A B ST R A C T
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lS
e r3 26 C
ys Pol
ym
o rph
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,
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okin
g,Pol yc
h
lori
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ph
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(
PC
B)
Expo s u r e, a nd
Ri
sk
of
Br e a stC
a n c e r(
U
nd
e r th
ed ir e c
ti
o n of
DR
RO
B E RT C
.M
I L L I KA N
)
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m
a n 8-o x ogu an
i
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h O G G
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r epai
r s8
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in e r e s
i d
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D
N A
th
at c a u s eG
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tr a n s v e r si
o nm
utati
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e e x am in e
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g l
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)
at c od
o n3 2 6
of
th
eh O G G l g
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i
sk
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r e a st c a n c e rin
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f A fi i
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e als o
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ci
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ych l
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ph
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PC B
)
e xpo s u r ein
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hO G G
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6
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a n2 0
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)
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R,1
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9
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e xpo s u r e a nd h O G G l 32 6 g
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18
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2 7R
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F I
G
UR
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g
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8
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O G G
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8
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6
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8
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l lele a n d ge n ot y pef
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p
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If
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l3
26
a n db
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i ed
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T
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O G G
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Bs, hO G G
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ge n ot y pea nd br e a st c a n c e r, o v e r al l a n d str at i f ied b y r a c e 2
5
T ab le
V
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s fo r l ip
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u sted PC
B le v els, hO G G
l3
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e n ot yp
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br e a st c a n c e r, low
tom
ode r ate a nd
hi g
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e n e r sA C
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I
w
o ul d
li k
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Dr . L o ui
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e rtM
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Dr. T em
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topeK
ek
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m
i
t te e and
spe ntm
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our s answ
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g
i
vi
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i l l i k
a n a nd D
r.K
ek
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a v e pr o vi d
ed
thef
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ati
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ch
e al
th e xpo s u r eM
yd
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si
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ci
e n c ei
n Pub l i
c H e al
th is ad i
r e ctr e s u
lt
of
th
ei
rd
e si
r e tof
o ste r the ed
uc ati
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al l
th
o s ew h
ow
o rk
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th
em
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ei
rh
a rd w
o rk
a nd d
ed i
c atio nh
a v e s e r v ed
tob
e a n e x c el l
e nt r ol
em
od
el
o v e r the pa stf
o u rye a r s
D
r.B
al l w
a si
n str um
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nm
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oi
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y to th
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n vi
r o nm
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ci
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d
Eng i
n e e rin
g
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a rtm
en t. He rg
e n e r osit
y w i
th h
e r ti
m
e,h
e rw i l l i
ng
n e s stow
o rk
wit
h
m
yi
n te r e sts, and h
e rh
um
orw
e r eh
e rb
e st a s s ets tom y
ed
u c ati
o n al
e xp
e ri
e n c e.Sh
ew
a sm
y pr opo n e nt.I
w
o uld
al
s ol i k
e to tha nk m
y c o-w
o r
k
e r sfr
om
th
el
ab f
o r al
l th
ei
rh
el
p a nd
f l
e xi b i l i
ty
.A l l i
s o n Eato nh
a sb
e e n ap
ri
c el
e s s r e s o u r c e a nd fr i
e nd
.S
a r aD
u ck w
o rth
tr a
i
n ed i
n th
eH i g h
-t
h
r o ugh p
utG
e n oty p i
ng
Lab b y h
el p i
ng
m
ew i
th
th
eg
e n oty p i
ng
of
h O G G l
.S
c ot tW
i
nk
el
,J
o nP l
aye r,L
om
aT
em
pe st, and K
ri
sti
nH
e a rd h
a v e of f
e r ed
p
r of
e s si
o n al
a nd
c oU
egi
al
s up
po rti
n thel
ab
.I a
m m
o st ap p
r ecia
ti
v e of
m
y m
oth
e r,S
us an Hal l
,f
o rh
erp
er s o n al
s up p
o rt a nd
th
ee x a
m
pl
e sh
e s etf
o rm
e top
e r s e v e r e.A
nd
Iw
o ul d l i k
e to th
a nk S
c ot t Bow
e n,m y f
iance,I N T R O D C U C T I O N
T h
em
o st c om
m
o n c a n c e rd i
agn o s ed i
nw
om
e nl i
vi
ng
th
eU
ni
ted S
tate si
sb
r e a stc a n c e r.
I
n2
00 3
, a n e sti
m
ated
2 1 1,3 0
0 w om
e nw i l l b
ed i
ag
n o s ed w i
th b
r e a st c a n c e r and
a n e st
i m
ated
3 9,8 0
0w i l l d i
efr
om
th
ed i
s e a s e(
J
em
al
, et al
,2 0 0 3
)
. Th
eh i
gh i
nci d
e n c ea n
d m
o r tal i
t y r atem
ak
e sb
r e a st c a n c e r a tr em
e nd
o u s pub l i
ch
e al
th
c o n c e r n .T h
e s e a r ch f
o r n ew d i
agn o sti
c to ol
s a nd
tr e atm
e n tsf
o rb
r e a st c a n c e ri
s a s e s s e n ti
al
a s t
h
e s e ar ch f
o r c a u s e s a nd
ri
sk f
a cto r sf
o r th
ed i
s e a s e.O
n e pr op
o s ed m
e ch
a ni
sm
of
b
r e a st c a n c e ri
ni
ti
ati
o ni
s th
ati
t r e s ul
tsfr
om
DN A d
am
age c a u s ed b
yb
ulk y
ad d
u cts o ro x
i d
ati
v ed
am
ag
efr
om
e n vi
r o nm
e ntal
e x po s u r e s. Tak i
n g th
eh
y poth
e si
s a stepf
u rth
e r ,de
f
i ci
e nt DN A
r epai
rm
e ch
a ni
sm
sm
ayi
n c r e a s e c a n c e r ri
sk
, a sd
am
aged D N A
r epl i
c ate s,m i
s-p
ai
r s a nd
r e s ul
tsi
nm
utati
o n s Th
ep
u rp
o s e of
th i
s r e s e a r ch i
s to exam
i
ne th
er e
l
ati
o n sh i p
sb
etw
e e n pol
ym
o rp h i
c DN A
r epai
r ge n e sw i
th
al
te r ed
r ep
ai
r a cti
vi
ti
e s,e n vir o n
m
e ntal e xpo s u r e s and
c a n c e r risk
.T h
em
utati
o n s r e s ul
ti
n gfr
om
o xi d
ati
v ed
am
ag
e a r e th
o ugh
t to pl
ay a ni m
po rta ntr o
l
ei
n c a n c e rd
e v el
op
m
e nt(
Po ul
s e n , et al
,19 9 8
)
,i
ncl
ud i
ngb
r e a st c an c e r. R e a cti
v eo xy ge n spe c
i
e s(
RO S
)
fo rm
ed fr om
e xpo s u r e to x e n ob i
oti
c s a nd e ndoge n o u s c el lula rm
etab
ol i
sm
pr od
u c e o xi d i
z ed
gu a ni
n e r e si d
u e s(
Fi
gu r e s la nd
2)
th
at a r eh i gh l y
m
utage ni
c(
B oi
te u x a nd
Rad i
c el l
a, 19 9 9
)
.O
n e stud
y sh
ow
ed
th
atm
al i g
n an tb
r e a st ti
s s u eh
ad 9
.7 6
-
f
ol d h i
g
h
e rl
e v el
s of
o xi d i
z ed g
u a ni
n e r e si d
u e s,8
-h
y
d
r o xy
-2
'
d
eoxy g
u a n o si
n e(
8
-O H d G
)
th
a n n o rm
al b
r e a st ti
s s u e(M
u s a r r at, et al
,19 9 6
)
.A
noth
e r stud
y r epo rted
s
i
gni
f ic a ntl y h i gh
e rl
e v el
s of
8-O H d G i
t
h
a ni
n th
e c o ntr ol
s, th
o ugh
th
e stand
a rd d
e vi
ati
o n of
c o n c e ntr ati
o n sw
e r e wi d
e(
Li
, et al
,2 0 0 1
)
.O f
c o u r s e, t
h
e el
e v ated l
e v el
s of
o xi d
ati
v ed
am
age c o ul d b
e a c a u s e o r a r e s ul
to
f
t
h
e c a n c e r.To
d
e s c ri b
e th
em
e ch
a ni
c s of
th
em
utag
e n esi
s, gu ani
n ep
r ef
e r s top
ai
rw i
th
cyto s
i
n e,
b
ut o xi d i
z ed
gu a ni
n e of
te nm i
s-pa
i
r s wi
th
ad
e ni
n e. Th
e r e s ulti
ng
str a nd h
a s a na
d
e ni
n e op po si
te th
e o xi d i
z ed
gu ani
n e. Du ri
n g th
e n e x t r ep l i
c ati
o n cycl
e, th
em i
s-p
ai
r ed
a
d
e ni
n ew i l
l c a u s e a tr a n s v e r si
o nm
utati
o ni
n th
ed
a u gh
te r str and
,w h
e n th
e ad
e ni
n e th
e npa
i
r s wi
th
a th
ym i
n e(
B
r u n e r, et a
l
,2 0 0 0
)
.O
xi d i
z ed
gu a ni
n e s s u ch
a s8
-
h
y
d
r o xy gu a ni
n e(
8
-O H
-G
) (
Boi
te u x and R
ad i
c el l
a,2 0 0 0
)
and 8
-o x ogu an
i
n e(
8
-o x -o -
G
) (
N
a sh
, et al
,1
99
7)
c a u s e th
e s eG
:C
toT
:A
tr a n s v e r si
o n sd
u ri
ngD N A
r epl i
c ati
o n(
Boi
te u x and
R a
d i
c el l
a,19 9 9
)
G
:C
to T :A
tr an s v e r si
on s ar e th
e se c ond
m
o st c om
m
o n ty p
e of
s om
ati
cm
utati
o nf
o u nd i
nh
um
a n c a n c e r s(
B
r u n e r, et al
, 20 0 0
)
. Th
us, r epai
r of
o xi d
ati
v ed
am
ag
ei
s e s s e nti
al
tom
ai
ntai
n th
ei
ntegri
t y of
DN A
.M
o st o xi d
ati
v eD N A d
am
agei
s r ep
ai
r ed
vi
a th
em
ul
ti
-p
r otei
n,b
a s e e x ci
si
onr epa
i
r(
B ER
)
path w
ay a s sh
ow
ni
nF i
gu r e3
(
N i l
s e n a nd
BCr ok
a n , 20 0
1)
. Th
eh
um
a nB
E Rpat
h w
ay u t i li
z e s the 8-o x ogu a n
i
n e g l yc o sy la s e/
apu ri
ni
cl
ya s e(
h O G G
l)
to r em
o v eo x
i d i
z ed p
u ri
n eb
a s e si
nD N A
a nd
c r e ate a n ab
a si
c si
te(
D h
e ri
n , et al
, 19 9 9
)
. Th
eh O G G l g
e n eh
a s a n o n -sy
no ny m
o u s si
ng l
e n u cl
e oti d
e pol
ym
o rp h i
sm
(
SN P
)
at c od
o n32 6
th
at c a u s e s a ch
angefr
om
aS
e r(
T C C
)
to aC
ys(
TG C
)
r e si d
u e. Tw
o stud i
e s r ep
o rted
r e
d
u c ed
e n zym
ati
c a cti
vi
ty f
o r th
eC
ys al l
el
e(
K
oh
n o , et al
,19 9 8
;D h
e ri
n, et al
,19 9 9
)
T
h i
s s ug ge sts th
at ah O G G
lC
ys3 26
ge n oty pem
ayl
e ad
to e x c e ssi
v e am
o u nts of
o xi d i
z ed
Expo s u r e to ciga r et te s
m
ok e and polych lo rin ate d b i p he n yls(
P C Bs)
c an le ad too x
i d
ati
v ed
am
age of
DN A i
n th
eh
um
a nb
od
y. Both
ch
em
i
c al
s al
s o ha v ep l
a u si b
lem
e cha ni
sm
s fo ri
n v ol
v em
en ti
n c a n c e rd
e v elopm
e nt. Th
u s,i
nd
ivi d
u alsw i
th
def
i ci
e ntr epa
i
rm
e chanism
sm
ayb
em
o r e s u s c ep
ti b l
e to c anc e ri f
e xpo s u r e sb
u r de n th
ei
r r epai
rc apa c
i
t y.C i g
a r et te sm
ok
e c o ntai
n s ch
em i
c al
s th
at yi
el d
RO S
in viv o and l
e ad
tof
o rm
ati
o n of 8
-O H
-G
r e si d
u e s, e s
p
e ci
al l
yi
nl
u n g ti
s s u e(
A
s am
i
, et al
, 19 9 7
)
.H
ow
e ver,b
r e a st c a n c e r studi
e s tod
ate r ep
o rt a r a ng
e of p
o si
ti
v e toi
n v e r s e a s s o ci
ati
o n sb
et we e nc
i
ga r et te sm
ok i
ng a nd b
r e a st c an c e r(
T e r ry and
Roh
a n , 2 00
2)
. Li p
op h i l i
cP C
Bs c a u s eR
O S
to a c c um
ulatei
nf
at ty tis s u e s,i
n cl
udi
n g br e a st ti
s s u e(
O
ak l
ey, et al
, 1 99
6)
.Ho
w
e v e r,m
o st stud i
e sh
a v e sh
ow
n n o o v e r al l
a s s o ci
ati
o nw i
th b
r e a st c a n c e r ri
sk
a nd
s e r u
m
PC
Bl
e v el
s(W
ol f f
, et al
, 19 9 3
; Kri
ege r, et al
,19 9
4 ;C
al l
e, et al
,2 0 0 2
)
.T
o e v al
u ate th
e a s s o ci
ati
o n of h O G G
l3 2 6 g
enot yp
e sw i
th
e n vi
r o nm
e ntal
e x po s u r e s to ci ga r et te s
m
oke a nd
PC
Bs, a ndb
r e a st c a n c e r ri
sk
,w
e ge n oty ped D N A
s a
m
p le s a nd
uti
li
z ed d
atafr
om
th
eC
a r ol i
n a Br e a stC
a n c e rS
tud
y(
C
BC S
)
.C
BC S i
s ap
op
ul
ati
onb
a s ed
c a s e-c o ntr ol
stud
y o
f A fr i
c a nA
m
eri
can a nd
n on-A fr i
c a nA
m
e
ri
c a nw
om
e ni
nN
o rth C
a r ol i
n a.W
e ge n ot yp
ed
2 0 2 5 c a s e s a nd
1 81
3 c o ntr ol
sfr
om
th
eC
BC S
a n
d
a n al
yz ed
po s si b l
e a s s o ci
ati
o n sw i
th
e x ta ntd
ata o n sm
ok i
n g a nd
PC B
e xp
o s u r e s.S
E L EC T
IV E R
EV I
EW
h O G G
lT h
eh
um
a n8
-o x og
u a ni
n e gl
yc o sy l
a s e/
ap
uri
ni
cl
ya s e,(
h O G G l
)
g
eneh
asb
e e nm
ap ped
to ch
r om
o s om
e3
, r egi
o n2
5(
Rad i
c el l
a, et al
, 19 9 7
)
o r2 6
(
Sh i
nm
u r a, et al
,fr equ e ntl y
m
is sing fr om
the chr om
o s om
al c o nte nts of
tum
o r c el l
s,i
n clu
d i
ngb
r e a st c a n c e rc e
l l
s(
Br u n e r, et al
, 2 00 0
)
T he a s s o cia
tio nb
etw
e e n thi
sl
o s s of h
ete r o zyg
o sit
y(
LO H
)
of
3 p 2 5-2
6
a nd
tum
o ri ge n e s
i
s c o ul din
d
ic ate th
ei m
po rta n c e of
hO G G
lin
m
ai
ntai
nin
g th
eD N A
stab i l i
t y th
ati
s ub i
qui
to u sl
ym is s
in
gfr
om
a n e upl
oi d
c a n c e r c el ls
.Du r
i
n g tr a n sl
atio n ,h O G G l m
RN A i
s spl i
c ed
to c r e ate tw
oi
s of
o rm
s of
th
epr ote
i
n , al
ph
a-
h O G G l
a nd b
eta-h O G G
l.
A l
ph
a-
h O G G
l, the
d
om
in a
nti
s of
o rm
,i
sc o n str u cte
d
of 34
5 am i
n o a ci d
s a nd i
sd
e sti
n ed f
o r th
e n u cle u s,w
hi l
eb
eta-
h O G G
Ii
s 4 2 4a
m in o
a ci dsin
le ngth
a nd i
sd
e stin e
d f
o r them i
to ch
o nd
ria
(
Boi
te u x a nd
Rad i
c el la, 1 99 9
)
F i g
u r e 4i l l
u str ate sh
ow
the al p h
a and b
etai
s of
o rm
s sh
a r e th
ef
i r st six
e x o n s,w h i
ch
cor r e spo n
d
s to th
ef
i r st3 16
am
i
n o a ci d
s(
B
oi
teu x and R
ad i
c el l
a,19 9 9
)
.A l p h
a-
h O G G l
e n
d
sw i
th e x o n s e v e nw h i l
eb
eta-h O G G
Iin c o r
po r ate s a
d i f f
e r e nt e x o nd
ow
n str e am
th
atr ep
l
a c e s e x o n s e v e n(
Boi
te u x and
Rad i
c el l
a,19 9 9
)
.h
O G G
l r emo v e s o xi d i
z ed
gu a nin e r e s
i d
u e sfr
om
DN A
. I t spe ci fi
c al l
y r e c ogniz e s
a n o x
i d i
z ed g
u anin e o
pp
o si
te a cyto si
n e o r a th
ym
in e
(
S h i
nm
u r a, et al
,19 9 7
;R
ad ic e
l l
a,et a
l
. ,19
97
)
. Th
eh O G G
lp
r otein
e xh i b i
ts th
eh
el ix
-h
air
p i
n-h
el i
x(
H h H
)
str u ctu r e thati
sco
m m
o n top
r otein s o
f
theb
a s e-e x c
is
io n
r epai
rf
am i l
y(
N
a sh
, et al
, 19 9
7)
.I
n th
eH h H
s
i
te, the c atal
y tic
glu
tam i
n e 24 9
at tr a cts th
e ele
ctr o n s of
theC
-1 p
o si
ti
o n of
thede o xyr
i b
o s e s ug
a r , a nd
c a u s e s th
e r ele a s e o
f 8
-o x o-
G
(
Fi
gu r e s
5
a nd 6
) (
Br u n e r, et al
,2 0 0 0
)
h O G G
l at ta ch
e sd i
r e ctl
y to th
e DN A
str a nd
c o ntain
i
ng
th
e 8 -o x -o-G
, c
le a v e s
thed
am
aged
r e si d
u e(
Br u n e r, et al
.,2 0 0 0
)
a nd l
e a v e s a n ap
u ri
ni
c si
te vi
ab
eta-e
l i
m
in a
tio n(
R
ad i
c el l
a, e^a/
.,19 9 7
)
.T
h
eh O G G
l ge n ei
s pol
ym
o rph i
c at s e v e r al
sit
e s.h O G G l S
e r3
26 C
ysh
a sb
e e na nd, the r efo r e, o n l y af fe cts the structur e of al p ha
-h O
G
G l, the iso for
m
that loc aHze s tot
h
e n u cl
e u s.A
ni
ni
ti
al f
u nc ti
o n al
stud
y of S
e r3
26 C
ys r epo rted
th
at a nE
c ol
ic o
m
p lem
e ntatio n a s s ay sh
ow
ed
a si
gni f
i c an td
e c r e a s ei
n r epai
r a cti
vi
t y of C
ys3
2 6c o
m
pa r ed
toS
e r3
26
(
K
oh
n o, et a
l
., 1
9 9 8
)
.K
oh
no, et a
l
, s ep
a r atel
y ad d
ed h O G G l S
e r3 2 6 a nd
C
ys 3 2 6 to am
utan t E c ol
i str ai
n that la cked
th
eh O G G
lh
om
ol
ogsM
utM
a n dM
utY wh i
ch
n o rm
al l y r epai
r 8-O
H-G
, a n
d m
e a s u r ed
th
e ge n e r ati
o n of
spo nta n e o u sm
utati
o n si
n th
e E. c ol i
c ol
o ni
e s a s ai
nd i
c ato r of p
r otei
n a cti
vi
t y.A
n oth
er stud y
m
e a s u r ed h O G G
l 3 2 6 r epai
r ef fi
ci
e n cyb
ym
e a s u ri
ng th
e r ate of
e x ci
si
o n of
8-O
H -
G
fr
om g
am m
a-i
r r a
d i
ated
DN A
, a nd
sh
ow
ed
a 1.6
-
f
old l
ow
e r e x ci
si
o n r atef
o r t he v a ri
a ntC
ys 3 26
;h
ow
e v e r , di f f
e r e n c ei
n e x ci
si
o n r ate s of
t he tw
o pr otei
n sw
e r e n ot stati
sti
c al
l ys
i
gni f
i c a n t(
Dh
e ri
n , et a/
,1
9 9
9)
.M
o st r e c e nt l y, D r. Ju n
N
ak
am
u r a a nd D
r.J
am
e sSw
e nb
e rg
, at th
eU
ni
v e r si
ty
of N
o rth C
a r ol i
n a atC h
ap
el H i l l
,d
e v el
op
ed
an a ss ay th
atm
e a s t i r e s apu ri
ni
c si
te s a s ani
nd i
c ati
o n of p
r otei
nf
u n cti
o n . Th
ei
rd
ata(
u npu
b l i
sh
ed
)
,s up po rt a
d
e c r e a s ed
a cti
vi
ty
of h O G G
lC
ys 3 2 6 Th
u s, th
r e ed i f f
e r e nt a s s ay
s r epo r td
e c r e a s ed
r ep
ai
r a cti
vi
t yf
o rh O G G
lC
ys3
26
; yet, n o n e of
th
e a s s aysm
e a s u r ed h
um
a n invitr o r e
p
ai
r.Po
l
ych l
o r in ated
B iph
e nyl
sP
ol y
chl
o ri
n atedb i
ph
e ny l(
PC
B)
d
efi n e s af
am
i l
y of ch lo ri
n ated
o rg
a nicc o
m
po u nd
s.T h
em
ol
e c ul
a r sk
el
eto ni
s c om
pri
s ed
of
tw
ob
enz e n e ri
ng
s(
F i
gu r e7
)
. Tw
oto te n c
h l
o ri
ne atom
s c a n rep l
a ce th
eh y d
r og
e n atom
s of
th
eb i p h
e ny l
stru ctu r e to c r e ate apo
l
ych l
ori
n ated b i p h
e nyl
.T h
e ch l
ori
n e atom
s c a n a r r a ng
e a r o u nd
th
e c or eri
n gsi
n2 09
pr ope rtie s. Fo r e x a
m
p le, s om
e c o nge n e r s a ct a s cyto chr om
e P 4 5 0i
nd
u c e r s, a nd /o rh
a v ee str oge n
i
c ef
fe cts, a nd /o r
e x hi b i
td i
o xi
n-l
i k
e a ct
i
vit
y(M
oysi
ch
et al
, 19
99
)
. Th
e r a ngeo
f b i
ol
ogic a
l a cti
vit
yi
s c e rtain
l
y a s o u r c e of h
e al
th c o n c e r n s Th
ey
a r e c o n si d
e r ed
tob
e ah
e alt
h h
a z a rd b
y th
e Envir o n
m
e ntal
Pr ote cti
o nA
ge n cy and h
a v eb
e e nl i
nk
ed
to s om
ef
o rm
s of
c a n c e r.M
etabol ism
of PCB s c a n le ad
to fo rm
atio n of RO S
a nd o xid
ativ ed
am
age.S
pe ci fi
c al l
y, cy to c
hr o
m
eP
450 w
i l l m
etab
ol i
z e PC
Bsw it
h f
ew
e r ch l
o rin e a
tom
sto R
O S
(
O
ak l
ey, et al
,19 9 6
)
.Fr o
m
the 19
3 0'
s to the
la
te 1 9 7 0's, PC
Bs we r e u s ed i
nm
an uf
a ctu ri
n g. Th
eyw
e r ep
ri
m
ari l y
u s ed i
n el
e ctri
c al
eq
ui p
m
entb
e c a u s e of
thei
rf l
am
e-r e s
i
sta nt n atu r e.C h l
o rin
eato
m
s repla c e
thef la
m
m
ab l
eh
yd
r oge n atom
s o n th
eb i
ph
enyl
ri
ng
to yi
el d
a n o n¬f la
m m
ab l
e s ub
sta n c e th
atw
a sfr
eq
ue ntl
y u s ed i
n tr an sf
orm
e r s, h yd
r a ul i
cf lu
i d
s,r efr i ge r ato r
m
oto r s, he atin
g c oi
ls, ai
r c o nd
i t io n e r s a nd
ty
pew
rit
e r sA
c c o rd i
ng
to theO h io
S
tateU
niv e r s
i
ty Exte n sio n
w
eb
si
te, PC B
sw
e r e u s ed
tom
a n uf
a ctu r e c e r e al b
o x e s,p
la s
tic s
, v a r ni
sh
e s a nd
pai
nts, tol i
st o nl y af
ew
c om
m
o n pr od
u cts(
h
ttp
://oh i
ol i
n e.o s u .ed
u/
cd
-
f
a ct/ 0
20
1.
h
tm l
)
.I
n 19
79
,P C
Bsw
e r eb
a nned fr
om
al l
m
a n uf
a ctu rin
gin
theU
ni
ted S
tate s.H
ow
e v e r, PC
Bsp
e r sis
tin
th
e e n vi
r o nm
e nt. Th
ey a r e n ot r e ad i l
yb
r ok
e nd
ownb
ym
ic r o o rga ni
sm
s o r ot he r e n vir o n
m
e ntal
pr o c e s s e s, a nd
th
ey a r ein c r e a s
in
gin
thee n v
ir o n
m
e nt a sla n
d f
il l
sl
e a ch
PC
Bsi
nto gr o u nd w
ate r. Th
e s el i
pi d
-s o
lu
b l
e c
h
em i
c al
sa r e a
b
s o rb
ed
a nd p
a s s ed
up
thr o u
gh
thef
o od
ch
ai
n.O
n e of
th
eb i g g
e st c o n c e r n s o v e rTobac co
Sm
okeTo
b
a c c o sm
ok
e c o ntai
n s n um
e r o u s c a r ci
n oge n si
n cl
ud i
n g pol y
cycl i
c a r om
ati
ch y
d
r o c a rb
o n s(
P AH
s)
, a r om
ati
c am
i
n e s, and N
-n
i
tr o s am i
n e s(
Te r ry a nd
Roh
a n, 2 00
2)
T he U pop
h i l i
c c a r ci
n ogen si
n th
e sm
ok
e c a nb
e tr a n spo rted
a c r o s s th
e al
v e ol
a rm
em
b
r a n einto the b
l
o od str e am
a nd
, e v e ntu all
y, sto r ed i
n ad i
po s e tis s u e s s u ch
a si
n t heb
r e a st(
Te r ry a nd R
oh
a n, 20 0
2)
. Th
e s e c a r ci
n og
e n s oft
e nf
o rm b
ul k
y ad d
u cts o n th
e DN A
w
hi
ch c a nl
e ad d i
r e ctl
y tom
utati
on s.M
etab
ol i
sm
of
s om
e ch
em
i
c al
si
n tob
a c c o sm
ok
ef
o rm R O S
a nd
r e sult
i
nf
o rm
ati
o n of
oxi d i
z eg
u a ni
n es.T h
u s, ap l
a u si b l
ep
ath w
ay
e xi
stsf
o r a n a s s o ci
ati
o nb
etw
e e n ci g
a r et te sm
ok i
ng and b
r e a st c an c e r.H
ow
e v e r, r e s ul
tsl i
nk i
ngb
r e a st c a n c e r to ci g
a r et te sm
ok i
n gh
a v eb
e e nin c o n s
i
ste nt(
Te r ry&
Roh
an , 2 0 0 2)
.A
r e c e ntl i
te r atu r e r e vi
ew
c om p i
led d
atafr
om
popu lat
i
o n-b
a s ed
c a s e-c o ntr ol
s stud i
e s of
ci
ga r et te s
m
oki
ng a nd b
r e a st c a n c e r ri
sk
,l i
ket
h
eC
a r ol i
n aB
r e a stC
a n c e rS
tud
y.F
o r sm
ok i
ng d
u r ati
o n of
sm
ok i
ngf
o rm
o r e th
a n2 0
ye a r s, t
h
e c om
pi l
ati
o nl i
sted
o n e stud
y th
at r ep
o rted
a pr ote cti
v e a s so ci
atio n(
O
R0 7
,9 5
% C
I0
.5-
0
.9
)
, o n e stud y w i t
h
n o a s s o ci
ati
o n(
O R
1.2,
9
5%
C
I 0.8
- 1
.7
)
, a nd
twostu
d i
e sw i
th
po si
ti
v e a s s o ci
ati
o n s(
O
R 1.3
, 9 5% C
I 1.1
-1.
8
;O
R 1 6, 9 5
% C
I 1.1-2
.3
)
F I
G
UR
ES
F i gu r e 1. De o x y
g
u a n o sin eO
H N
H
2N
N
'
N H
-
N
:
0
\
d R
F i
g
u r e2
.8
-o x -o-
d
J
^
B ifu n ctio n al D M A« oxor.* 1 olv c o sv la s e
3'
-B-lv a s e
J
3'-tri m min
g b y
^
\
. H A PI o r PolBJ
J
m
U
1
PolBX R C C 1
i
i
s
m
I
Lialllc xPoip
X R C C1
F i g
u r e3
.Sh
or tP
atch B
a s eE
x ci
si
onR
ep
ai
rP
ath w
ay
h O G G
l is ab
if
u n cti
o n al D N A
gl
yc o syl
a s ei
n v ol
v ed
in th
ef
ir st step
.(
A dapted fr om N i ls e n a nd Kr oka n, 2 0 0 1)
Mig f i
. _
—A
A
1 I 2 ; 3
.
h
i
i
i
l
r
F i gu r e
4
.h O G G l E
x onsO
n e toE i
gh
t and S
pl i
ci
ngA
r r ang
em
ent10
G ln3 1 S G ly 4 2
Phe3 1 9 -•
E
l ♦- C ys2 5 3
Val 2 5 0
As n1 5 1
H IS2 7 0
^
®
j
si
^
4
As nI SO / / G lr\2 4 9
» As n1 4 9
9
n
T yr2 0 3€
Arg 1 5 4 Afg 2 0 4
3'
F i g
ur e5
.8
-o x o-G
u ani
n ei
nD N A S
tra nd
a nd h O G G l I
nte r acti
o nW
i
th N
um
b
e r ed
A
m
i
n oA
ci d
si
nB l
ue(
A dapted fr o m Br u n e i,eta l , 2 00 0)
<
>;
F
i gur e6
.3
-D
view o
f
liO G G l
at ta ch i
ng
to8
-o x o-G
8
-o x -o-
G
, r e
d
,i
n th
e c e nte r of
th
ep
ictu r e isf l
ip p
ed
o ut of
th
eh
el i
x a nd
si
tsi
n th
e acti
ve si
te of h O G G l
,p
urp l
e ri b b
o n s a nd
r ed
a r r ow s1 1
^ '
n^ (I O-n)
Po l ych lo rin ated B ip he ny l
(
P C B)
M
A T E R I A L S A N D
M
E T
H
O D S
S
tud
y Pop ul
atio nD eta
i l
s of th
eC
BC S
stud
y popul
ati
o n a nd
r e s ul
ts spe ci f i
c al l
y c o n c e r ni
ng sm
ok i
nga n
d
PC
B e xp
o s u r ew
e r ep
r e vi
o u sl
y r epo rted
( M
i l l i k
a n, et a
l
, 19 9 8
;M
i l l i k
a n , et ah
,2 0 0 0
)
Bri
ef l y
, t
h
eC
BC S i
s ap
op
ul
ati
o n -b
a se
d
c a s e-c o ntr o
l
stud y
th
at e n r ol l
ed
a total
of
4 3
3
3w
om
e nfr
om
2 4 c o u n ti
e si
n e a ste r n a nd
c e ntr al N
o rth C
a r ol i
n a, age s2 0
-7
4,
w i
th
i
n v a si
v e a nd i
n-si
tub
r e a st ca n c e r and m
atch i
ng
c o ntr ol
s(
N
ew
m
a n, et al
, 19 9 5
)
.A g
oal
o
f
th
eC
BC S w
a s to o v e r-s a
m
p leA fr i
c a nA
m
e ri
c anw
om
en a nd w
om
e n u nd
e r th
e age of
5 0. Ra c e
w
a s s elf
r epo rted
. Th
e 2%
wh
o r ep
o rted N
ati
v eA
m
e ri
c a n o rH i
spa ni
c we r ec atego r
i
z ed
a s n o n-A fr i
c a nA
m
e ri
c a n.
C
a s e sw
e r ei d
en ti f
i ed
th
r o ugh
a r api d
a s c e rtai
nm
e nt sy
stem
i
n c o op
e r ati
o n wi
th
the
N
o rth C
a r ol i
n aC
e ntr alC
a n c e r Regi
stry a nd h
ad
af
i r std i
agno si
s a nd h i
stol
og i
c al l yc o n
f
i rm
ed i
n v a si
v e o r in situb
r e a st c a n c e r .C
o ntr ol
sw
e r ei d
e nti f
i ed
u si
ng
th
eD i
vi
si
o no
f
M
oto rV
eh i
cl
e sl i
stsf
o rw
om
e n u nd
e r age6
5 a nd
M
ed i
c a r eb
e n ef
i ci
a ryl i
stsf
o rwo
m
e n6
5 ye a r s a nd
o v e r.T h
e o v e r al l
r e spo n s e r atew
a s7
6.0
%
f
o r c a s e s a nd
5 5.0 % f
o rc o ntr o
l
s. En r ol l
ed w
om
e nw
e r ei
nte r vi
ew
ed
ath
om
eb
y a tr ai
n ed
n u r s e, a nd 89
.0%
of
r es
p
o nd i
ng c a s e s and 9 0
.0
%
of
r e spo n
di
ng c o ntr ol
s ga v e a 30
-m
L b l
o o
d
s am
p l
e at th
et
i m
e of
th
ei
nte r vi
ew
. Th
ei
nte r vi
ew i
n cl
ud
ed q
u e sti
o n s ab
out sm
ok i
ng h i
story
a nd
oth
e rr
i
sk f
a cto r sf
o rb
r e a st c a n c e r s u ch
a sf
am i l
y, r epr od
u cti
v e a nd m
e n str u al h i
sto ry
,h
o rm
on eu s e, alc o
h
ol
u s e, a nd
s o ci
o -d
e
m
ogr ap hi
c ch
a r a cte ri
sti
c s(
N
ew
m
a n, et al
, 1 9 95
)
. Bl
o od
1 3
to obtain D
N A
fr om
peri p he r al b lo od l ym
p hcyte s. Pa rtici pan tsw
ho ga v e b lo od s am
ple sf
or DN A
a nd
PC
Bs a nd
th
o s ew h
od i d
n otw
e r e n ot stati
sti
c all
yd
if f
e r e ntw i
th
r e sp
e ct toage a n
d
a v a ri
ety of b
r e a st c a n c e rri
sk f
a cto r s.G
e n oty p ing Pr o c edu r e sFo r r e vie
w
,i
n T aqm
a n^ ' ^
che
m i
stry , a s e cti
o n of
DN A
s u r r o undi
ng
th
eS
N P of
i
nte r e sti
s am
pl i f
i ed w i
th
pri m
e r s a nd p
r ob
e si
n ah
ot-start P
C R
a s say
. Tw
oSN P
-s
p
e ci f
i co
l i g
o-pr o
b
e s a r el
ab
el
ed w i
th d i f f
e r e ntd
ye s.A ft
e r th
e PC R
pr o c e s s, th
ep
r ob
e s sh
o ul d
f l
u o r e s c ei
n o n e of
th
r e ew
ays : al l
of
o n ed
ye,i
nd i
c ate s ah
om
o zy g
ou s ge n ot yp
ef
o ra
l
lel
e 1 ; al l
of
th
e oth
e rd
ye,i
nd i
c ate s ah
om
o zy go u sg
e n ot y pef
o r al l
el
e2
; o rb
oth
pr ob
e sf l
u o r e s ci
ng,i
nd i
c ate ah
ete r o zyg
o u s ge n ot yp
e.Fo r t
h
eh O G G
l3 2
6 Taqm
a n ^ ' ^a s s ay, P
C R
pri m
e r s and p
r ob
e sw
er ed
esi
gn ed
u s
i
ng
Pri
m
e rE
xp
r e s s^ ' ^ s oft w
a r e(
A p p l i
ed B i
o system
s)
.A
s s ayd
e si
gn a nd
c o nd i
ti
o n swe r e
b
a s ed
o n th
e al l
el i
cd i
s cri m i
n ati
o n pr oto c ol fr
om A p p l i
ed B i
o system
s.O l i
go n u cl
e oti d
ed
e si g
nw
a sb
a s ed
o n th
eG
e nb
a nk
DN A
s eq
u e n c e, a c c e s si
o n n um
b
e rA
F 0 8 8 2 8 2(
h
tt p ://
n cb i
.nl m
.n
i h
.go v)
. Th
eS
e r3 26
(
C
)
al l
el
epr o
b
ew
a sl
ab
el
ed
o n th
e5
'e n
d w i
th
th
eV I C
r epo rte rd
ye a nd
c o ntai
n ed
th
ef
ol l
ow i
n g n u cl
e oti d
e s eq
u e n c e,5
'
-C G -C -C A A
TC C C G C C A
-3'
, w
i
th
a7 1
.4 % G
-
C
c o nte nt a nd 6
5.
6
°
C m
el
ti
ng tem p
e r atu r e.T h
eD N A C
ys3 2 6
(
G
)
al l
el
ep
r ob
ew
a sl
ab
el
ed
o n th
e5
' e nd
wi
th
th
e6
-F A
M
r ep
o rte rd
ye a nd
c o ntai
n ed
th
ef
ol l
ow i
ng
n u cl
e oti d
e s eq
u e n c e5
'-C C A A T G C C G C C A T G
-3
' ,w i
th
a
6
4.3 % G
-
C
c onte nt an
d 6 6
.5
°
C
m
el
ti
ng tem
pe r atu r e. Both p
r ob
e sw
e r em
i
n o rg
r o o v eb i
nd i
ng
(M
G B
)
a nd
u s ed
a n o n-f l
u o r e s c
i
ng qu e n ch
e r(
N
FQ )
o n th
e3
'
e n
d
.F
o rwa rd
a nd
r e v e r s e p
ri
m
e r sw
e r e u s ed
to am
pl i f
y th
e r egi
o n s u r r o u nd i
ng
th
eS
e r32 6 C y
s1 4
s equ e n c e fo r t
h
e fo rw
a rd pr im
e rw
a s 5'-C C C
TC C
TA C A G G
TG C
TG
T TC A
-3'w i
th
am
el
ti
n g tem
p
e r atu r e of
5 8.8
°C
and
a5
7.1
%
G
-
C
c o nte nt Th
e nu cl
e oti d
e s equ enc e
f
o rt
h
e r e v e r s e pri m
e rw
a s5
'-G A A C C C T
TT C
TG C G C T T
T -3
'
w i
th
am
el
ti
n g tem
pe r atu r e of
S S.
O
^
C
wi
th a 5 2.6%
G
-
C
c o nte nt.
P C
R r e a cti
o n sw
e r e pe rf
o rm
ed i
n a 1 5.0
ul
r e a cti
on v ol
um
e u si
ng t heh
ot-sta r t
f
o rm
at.T h
e r e a cti
on c om
p
o n e n tsw
e r e a sf
ol l
ow
s :0
.7 X T
aq
m
a nU
ni
v e r s al
PC
RM
a ste rM
i
x , 90 0
nM
of
e a ch p
ri
m
e r,2 0
0nM
of
e a ch
pr ob
e, and I S
.Ong of
genom i
c DN A
.P
ri m
e r s a nd
pr ob
e sw
e r em
a n uf
a ctu r ed b y A p p l i
ed B i
o sy
stem
s(
F
o ste rC i
ty
,C A
)
.T h
eP
C R w
a s r un on a Pe rk i
nE l
m
e rG
e nA
m
p®
9 7 0 0 th
e rm
o cycl
e r usi
ng
th
e9 6 0
0m
od
eu n
d
e r th
ef
ol l
ow i
ng
cond i
ti
o n s :1
cycl
e of 5 0
.0
°C f
o r2 m i
n ute s(
A
m
p E
r a s e® U N G
A
cti
v ati
o n)
, 1 cycl
e of
9 5.0
°
C f
o r 10 m i
n ute s(
A m
pl i
TaqG
ol d A
cti
v ati
o n)
,f
ol l
ow
ed b y
3
5 cycl
e s of
9 1.0 °C f
o r 1 5 s e c o nd
s(
d
e n atu r e)
a nd
58
.5
°C f
o r 1m
i
n ute(
a n n e al/
e x te nd
)
.T h
efl
u o r e s c enc e v al
u e s a nd
al l
el
e c al l
sw e r ed
ete rm i
n ed
u si
ng th
eA
pp l i
ed
Bi
o system
s'
7 7 0 0 syste
m
a nd th
ei
r s of
tw
a r e.S
ynt heti
c ol i
gonu cl
e oti d
e sf
r om
th
eN
u cl
ei
cA
ci d
sC
o r eF
a ci l i
ty at th
eU
ni
v e rsi
ty
of N
o rth C
a r ol i
n a(
C h
ap
el H i l l
,N C
)
th
atw
e r ed
e si g
ned
tom
atch
th
el
e ng
th
and S N
P-sp
e ci f
i c s eq
u e n c e of
th
ePC R
am
p l i
co nw
e r e u s ed f
o rp
o si
ti
v ec o ntr o
l
s. Fo rS
e r3
2 6 th
e 8 3b
a s epa
i
r(
b
p)
n u cl
e oti d
e s equ e n c ew
a s5
'
-C -C -C
TC C
TA C A G G T G C
TG T T C A G T G C C G A C C T G C G C C A
A
TC C C G C C A
TG C
TC A G G A G C C A C C A G C A A A G C G C A G A A A G G G
T TC C
-3
'
Fo r
C
ys3 26
th
e s equ e n c ew
a si d
e nti
c al
to th
e pr e vi
o u s s equ e n c ew i
th
aG
s ub
sti
tuted f
o rC
_atbp 4 0
.S
am
p l
es th
at co ul d
n otb
e s c o r ed w
e re r ep
e ated
.U
n r e ad
ab l
e r e sul
ts on th
e th i
rd
atte
m
p
t,(
N
=
2 5
)
,
w
e r e s c o r ed
a s"