss-of-function variants should be protected fromtobacco-related lung cancer for several reasons.First, they should be less likely to be a smoker.Second, if they do smoke, they should consumefewer cigarettes. Hepatic CYP2A6 and lungCYP2A13 (also genetically polymorphic) meta-bolically activate the tobacco-smoke N-nitros-amine procarcinogen NNK to the carcinogenicform. NNK is a component of tobacco smokethat causes lung cancer [107–109]. The capacity ofenzymes to activate chemical carcinogens hasbeen recognized as one of the determinants ofcancer risk [110]. Individuals who have CYP2A6decrease- or loss-of-function variants may be lessefficient at activating tobacco-smoke procarcino-gens. Accordingly, less liver NNK activation mayallow for increased circulatory levels and conse-quent activation at the site of interest, possiblyby lung CYP2A13 [81], which has also been asso-ciated with lung cancer [111]. Thus, the role ofgenetic variation in CYP2A6 and lung cancermay be a balance of relative risks. Consistentwith this, in several case–control studies,CYP2A6 has been associated with increased [112],reduced [100,101,113,114] and no risk [111,115,116] of




lung cancer. Methodological differences andstatistical power may be issues, particularly instudies in Caucasians. Further research isneeded to clarify the relationship of theCYP2A6 genotype with the risk of lung cancer.For a more extensive review of CYP2A6 geno-type as a potential determinant of cancer risksee Kamataki et al. [113].

ConclusionThe high interindividual variability in CYP2A6protein levels and capacity of nicotine C-oxida-tion was first demonstrated in human livers andsuggested a highly polymorphic CYP2A6 gene.This has been confirmed, and the past 20 yearshave seen substantial advances in the identifi-cation and characterization of CYP2A6 geneticpolymorphisms. Several human nicotinepharmacokinetic and CYP2A6 genetic studieshave demonstrated the contribution of multipleincrease-, decrease- and loss-of-function poly-morphisms in CYP2A6 to the variability innicotine metabolism. Genetic variation inCYP2A6 influences nicotine pharmacokineticsand, accordingly, is associated with alteredsmoking behavior and lung cancer risk. Theoutcomes of clinical or experimental studiesexamining smoking or using nicotine, may beaffected by variation in rates of nicotine metab-olism. Thus, understanding the sources of vari-ation, such as variation in CYP2A6, can onlyimprove data interpretation.

Future perspectiveMany avenues of investigation into CYP2A6polymorphisms and their impact on nicotinepharmacokinetics remain. In the immediatefuture, characterization of the haplotype, fre-quency and functional impact of knownCYP2A6 SNPs would be valuable. In addition,identifying novel CYP2A6 polymorphisms innon-Caucasian and non-Asian populations isanother obvious area of pursuit, and over the lastfew years, such work has begun. Gene–environ-ment and gene–gene interactions will becomeincreasingly important in understanding thecontribution of CYP2A6 genotype to nicotineC-oxidation in the context of other dynamic andmalleable variables. Potential biological targetsthat may substantially contribute to variability innicotine C-oxidation would be proteins thatregulate the expression of CYP2A6.

On a different note, significant advances inthe understanding of the implications ofimpaired CYP2A6 activity on smoking

behaviors, cessation success, cancer risk andnicotine levels obtained from pharmaceuticalsources are also required. Currently, the bal-ance of evidence is clear, CYP2A6 genotype isassociated with cigarette consumption; more-over, the biological rational is logical. Slowernicotine elimination requires less frequent self-administration and, as a result, those withimpaired CYP2A6 activity smoke fewer ciga-rettes. In addition to smoking maintenancebehavior, several association studies have dem-onstrated links between CYP2A6 genotype andsmoking acquisition in adolescents. Howimpaired CYP2A6 activity, as predicted byCYP2A6 genotype, mediates these effectsremains to be determined. Likewise, CYP2A6genotype has been associated with the likeli-hood of smoking cessation success in adults;but the biological underpinning for this obser-vation requires further investigation. The bio-logical rationale behind increased cessation forthose with impaired CYP2A6 activity followingnicotine patch treatment is reasonable as theyobtain higher nicotine plasma levels, whichhave been shown to enhance cessation.Pharmacological manipulation (i.e., inhibitionof CYP2A6) that results in slower rates of nico-tine C-oxidation could be useful both as aresearch tool and as a novel treatment approachto smoking cessation. Thus, mimicking geneti-cally impaired CYP2A6 activity may be an areaof research related to a potential therapeuticapplication. As discussed, the association ofCYP2A6 genotype with tobacco-related lungcancer is still in the early stages of research. Thebalance of relative risks, due to the involvementof multiple genetically variable enzymatic acti-vation and inactivation pathways in multipletissues remains to be studied in more depth.Overall, there are numerous exciting avenues ofresearch into CYP2A6 genetic variation and theresulting pharmacological and toxicologicalimpacts to be explored.

AcknowledgementsWe thank Nael Al Koudsi, Man Ki Ho, Jibran Khokhar andEric Siu for their careful revision of this manuscript.

Financial disclosureRachel Tyndale holds shares in Nicogen Inc., a companyfocused on creating novel smoking cessation treatments. Nofunding for this manuscript was received from Nicogen. Thisstudy was supported by the Centre for Addiction and MentalHealth, Canadian Institute of Health Research (CIHR)grant MOP53248, Public Health Services Grants



1398

Executive Summary

• Most nicotine (70–80varies between individ

• CYP2A6 is the human

• CYP2A6 is polymorphits expression.

• Polymorphisms in the

• The ratio of trans-3´-h

• Individuals homozygoexposure to nicotine a

• Multiple CYP2A6 decC-oxidation of nicotin

• Other sources that coother enzymes.

• There are differences

• Decrease- or loss-of-fconsumption, decreastobacco-related cance

• The outcomes of clininicotine metabolism adata interpretation.

DA020830, a Canada Research Chair in Pharmacogenet-ics, CIHR-Tobacco Use In Special Populations and CIHR-Student Program In Interdisciplinary Capacity Enhance-ment scholarships. The authors have no other relevant affili-ations or financial involvement with any organization or

entity with a financial interest in or financial conflict withthe subject matter or materials discussed in the manuscriptapart from those disclosed.

No writing assistance was utilized in the production ofthis manuscript.

%) is metabolically inactivated to cotinine; however, the rate and extent of nicotine’s C-oxidation to cotinine uals and ethnic groups.

hepatic drug-metabolizing enzyme that mediates more than 90% of cotinine formation.

ic, which means that there is variation in the DNA sequence for this gene and the regions controlling

gene can result in higher or lower enzyme levels and/or decrease- or loss-of-function of the enzyme.

ydroxycotinine:cotinine is a proxy measure of CYP2A6 activity and can be used for in vivo phenotyping.

us for the deletion of the CYP2A6 gene produce no functional enzyme and have a 3.6-fold higher systemic fter oral administration and produce relatively little cotinine.

rease- or loss-of-function polymorphisms have been demonstrated to alter disposition kinetics and impair e in vivo.

ntribute to the variability seen in nicotine C-oxidation include gender, smoking status, ethnicity and

in the CYP2A6 allele frequencies across ethnic groups.

unction CYP2A6 polymorphisms have been associated with altered smoking initiation, decreased cigarette ed likelihood of being a current dependent smoker and increased success with cessation, and risk for rs.

cal or experimental studies examining smoking, or using nicotine, may be affected by variation in rates of nd/or cotinine formation. Understanding the sources of variation, such as variation in CYP2A6, will improve

BibliographyPapers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers.1. Henningfield JE, Miyasato K, Jasinski DR:

Abuse liability and pharmacodynamic characteristics of intravenous and inhaled nicotine. J. Pharmacol. Exp. Ther. 234(1), 1–12 (1985).

2. US Department of Health, Education, and Welfare: Smoking And Health. Report Of The Advisory Committee to the Surgeon General Of The Public Health Service. In: PHS Publ. No. 1103. USDHEW, Washington, DC, USA (1964).

3. Schneider NG, Popek P, Jarvik ME, Gritz ER: The use of nicotine gum during cessation of smoking. Am. J. Psychiatry 134(4), 439–440 (1977).

4. Puska P, Bjorkqvist S, Koskela K: Nicotine-containing chewing gum in smoking cessation: a double blind trial with half year follow-up. Addict. Behav. 4(2), 141–146 (1979).

5. Levin ED, Conners CK, Sparrow E et al.: Nicotine effects on adults with attention-deficit/hyperactivity disorder. Psychopharmacology (Berl.) 123(1), 55–63 (1996).

6. Potter AS, Newhouse PA: Effects of acute nicotine administration on behavioral inhibition in adolescents with attention-deficit/hyperactivity disorder. Psychopharmacology (Berl.) 176(2), 182–194 (2004).

7. Engeland C, Mahoney C, Mohr E, Ilivitsky V, Knott VJ: Acute nicotine effects on auditory sensory memory in tacrine-treated and nontreated patients with Alzheimer’s disease: an event-related potential study. Pharmacol. Biochem. Behav. 72(1–2), 457–464 (2002).

8. White HK, Levin ED: Chronic transdermal nicotine patch treatment effects on cognitive performance in age-associated memory impairment. Psychopharmacology (Berl.) 171(4), 465–471 (2004).

9. Vieregge A, Sieberer M, Jacobs H, Hagenah JM, Vieregge P: Transdermal nicotine in PD: a randomized, double-blind, placebo-controlled study. Neurology 57(6), 1032–1035 (2001).

10. Lemay S, Chouinard S, Blanchet P et al.: Lack of efficacy of a nicotine transdermal treatment on motor and cognitive deficits in Parkinson’s disease. Prog. Neuropsychopharmacol. Biol. Psychiatry 28(1), 31–39 (2004).

11. Orth M, Amann B, Robertson MM, Rothwell JC: Excitability of motor cortex inhibitory circuits in Tourette syndrome before and after single dose nicotine. Brain 128(Pt 6), 1292–1300 (2005).

12. Howson AL, Batth S, Ilivitsky V et al.: Clinical and attentional effects of acute nicotine treatment in Tourette’s syndrome. Eur. Psychiatry 19(2), 102–112 (2004).

13. Ingram JR, Rhodes J, Evans BK, Thomas GA: Preliminary observations of oral nicotine therapy for inflammatory



bowel disease: an open-label Phase I–II study of tolerance. Inflamm. Bowel Dis. 11(12), 1092–1096 (2005).

14. McGrath J, McDonald JW, Macdonald JK: Transdermal nicotine for induction of remission in ulcerative colitis. Cochrane Database Syst. Rev. CD004722 (2004).

15. Benowitz NL, Jacob P 3rd: Metabolism of nicotine to cotinine studied by a dual stable isotope method. Clin. Pharmacol. Ther. 56(5), 483–493 (1994).

16. Messina ES, Tyndale RF, Sellers EM: A major role for CYP2A6 in nicotine C-oxidation by human liver microsomes. J. Pharmacol. Exp. Ther. 282(3), 1608–1614 (1997).

•• Experimentally demonstrates, through pharmacological and immunological inhibition studies, that CYP2A6 is the enzyme responsible for the majority of nicotine’s C-oxidation in human liver microsomes.

17. Nakajima M, Yamamoto T, Nunoya K et al.: Role of human cytochrome P4502A6 in C-oxidation of nicotine. Drug Metab. Dispos. 24(11), 1212–1217 (1996).

•• Experimentally demonstrates, through pharmacological and immunological inhibition studies, that CYP2A6 is the enzyme responsible for the majority of nicotine’s C-oxidation in human liver microsomes.

18. Yamano S, Tatsuno J, Gonzalez FJ: The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 29(5), 1322–1329 (1990).

• First paper to describe a genetic polymorphism in CYP2A6.

19. Nakajima M, Yamamoto T, Nunoya K et al.: Characterization of CYP2A6 involved in 3´-hydroxylation of cotinine in human liver microsomes. J. Pharmacol. Exp. Ther. 277(2), 1010–1015 (1996).

• Authors experimentally determined that cotinine’s hydroxylation to trans-3´-hydroxycotinine is mediated exclusively by CYP2A6 in human liver microsomes.

20. Hoffman SM, Nelson DR, Keeney DS: Organization, structure and evolution of the CYP2 gene cluster on human chromosome 19. Pharmacogenetics 11(8), 687–698 (2001).

21. Fernandez-Salguero P, Hoffman SM, Cholerton S et al.: A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles. Am. J. Hum. Genet. 57(3), 651–660 (1995).

22. Solus JF, Arietta BJ, Harris JR et al.: Genetic variation in eleven Phase I drug metabolism genes in an ethnically diverse population. Pharmacogenomics 5(7), 895–931 (2004).

23. Kiyotani K, Fujieda M, Yamazaki H et al.: Twenty one novel single nucleotide polymorphisms (SNPs) of the CYP2A6 gene in Japanese and Caucasians. Drug Metab. Pharmacokinet. 17(5), 482–487 (2002).

24. Haberl M, Anwald B, Klein K et al.: Three haplotypes associated with CYP2A6 phenotypes in Caucasians. Pharmacogenet. Genomics 15(9), 609–624 (2005).

25. Benowitz NL, Griffin C, Tyndale R: Deficient C-oxidation of nicotine continued. Clin. Pharmacol. Ther. 70(6), 567 (2001).

26. Kitagawa K, Kunugita N, Katoh T, Yang M, Kawamoto T: The significance of the homozygous CYP2A6 deletion on nicotine metabolism: a new genotyping method of CYP2A6 using a single PCR-RFLP. Biochem. Biophys. Res. Commun. 262(1), 146–151 (1999).

27. Xu C, Rao YS, Xu B et al.: An in vivo pilot study characterizing the new CYP2A6*7, *8, and *10 alleles. Biochem. Biophys. Res. Commun. 290(1), 318–324 (2002).

28. Fukami T, Nakajima M, Yoshida R et al.: A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo. Clin. Pharmacol. Ther. 76(6), 519–527 (2004).

29. Oscarson M, McLellan RA, Gullsten H et al.: Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism. FEBS Lett. 460(2), 321–327 (1999).

30. Kitagawa K, Kunugita N, Kitagawa M, Kawamoto T: CYP2A6*6, a novel polymorphism in cytochrome p450 2A6, has a single amino acid substitution (R128Q) that inactivates enzymatic activity. J. Biol. Chem. 276(21), 17830–17835 (2001).

31. Daigo S, Takahashi Y, Fujieda M et al.: A novel mutant allele of the CYP2A6 gene (CYP2A6*11) found in a cancer patient who showed poor metabolic phenotype towards tegafur. Pharmacogenetics 12(4), 299–306 (2002).

32. Fukami T, Nakajima M, Higashi E et al.: Characterization of novel CYP2A6 polymorphic alleles (CYP2A6*18 and CYP2A6*19) that affect enzymatic activity. Drug Metab. Dispos. 33(8), 1202–1210 (2005).

33. Fukami T, Nakajima M, Higashi E et al.: A novel CYP2A6*20 allele found in African–American population produces a truncated protein lacking enzymatic activity. Biochem. Pharmacol. 70(5), 801–808 (2005).

34. Oscarson M, McLellan RA, Asp V et al.: Characterization of a novel CYP2A7/CYP2A6 hybrid allele (CYP2A6*12) that causes reduced CYP2A6 activity. Hum. Mutat. 20(4), 275–283 (2002).

35. Pitarque M, von Richter O, Oke B et al.: Identification of a single nucleotide polymorphism in the TATA box of the CYP2A6 gene: impairment of its promoter activity. Biochem. Biophys. Res. Commun. 284(2), 455–460 (2001).

36. Benowitz NL, Swan GE, Jacob P 3rd, Lessov-Schlaggar CN, Tyndale RF: CYP2A6 genotype and the metabolism and disposition kinetics of nicotine. Clin. Pharmacol. Ther. 80(5), 457–467 (2006).

37. Nakajima M, Fukami T, Yamanaka H et al.: Comprehensive evaluation of variability in nicotine metabolism and CYP2A6 polymorphic alleles in four ethnic populations. Clin. Pharmacol. Ther. 80(3), 282–297 (2006).

•• Amalgamates CYP2A6 genotype data from four different ethnic groups. It provides up-to-date allele frequencies and demonstrates some associations between specific CYP2A6 alleles and cotine:nicotine (COT:NIC) phenotypes.

38. Wang J, Pitarque M, Ingelman-Sundberg M: 3´-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression. Biochem. Biophys. Res. Commun. 340(2), 491–497 (2006).

39. Mwenifumbo JC, Lessov-Schlaggar CN, Zhou Q et al.: Identification of novel CYP2A6*1B variants: the CYP2A6*1B allele is associated with faster in vivo nicotine metabolism. Clin. Pharmacol. Ther. (2007) (Epub ahead of print).

40. Rao Y, Hoffmann E, Zia M et al.: Duplications and defects in the CYP2A6 gene: identification, genotyping, and in vivo effects on smoking. Mol. Pharmacol. 58(4), 747–755 (2000).

41. Fukami T, Nakajima M, Yamanaka H et al.: A novel duplication type of CYP2A6 gene in African–American population. Drug Metab. Dispos. 35(4), 515–520 (2007).

42. Mwenifumbo JC, Myers MG, Wall TL et al.: Ethnic variation in CYP2A6*7, CYP2A6*8 and CYP2A6*10 as assessed with a novel haplotyping method. Pharmacogenet. Genomics 15(3), 189–192 (2005).



43. Al Koudsi N, Mwenifumbo JC, Sellers EM et al.: Characterization of the novel CYP2A6*21 allele using in vivo nicotine kinetics. Eur. J. Clin. Pharmacol. 62(6), 481–484 (2006).

44. Pitarque M, von Richter O, Rodriguez-Antona C et al.: A nicotine C-oxidase gene (CYP2A6) polymorphism important for promoter activity. Hum. Mutat. 23(3), 258–266 (2004).

45. von Richter O, Pitarque M, Rodriguez-Antona C et al.: Polymorphic NF-Y dependent regulation of human nicotine C-oxidase (CYP2A6). Pharmacogenetics 14(6), 369–379 (2004).

46. Benowitz NL, Perez-Stable EJ, Fong I et al.: Ethnic differences in N´-glucuronidation of nicotine and cotinine. J. Pharmacol. Exp. Ther. 291(3), 1196–1203 (1999).

47. Benowitz NL, Perez-Stable EJ, Herrera B, Jacob P 3rd: Slower metabolism and reduced intake of nicotine from cigarette smoking in Chinese–Americans. J. Natl Cancer Inst. 94(2), 108–115 (2002).

48. Shimada T, Yamazaki H, Guengerich FP: Ethnic-related differences in coumarin 7-hydroxylation activities catalyzed by cytochrome P4502A6 in liver microsomes of Japanese and Caucasian populations. Xenobiotica 26(4), 395–403 (1996).

49. Fuhr U, Jetter A, Kirchheiner J: Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the ‘cocktail’ approach. Clin. Pharmacol. Ther. 81(2), 270–283 (2007).

50. Yamazaki H, Inoue K, Hashimoto M, Shimada T: Roles of CYP2A6 and CYP2B6 in nicotine C-oxidation by human liver microsomes. Arch. Toxicol. 73(2), 65–70 (1999).

51. Hukkanen J, Jacob P 3rd: Benowitz NL: Metabolism and disposition kinetics of nicotine. Pharmacol. Rev. 57(1), 79–115 (2005).

52. Dempsey D, Tutka P, Jacob P, 3rd et al.: Nicotine metabolite ratio as an index of cytochrome P450 2A6 metabolic activity. Clin. Pharmacol. Ther. 76(1), 64–72 (2004).

• Demonstrates that both the salivary and plasma metabolic 3HC:COT correlated very well with the total clearance of nicotine and helped establish the ratio as a proxy measure of in vivo CYP2A6 activity.

53. Yamanaka H, Nakajima M, Nishimura K et al.: Metabolic profile of nicotine in subjects whose CYP2A6 gene is deleted. Eur. J. Pharm. Sci. 22(5), 419–425 (2004).

• Highlights the impact that CYP2A6 has on the profile of nicotine and its urinary metabolites through the dramatically different proportions of metabolites excreted in persons homozygous for the CYP2A6 gene deletion.

54. Malaiyandi V, Lerman C, Benowitz NL et al.: Impact of CYP2A6 genotype on pretreatment smoking behaviour and nicotine levels from and usage of nicotine replacement therapy. Mol. Psychiatry 11(4), 400–409 (2006).

•• Demonstrates that the CYP2A6 genotype is associated with CYP2A6 activity, as assessed by 3HC:COT from baseline cigarette consumption, as well as the level of plasma nicotine achieved from the transdermal patch during smoking abstinence.

55. Lerman C, Tyndale R, Patterson F et al.: Nicotine metabolite ratio predicts efficacy of transdermal nicotine for smoking cessation. Clin. Pharmacol. Ther. 79(6), 600–608 (2006).

• A clinical trial that found CYP2A6 activity, as assessed by 3HC:COT, was a determinant of successful smoking cessation in a nicotine-replacement therapy.

56. Levi M, Dempsey DA, Benowitz NL, Sheiner LB: Population pharmacokinetics of nicotine and its metabolites I. Model development. J. Pharmacokinet. Pharmacodyn. 34(1), 5–21 (2007).

57. Lea RA, Dickson S, Benowitz NL: Within-subject variation of the salivary 3HC/COT ratio in regular daily smokers: prospects for estimating CYP2A6 enzyme activity in large-scale surveys of nicotine metabolic rate. J. Anal. Toxicol. 30(6), 386–389 (2006).

58. Levi M, Dempsey DA, Benowitz NL, Sheiner LB: Prediction methods for nicotine clearance using cotinine and 3-hydroxy-cotinine spot saliva samples II: model application. J. Pharmacokinet. Pharmacodyn. 34(1), 23–34 (2007).

59. Mwenifumbo JC, Sellers EM, Tyndale RF: Nicotine metabolism and CYP2A6 activity in a population of black African descent: impact of gender and light smoking. Drug Alcohol Depend. 89(1), 24–33 (2007).

60. Swan GE, Benowitz NL, Lessov CN et al.: Nicotine metabolism: the impact of CYP2A6 on estimates of additive genetic influence. Pharmacogenet. Genomics 15(2), 115–125 (2005).

61. Yang M, Kunugita N, Kitagawa K et al.: Individual differences in urinary cotinine levels in Japanese smokers: relation to genetic polymorphism of drug-metabolizing enzymes. Cancer Epidemiol. Biomarkers Prev. 10(6), 589–593 (2001).

62. Byrd GD, Chang KM, Greene JM, deBethizy JD: Evidence for urinary excretion of glucuronide conjugates of nicotine, cotinine, and trans-3´-hydroxycotinine in smokers. Drug Metab. Dispos. 20(2), 192–197 (1992).

63. Neurath GB, Dunger M, Orth D, Pein FG: Trans-3´-hydroxycotinine as a main metabolite in urine of smokers. Int. Arch. Occup. Environ. Health 59(2), 199–201 (1987).

64. Peamkrasatam S, Sriwatanakul K, Kiyotani K et al.: In vivo evaluation of coumarin and nicotine as probe drugs to predict the metabolic capacity of CYP2A6 due to genetic polymorphism in Thais. Drug Metab. Pharmacokinet. 21(6), 475–484 (2006).

•• Demonstrates a clear association between CYP2A6 genotype and nicotine C-oxidation phenotype, as assessed by COT:NIC, in a nonsmoking Thai population.

65. Nakajima M, Kwon JT, Tanaka N et al.: Relationship between interindividual differences in nicotine metabolism and CYP2A6 genetic polymorphism in humans. Clin. Pharmacol. Ther. 69(1), 72–78 (2001).

66. Prather RD, Tu TG, Rolf CN, Gorsline J: Nicotine pharmacokinetics of Nicoderm (nicotine transdermal system) in women and obese men compared with normal-sized men. J. Clin. Pharmacol. 33(7), 644–649 (1993).

67. Zeman MV, Hiraki L, Sellers EM: Gender differences in tobacco smoking: higher relative exposure to smoke than nicotine in women. J. Womens Health Gend. Based Med. 11(2), 147–153 (2002).

68. Benowitz NL, Lessov-Schlaggar CN, Swan GE, Jacob P 3rd: Female sex and oral contraceptive use accelerate nicotine metabolism. Clin. Pharmacol. Ther. (2006) 79(5), 480–488.

• Demonstrates that women have faster nicotine clearance compared with men.



69. Dempsey D, Jacob P 3rd, Benowitz NL: Accelerated metabolism of nicotine and cotinine in pregnant smokers. J. Pharmacol. Exp. Ther. 301(2), 594–598 (2002).

70. Benowitz NL, Jacob P 3rd: Nicotine and cotinine elimination pharmacokinetics in smokers and nonsmokers. Clin. Pharmacol. Ther. 53(3), 316–323 (1993).

71. Benowitz NL, Jacob P 3rd: Effects of cigarette smoking and carbon monoxide on nicotine and cotinine metabolism. Clin. Pharmacol. Ther. 67(6), 653–659 (2000).

72. Schoedel KA, Sellers EM, Palmour R, Tyndale RF: Down-regulation of hepatic nicotine metabolism and a CYP2A6-like enzyme in African green monkeys after long-term nicotine administration. Mol. Pharmacol. 63(1), 96–104 (2003).

73. Denton TT, Zhang X, Cashman JR: Nicotine-related alkaloids and metabolites as inhibitors of human cytochrome P-450 2A6. Biochem. Pharmacol. 67(4), 751–756 (2004).

74. von Weymarn LB, Brown KM, Murphy SE: Inactivation of CYP2A6 and CYP2A13 during nicotine metabolism. J. Pharmacol. Exp. Ther. 316(1), 295–303 (2006).

75. Hakooz N, Hamdan I: Effects of dietary broccoli on human in vivo caffeine metabolism: a pilot study on a group of Jordanian volunteers. Curr. Drug Metab. 8(1), 9–15 (2007).

76. Anderson GD, Rosito G, Mohustsy MA, Elmer GW: Drug interaction potential of soy extract and Panax ginseng. J. Clin. Pharmacol. 43(6), 643–648 (2003).

77. Schoedel KA, Hoffmann EB, Rao Y, Sellers EM, Tyndale RF: Ethnic variation in CYP2A6 and association of genetically slow nicotine metabolism and smoking in adult Caucasians. Pharmacogenetics 14(9), 615–626 (2004).

•• Case–control study demonstrating that Caucasians with decrease- or loss-of-function CYP2A6 alleles are less likely to be smokers and consume fewer cigarettes.

78. Saeki M, Saito Y, Jinno H et al.: Genetic variations and haplotypes of UGT1A4 in a Japanese population. Drug Metab. Pharmacokinet. 20(2), 144–151 (2005).

79. Bao Z, He XY, Ding X, Prabhu S, Hong JY: Metabolism of nicotine and cotinine by human cytochrome P450 2A13. Drug Metab. Dispos. 33(2), 258–261 (2005).

80. Koskela S, Hakkola J, Hukkanen J et al.: Expression of CYP2A genes in human liver and extrahepatic tissues. Biochem. Pharmacol. 57(12), 1407–1413 (1999).

81. Su T, Bao Z, Zhang QY et al.: Human cytochrome P450 CYP2A13: predominant expression in the respiratory

tract and its high efficiency metabolic activation of a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Cancer Res. 60(18), 5074–5079 (2000).

82. Gourlay SG, Benowitz NL: Arteriovenous differences in plasma concentration of nicotine and catecholamines and related cardiovascular effects after smoking, nicotine nasal spray, and intravenous nicotine. Clin. Pharmacol. Ther. 62(4), 453–463 (1997).

83. Miksys S, Lerman C, Shields PG, Mash DC, Tyndale RF: Smoking, alcoholism and genetic polymorphisms alter CYP2B6 levels in human brain. Neuropharmacology 45(1), 122–132 (2003).

84. Gervot L, Rochat B, Gautier JC et al.: Human CYP2B6: expression, inducibility and catalytic activities. Pharmacogenetics 9(3), 295–306 (1999).

85. Gonzalez FJ, Crespi CL, Czerwinski M, Gelboin HV: Analysis of human cytochrome P450 catalytic activities and expression. Tohoku J. Exp. Med. 168(2), 67–72 (1992).

86. Lee AM, Jepson C, Shields PG et al.: CYP2B6 genotype does not alter nicotine metabolism, plasma levels, or abstinence with nicotine replacement therapy. Cancer Epidemiol Biomarkers Prev. 16(6), 1312–1314 (2007).

87. Russell MA, Wilson C, Patel UA, Cole PV, Feyerabend C: Comparison of effect on tobacco consumption and carbon monoxide absorption of changing to high and low nicotine cigarettes. Br. Med. J. 4(5891), 512–516 (1973).

88. Kozlowski LT, Jarvik ME, Gritz ER: Nicotine regulation and cigarette smoking. Clin. Pharmacol. Ther. 17(1), 93–97 (1975).

89. Benowitz NL, Jacob P 3rd: Nicotine renal excretion rate influences nicotine intake during cigarette smoking. J. Pharmacol. Exp. Ther. 234(1), 153–155 (1985).

90. Zhang W, Kilicarslan T, Tyndale RF, Sellers EM: Evaluation of methoxsalen, tranylcypromine, and tryptamine as specific and selective CYP2A6 inhibitors in vitro. Drug Metab. Dispos. 29(6), 897–902 (2001).

91. Sellers EM, Kaplan HL, Tyndale RF: Inhibition of cytochrome P450 2A6 increases nicotine’s oral bioavailability and decreases smoking. Clin. Pharmacol. Ther. 68(1), 35–43 (2000).

92. Sellers EM, Tyndale RF, Fernandes LC: Decreasing smoking behaviour and risk through CYP2A6 inhibition. Drug Discov. Today 8(11), 487–493 (2003).

93. Gourlay SG, Benowitz NL, Forbes A, McNeil JJ: Determinants of plasma concentrations of nicotine and cotinine during cigarette smoking and transdermal nicotine treatment. Eur. J. Clin. Pharmacol. 51(5), 407–414 (1997).

94. Sachs DP: Effectiveness of the 4-mg dose of nicotine polacrilex for the initial treatment of high-dependent smokers. Arch. Intern. Med. 155(18), 1973–1980 (1995).

95. Malaiyandi V, Sellers EM, Tyndale RF: Implications of CYP2A6 genetic variation for smoking behaviors and nicotine dependence. Clin. Pharmacol. Ther. 77(3), 145–158 (2005).

96. Audrain-McGovern J, Al Koudsi N, Rodriguez D et al.: The role of CYP2A6 in the emergence of nicotine dependence in adolescents. Pediatrics 119(1), E264–E274 (2007).

97. O’Loughlin J, Paradis G, Kim W et al.: Genetically decreased CYP2A6 and the risk of tobacco dependence: a prospective study of novice smokers. Tob. Control 13(4), 422–428 (2004).

98. Iwahashi K, Waga C, Takimoto T: Whole deletion of CYP2A6 gene (CYP2A6AST;4C) and smoking behavior. Neuropsychobiology 49(2), 101–104 (2004).

99. Minematsu N, Nakamura H, Furuuchi M et al.: Limitation of cigarette consumption by CYP2A6*4, *7 and *9 polymorphisms. Eur. Respir. J. 27(2), 289–292 (2006).

•• Demonstrates that daily cigarette consumption is genetically modulated by CYP2A6 decrease- and loss-of-function alleles in a Japanese population.

100. Ariyoshi N, Kitada M, Kamataki T: Association between genetic polymorphism and lung cancer risk. Nippon Rinsho 60 (Suppl. 5), 46–49 (2002).

101. Fujieda M, Yamazaki H, Saito T et al.: Evaluation of CYP2A6 genetic polymorphisms as determinants of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers. Carcinogenesis 25(12), 2451–2458 (2004).

102. Strasser AA, Malaiyandi V, Hoffmann E, Tyndale RF, Lerman C: An association of CYP2A6 genotype and smoking topography. Nicotine Tob. Res. 9(4), 511–518 (2007).

103. Malaiyandi V, Goodz SD, Sellers EM, Tyndale RF: CYP2A6 genotype, phenotype, and the use of nicotine metabolites as biomarkers during ad libitum smoking. Cancer Epidemiol. Biomarkers Prev. 15(10), 1812–1819 (2006).



104. Gu DF, Hinks LJ, Morton NE, Day IN: The use of long PCR to confirm three common alleles at the CYP2A6 locus and the relationship between genotype and smoking habit. Ann. Hum. Genet. 64(Pt 5), 383–390 (2000).

105. Carter B, Long T, Cinciripini P: A meta-analytic review of the CYP2A6 genotype and smoking behavior. Nicotine Tob. Res. 6(2), 221–227 (2004).

106. Munafo M, Clark T, Johnstone E, Murphy M, Walton R: The genetic basis for smoking behavior: a systematic review and meta-analysis. Nicotine Tob. Res. 6(4), 583–597 (2004).

107. Hecht SS: Approaches to cancer prevention based on an understanding of N-nitrosamine carcinogenesis. Proc. Soc. Exp. Biol. Med. 216(2), 181–191 (1997).

108. Hecht SS: DNA adduct formation from tobacco-specific N-nitrosamines. Mutat. Res. 424(1–2), 127–142 (1999).

109. Hecht SS: Biochemistry, biology, and carcinogenicity of tobacco-specific N-nitrosamines. Chem. Res. Toxicol. 11(6), 559–603 (1998).

110. Perera FP: Environment and cancer: who are susceptible? Science 278(5340), 1068–1073 (1997).

111. Wang H, Tan W, Hao B et al.: Substantial reduction in risk of lung adenocarcinoma associated with genetic polymorphism in CYP2A13, the most active cytochrome P450 for the metabolic activation of tobacco-specific carcinogen NNK. Cancer Res. 63(22), 8057–8061 (2003).

112. Tan W, Chen GF, Xing DY et al.: Frequency of CYP2A6 gene deletion and its relation to risk of lung and esophageal cancer in the Chinese population. Int J. Cancer 95(2), 96–101 (2001).

113. Kamataki T, Nunoya K, Sakai Y, Kushida H, Fujita K: Genetic polymorphism of CYP2A6 in relation to cancer. Mutat. Res. 428(1–2), 125–130 (1999).

114. Miyamoto M, Umetsu Y, Dosaka-Akita H et al.: CYP2A6 gene deletion reduces susceptibility to lung cancer. Biochem. Biophys. Res. Commun. 261(3), 658–660 (1999).

115. London SJ, Idle JR, Daly AK, Coetzee GA: Genetic variation of CYP2A6, smoking and risk of cancer. Lancet 353(9156), 898–899 (1999).

116. Loriot MA, Rebuissou S, Oscarson M et al.: Genetic polymorphisms of cytochrome P450 2A6 in a case–control study on lung cancer in a French population. Pharmacogenetics 11(1), 39–44 (2001).

117. Ariyoshi N, Takahashi Y, Miyamoto M et al.: Structural characterization of a new variant of the CYP2A6 gene (CYP2A6*1B) apparently diagnosed as heterozygotes of CYP2A6*1A and CYP2A6*4C. Pharmacogenetics 10(8), 687–693 (2000).

118. Nakajima M, Yoshida R, Fukami T, McLeod HL, Yokoi T: Novel human CYP2A6 alleles confound gene deletion analysis. FEBS Lett. 569(1–3), 75–81 (2004).

119. Oscarson M, McLellan RA, Gullsten H et al.: Characterisation and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population. FEBS Lett. 448(1), 105–110 (1999).

120. Nunoya K, Yokoi T, Kimura K et al.: A new deleted allele in the human cytochrome P450 2A6 (CYP2A6) gene found in individuals showing poor metabolic capacity to coumarin and (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502). Pharmacogenetics 8(3), 239–249 (1998).

121. Nunoya K, Yokoi T, Takahashi Y et al.: Homologous unequal cross-over within the human CYP2A gene cluster as a mechanism for the deletion of the entire CYP2A6 gene associated with the poor metabolizer phenotype. J. Biochem. (Tokyo) 126(2), 402–407 (1999).

122. Nunoya KI, Yokoi T, Kimura K et al.: A new CYP2A6 gene deletion responsible for the in vivo polymorphic metabolism of (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride in humans. J. Pharmacol. Exp. Ther. 289(1), 437–442 (1999).

123. Ariyoshi N, Sekine H, Saito K, Kamataki T: Characterization of a genotype previously designated as CYP2A6 D-type: CYP2A6*4B, another entire gene deletion allele of the CYP2A6 gene in Japanese. Pharmacogenetics 12(6), 501–504 (2002).

124. Ariyoshi N, Sawamura Y, Kamataki T: A novel single nucleotide polymorphism altering stability and activity of CYP2A6. Biochem. Biophys. Res. Commun. 281(3), 810–814 (2001).

125. Yoshida R, Nakajima M, Watanabe Y, Kwon JT, Yokoi T: Genetic polymorphisms in human CYP2A6 gene causing impaired nicotine metabolism. Br. J. Clin. Pharmacol. 54(5), 511–517 (2002).

126. Yoshida R, Nakajima M, Nishimura K et al.: Effects of polymorphism in promoter region of human CYP2A6 gene (CYP2A6*9) on expression level of messenger ribonucleic acid and enzymatic activity in vivo and in vitro. Clin. Pharmacol. Ther. 74(1), 69–76 (2003).

127. Nurfadhlina M, Foong K, Teh LK et al.: CYP2A6 polymorphisms in Malays, Chinese and Indians. Xenobiotica 36(8), 684–692 (2006).

128. Gambier N, Batt AM, Marie B et al.: Association of CYP2A6*1B genetic variant with the amount of smoking in French adults from the Stanislas cohort. Pharmacogenomics J. 5(4), 271–275 (2005).

129. Huang S, Cook DG, Hinks LJ et al.: CYP2A6, MAOA, DBH, DRD4, and 5HT2A genotypes, smoking behaviour and cotinine levels in 1518 UK adolescents. Pharmacogenet. Genomics 15(12), 839–850 (2005).

130. Gyamfi MA, Fujieda M, Kiyotani K, Yamazaki H, Kamataki T: High prevalence of cytochrome P450 2A6*1A alleles in a black African population of Ghana. Eur. J. Clin. Pharmacol. 60(12), 855–857 (2005).

Website201. CYP2A6 allele nomenclature (2001)

www.cypalleles.ki.se/cyp2a6.htm


genetic variability in cyp2a6 and the pharmacokinetics of nicotine

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