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Y

U

-P

A

N

G

L I

N

. I

d

en t i f ic at io n an

d q

uan t i tat io n o

f

D N A

a

d d

u cts

f

r o

m

c a

l f

t

h y

m

u s

D

N

A

e x

p

o s e

d

to 1,2

-e

p

o x

y b

u ten e

(u

n

d

e r t

h

e

d i

r e ct

io

n o

f

J

a

m

es

A

.

S

w

e n

b

er

g

)

A b

s

t

r a c

t

T h i

s stu

d y

w

a s c o n

d

u cte

d

to c

h

a r a cte r

iz e

t

h

e

D

N

A

a

d d

u cts

f

o r

m

e

d

w

h

e n

a

d

e n

i

n e,

g

u a nos

i

n e or c a

l f

t

h y

m

u s

D

N

A i

s reacte

d

w

i

th 1,

2

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p

o x

y b

ute n e

(

E B

)

.

H

P L C

s e

p

a r at

i

o n c o u

p

l

e

d w

i

th

d

i

o

d

e

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y d

ete ctio n

w

a s e

m

p l

o

y

e

d

t

o

is o

l

a

t

e

t

h

e a

d d

u cts t

h

at

w

ere

f

or

m

e

d

.

T

w

o

E B

- a

d

e n

i

n e

p

r o

d

u cts

w

e r e

i

d

e nt

i

f

i

e

d

a s

N

^

-(

2-

h y d

ro x

y

-

3

-

b

ute n - l

-y l

)

a

d

e n

i

n e

(

E B-

A d

e

I

)

a n

d

N

^-

(

l

-

h

y

d

r ox

y

-

3

-

b

u te n -

2

-y

l

)

a

d

e n

i

n e

(

E B

-

A d

e

I I

)

b y

^

H

-

N

M

R

a n a

l y

s

i

s

.

T

w

o

g

uan

i

n e a

d d

u cts s

i

m

i l

a r to t

h

o s e

p

r e v

io u s

l y

c

h

a r a cter

iz e

d

a s

7

-

(

2

-h y d

rox

y

-

3

-

b

u te n-

l

-

y l)

g

u a n

i

n e

(

E B

-

G

u a

I

)

a n

d

7-

(

l

-

h y d

r o x

y

-

3

-

b

ut en -2

-y l

)

g

u anin e

(

E B-

G

u

a I I

) w

e re a

l

s o co

l l

e c te

d

.

A l l f

o u r

a

d

d

u cts

w

e r e u s e

d

a s r e

f

e r e n c e c o

m

p

o u n

d

s to

d

ete ct a n

d

q

u a nt

i

tate t

h

e

cor re s

p

o n

d i

n

g

a

d

d

u ct s

p

e c

ie s

f

or

m

e

d in

D

N

A in c u

b

ate

d

i

n v

i

tr o

w

i

t

h E B

.

E B

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d

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d E B

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u a n

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d

d

u cts

w

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d

i

n

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e

d

c a

l

f

t

h

y

m

u s

D

N

A

.

T h

e

f

o r

m

at

i

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f E B

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d

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d d

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(

1

.

0

x

1 0

'^

a n

d 2

.

7

x

10

'^

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A

d

e

I

a n

d

I I

p

e r a

d

e n

in e

, r e s

p

e ct

i

v e

l

y

)

in c a

l f

t

h

y m

u s

D

N

A

w

a s a

p p

r ox

im

ate

l

y

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h

t

h

at

o

f

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u a n

in e a

d d

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(

4

.

2

x

1 0

'^

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d 3

.

0

x

1 0

'^

E B

-

G

u a

I

a n

d I I

p

e r

g

u a n

i

n e,

r e s

p

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ti

v e

l

y

)

.

S i

n c e

1

,

3

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bu

ta

d i

e ne

(

B D

)

a n

d

i

ts

m

eta

b

o

l i

te s,

E B

a n

d

d i

e

p

ox

y

b

u ta n e,

i

n

d

u c e n e ar

l

y

e

q

u a

l

n u

m

b

e r s o

f p

o

in

t

m

u ta

ti

o n s a t

A

:

T

a n

d G

:C

b

a s e

p

a

i

rs, t

h

e

N

^

-s u

b

s

ti

tu te

d

a

d

e n

i

n e a

d d

u cts

m

a

y

r e

p

r e s e n t

i

m

p

o rta nt

p

r o

m

u ta

g

e n

ic

D

N

A

a

d d

ucts

i

nv o

l

v e

d

i

n

B D

-

i

n

d

u c e

d

m

u ta

g

e n e s

i

s a n

d

(2)

A

C K

N O

W

L E

D G

E

M

E

N

T S

I

w

o u

l d

l i

k

e to t

h

a n

k

D

r.

J

a

m

e s

A

.

Sw

e n

b

er

g f

o r

h i

s

g

u

i d

a n c e a n

d

s u

p p

or t t

h

ro u

g

h

o u t t

h i

s stu

d y

.

I

a

l

s o t

h

a n

k D

r.

B

a

l l

,

D

r.

G

o

l d

a n

d D

r.

B

o n

d

f

or

t

h

e

i

r v a

lu

a

b l

e a

d

v

i

c e on t

h i

s r e

p

o r t.

I

a

m

g

r ate

f

u

l

to

D

r.

R

a

m

i

a

h Sa n

g

a

i

a

h

a n

d

D

r.

A

s o

k

a

Ra n a s

in

g

h

e

f

o r t

h

e

i

r

in v a

l

u a

b l

e a s s

is

is

ta n c e

i

n t

h

e

p

e r

f

or

m

i

n

g

n u c

l

e ar

m

a

g

n et

i

c r e s o n a n c e

(

N

M

R

)

a n a

l

y

s

i

s a n

d m

a s s s

p

e ctr o

m

etr

y

, r e s

p

e ct

iv e

l

y

, a n

d

i

n ter

p

r etat

i

n

g

t

h

e re s u

l

t

i

n

g

s

p

e ctra

d

ata.

I

g

rate

f

u

l l

y

a c

k

n o

w

l

e

d

g

e

N

o v a

Sc

h

e

l l

er a n

d P

atr

ic

i

a

B

.

U p

to n

f

o r t

h

e

i

r

te c

h

n

i

c a

l

a s s

i

sta n c e a n

d

h

e

l

p

f

u

l

d

i

s c u s s

io n s

t

h

r o u

g

h

o ut t

h

e stu

d y

.

I

a

m

i

n

d

e

b

te

d

to

m

y

c o

l l

e a

g

u e s

f

o r t

h

e

i

r

h

e

l

p

a n

d

s u

gg

e st

i

o n s to t

h i

s

w

or

k

.

T h i

s

w

o r

k

w

a s s

p

o n s ore

d

i

n

p

a r t

b

y

a

g

r a nt

f

r o

m

t

h

e

C h

e

m

i

c a

l

M

a n u

f

a ctu r e r s

(3)

HI

T

A

B L E

O

F C

O N

T E

N

T S

P

a

g

e

L I

S

T

O

F T A B L E S

iv

L I ST O F F I G U R E S

v

L

I S T O

F

A

B B R

E V

I

A

T I

O

N

S

v

i

I

.

I

N

T

R

O

D U C

T I

O

N

1

I I

.

M

A T

E

R I A L S A

N

D

M

E T

H

O D S

4

A

.

C h

e

m

i

c a

ls

4

B

.

H

i

g

h P

e r

f

o r

m

a n c e

L i

q

u

i

d

C h

r o

m

ato

g

r a

p

h

y

(

H

P L

C

)

4

C

.

M

a ss

S

p

ectr o

m

etr

y

4

D

.

R

ea ct

io n o

f E B

w

i

t

h G

u a n o s

i

n e

5

E

.

R

e a ct

io n o

f E B

w

i

th A de n

i

n e

6

F

.

^

H

-

N

M

R S

p

e ctr a

D

ata

8

G

.

R

e a ct

i

o n o

f E B

w

i

t

h C

a

l f T h

y m

u s

D

N

A

8

H

.

G

u a n

i

n e

A

s s a

y

a n

d

A d

e n

i

n e

A

n a

l y

s

is

9

I I I

.

R E S U L T S

1 0

A

.

R

e a ct

i

o n o

f E B

w

i

t

h G

u a n o s

i

n e

1 0

B

.

R

e a ct

i

o n o

f E B

w

i

t

h A d

e n

in e

1 0

C

.

R

e a ct

i

o n o

f E B

w

i

th Ca

lf

T h

y m

u s

D

N

A

.

1 2

I V

.

D I SC U S SI O

N

1 4

V

.

R E F E R E

N

C E S

1 6

(4)

IV

L I

S

T

O

F

T

A

B L E

S

P

a

g

e

T

a

b l

e

1

.

M

e a s ure

d

v a

lu e

s o

f

X

„ iax ^^

^

^

n o

f

e a c

h E B

-a

d

en

i

n e a

d d

u ct , a n

d

t

h

e c o r r e s

p

o n

d i

n

g

e st

i

m

ate

d

a

bs o r

p

t

i

v

i

t

y

c o e

f f i

c

i

e n ts

i

n

p

are n t

h

e s

is a

t

d i f f

er e nt

p H

v a

l

u e s

2 0

T

a

b l

e

2

.

E

st

i

m

ate

d

a

m

o u nt o

f

p

u r

i

n e a

d d

u cts

i

n

N

T

H f

r o

m

E B

-e x

p

o s e

d D

N

A

a n

d

t

h

e

p

r o

p

o rt

io n o

f

t

h

e a

m

o u nt o

f E B

(5)

L

I

S T

O

F

F I

G U

R E S

P

a

g

e

F i

g

.

1

.

C h

r o

m

ato

g

r a

m

o

f

t

h

e

p

ro

d

u cts o

f

t

h

e r e a ct

i

o n o

f E B

w

i

t

h

g

uan o s

i

n e a

f

ter a c

i d h y d

r o

l y

s

i

s

2 3

F i g

.

2

.

A

b

s o r

p

t

i

o n s

p

ectr a o

f

g

uan

i

ne,

E

B -

G

u a

I

a n

d I I i

n

m

o

b i l

e

p

h

a s e

d

u r

i

n

g

t

h

e

H

P

L C

a n a

l y

s

is

w

i

t

h D A D

o n t

h

e

p

re v

i

o u s

f i g

u r e

2 4

F i

g

.

3

.

M

a s s s

p

e ctr a o

f E B

-

G

u a

I I f

r o

m

(

A

)

l i

q

u

i d

s e c o n

d

a r

y

i

o n

iz a

t

i

o n

m

a s s s

p

e ctr o

m

etr

y

a n

d

(

B

) M

S/ M

S

2 5

F i

g

.

4

.

C h

r o

m

ato

g

r a

m

o

f

t

h

e

p

ro

d

u cts o

f

t

h

e r e act

i

o n o

f E B

w

i

t

h

a

d

e n

in e

2 6

F i

g

.

5

.

A b

s or

p

t

i

o n s

p

e ctr a o

f

a

d

e n

i

n e ,

E B

-

A d

e

I

a n

d I I i

n

m

o

b i l

e

p h

a s e

d

u r

i

n

g

t

h

e

H

P

L

C

a n a

l y

s

i

s

w

i

t

h

D

A D

o n t

h

e

p

r e v

i

o u s

f i

g

u r e

2 7

F i

g

.

6

.

M

a s s s

p

e ctra o

f E B

-

A

d

e

I I f

r o

m

(

A

)

l i

q

u

i d

s e c o n

d

a r

y

i

o n

iz a

t

i

o n

m

a s s s

p

e ctro

m

etr

y

a n

d

(

B

)

M

S

/

M

S

2 8

F i

g

.

7

.

^

H

-

N

M

R

s

p

e ctra o

f E B

-

A

d

e

(

A

)

I

a n

d

(

B

)

I I

i

n

D

M

SO

-

d

^

2 9

F i

g

.

8

.

C

h

r o

m

ato

g

r a

m

o

f

n e utra

l

t

h

er

m

a

l h

y d

r o

l

y

s ate

f

r o

m

c a

l f

t

h

y m

u s

D

N

A

ex

p

os e

d

to

E

B

3 0

F i

g

.

9

.

C h

r o

m

ato

g

r a

m

o

f

a c

i d h

y d

ro

l

y

s ate

f

ro

m

c a

l f

t

h

y

m

u s

(6)

V I

L

I S

T

O

F

A

B

B R

E

V

I

A

T I

O N

S

B

D

1

,

3

-

b

u ta

d i

e n e

C Y

P 2

E

1 c

y

to c

h

r o

m

e

P 4 5 0 2 E 1

m

o n o o x

y

g

e n a s e

D

A

D

d

io

d

e

-ar r a

y d

ete ctor

D

E B

d

i

e

p

o x

y b

u ta n e

E B

1

,2

- e

p

ox

y b

u ten e

E B D

3

,

4

-e

p

ox

y

-

l

,

2

-

b

u ta n e

d io

l

E

B-

A d

e I

N

^-

(

2

-h y d

r ox

y

-

3

-

b

u te n-

l

-y

l

)

a

d

e n

i

n e

E B-

A d

e

I I

N

^-

(

l

-

h y d

r o x

y

-

3

-

b

u te n-

2

-y

l

)

a

d

e n

i

n e

E B

-

G

u a

I

7

-

(

2

-

h

y

d

r ox

y

-

3

-

b

u te n-

l

-y

l

)

g

u a n

i

n e

E

B-

G

u a

I

I

7

-

(

l

-

h

y

d

r ox

y

-

3

-

b

u te n-

2

-y

l

)

g

u a n

i

n e

F

R

fl

o

w

r ate

H

P L C

h

i

g h

p

er

f

o r

m

an c e

l i

q

u

i d

c

h

ro

m

ato

g

r a

p h

y

L S I

M

S

l i

q

u

i d

s e c o n

d

ar

y

i

o n

i

zat

i

o n

m

a s s s

p

e ctr a

M

S /

M

S

ta n

d

e

m

s

p

e ctr o

m

etr

y

N

M

R

n u c

l

e a r

m

a

g

n et

ic r e s o n a n c e

N

T

H

n e utr a

l

t

h

er

m

a

l h

y

d

ro

l

y

s ate

S

P

E

so

l i d

p

h

a s e e x tra ct

i

o n

T L

C

t

h i

n

l

a

y

e r c

h

ro

m

ato

g

r a

p

h y

(7)

I

N

T

R

O D U

C T

I

O N

1

^

-

B

u ta

d

i

e n e

(

B D

)

, a c o

l

o r

l

e s s

g

a s,

h

a s

b

e e n

m

a

i

n

ly

u s e

d

i

n

t

h

e

m

a n u

f

a ctu r e o

f

s

y

n t

h

et

i

c r u

b b

er.

E

m

i

s s

i

o n o

f

B D

m

a

y

o ccu r

d

ur

i

n

g

i

ts

m

an u

f

a ctu re, stora

g

e, tr an s

p

o rt, u s e o r

d

i

s

p

o s a

l

.

B D

h

a s a

l

s o

b

e e n

f

o u n

d

i

n

auto

m

o

b i l

e ex

h

au st

[

1

]

an

d

c

i g

a r et te s

m

o

k

e

[

2

]

.

B D

i

s

l i

ste

d

a s on e o

f 18 9

h

a z a r

d

o u s a

i

r

p

o

l l

u ta n ts

i

n t

h

e

19 9 0 C l

e a n

A i

r

A

c

t A

m

e n

d

m

e n ts

[

3

]

.

T h

e In te rna t

i

on a

l A g

en c

y f

o r

R

ese a r c

h

o n

C

an c e r

h

a s c

l

a s s

i f i

e

d B D

a s a

g

r o u

p

2 A

c a r c

i

n o

g

e n

[

4

]

w

i

th s u

f f i

c

i

e n t e v

i

d

e n c e

f

o r c a r c

i

n o

g

e n

i

c

i

t

y

i

n ex

p

er

i

m

e nta

l

a n

i

m

a

l

s,

b

u t

l i

m

i

te

d

ev

i d

e n c e

f

or c a r c

i

n o

g

e n

i

c

it

y

o

f

B D i

n

h

u

m

a n s.

E

p

i

d

e

m

i

o

l

o

g

i

c a

l

stu

d i

e s o

f

w

or

k

e r s e x

p

o s e

d

to

B D

p

ro v

i

d

e c o ntr o v e r s

i

a

l

e v

i

d

e n c e

w

i

t

h

the r e s u

lt

s s

h

o

w

i

n

g

t

h

e a s s o c

i

at

i

o n o

f

B D

e x

p

o s ur e

w

i

t

h

l y

m

p

h

at

i

c a n

d

h

e

m

ato

p

o

i

et

i

c c a n c ers

d

e s

p

i

te a n o v er a

l l

d

e

f i

c

it

i

n c a n c e r

m

o rta

l i

t

y

[

5

,

6

]

.

R

e c e nt

f i

n

d i

n

g

s

i

n r o

d

en t

i

n

h

a

l

atio n c a rc

i

n o

g

e n

i

c

i

t

y

stu

d i

e s o

f

B D

h

a v e

d

e

m

o n str ate

d

t

h

e str

i k i

n

g

l

y

h i

g

h

s e n s

i

tiv

i

t

y

o

f

m

i

c e to

B D

-

i

n

d

u c e

d

tu

m

o r s c o

m p

ar e

d

to r ats

[

7

-

9

]

.

L

o

w

ex

p

o s u re o

f B D

, eve n at

6

.

2 5

p p

m

,

i

n c r e a s e

d

i

n c

i

d

e n c e s o

f

tu

m

o r s

i

n

f

e

m

a

l

e

B 6 C 3 F 1

m

i

c e;

i

n c o n

t

r a st,

i

n c r e a s e

d i

n c

i

d

e n c e s o

f

tu

m

o r s

w

ere o n

l

y

s e e n

i

n

S

p

r a

g

u e-

D

a

w

l

e

y

r ats e x

p

o s e

d

to

10 0 0

to

8 0 0 0

p p

m

B D

.

T h

e

m

ar

k

e

d

s

p

e c

i

es

d i f f

ere n c e

i

n ca rc

i

no

g

en

i

c

p

o te n c

y

o

f B D

i

s n ea r

l

y

t

h

r e e o r

d

ers o

f

m

a

g

n

i

tu

d

e

h i

g

h

er

i

n

m

i

c e t

h

a n r ats, a n

d

h

e n c e

B D

i

s a

p

o te nt

c a r c

i

n o

g

e n

i

n

m

i

c e a n

d

a

w

e a

k

c a r c

i

n o

g

e n

i

n t

h

e r at.

A

c co r

d i

n

g

to s e v e r a

l

stu

d

i

e s o n t

h

e

m

eta

b

o

l i

s

m

o

f

B D

,

B D

i

s

f i

r st

m

eta

b

o

l i

z e

d

b y

c

y

to c

h

r o

m

e

P 4 5 0

2 E 1

m

o n o o x

y g

e n a s e

(

C

Y P 2 E 1

)

to

1

,

2

-e

p

o x

y b

ute n e

(

E B

)

, a n

d

t

h

e n

E B

c a n

b

e e

i

the r o x

i

d

i

z e

d f

u r

th

e r

t

o

d

i

e

p

o x

y b

uta n e

(

D

E B

)

o r

h y d

r o

l y

ze

d

v

i

a e

p

ox

i d

e

h y d

r o

l

ase to

3

-

b

u t

e n e- l ,2

-

d i

o

l

,

w

h i

c

h

c an

b

e

s u

b

s e

q

u e n

t

ly m

e

t

a

b

o

l i

z e

d

t

o

3

,

4

-e

p

o x

y

-

l

,

2

-

b

u t

(8)

P 4

5 0

[

1

0

- 1

3

]

. T

h

e e

l i

m

i

n at

i

o n o

f

B

D

m

a

y

e

i

t

h

e r

p

r o c e e

d

b

y

g

l

utat

h i

o n e

c o n

j

u

g

at

io

n to

E

B o r

D E

B, o r

b y h y d

ro

l y

s

i

s o

f E B

or

D E B

[

13

]

.

B D

m

uta

g

en

i

c

i

t

y

i

n t

h

e

p

re s e n c e o

f

m

eta

b

o

l i

c a ct

i

vat

i

o n

h

as

b

e e n

d

e

m

o n str ate

d i

n t

h

e

S

. t

y

p h i m

u r

i

u

m

a s s a

y

. T

h

e r e a ct

i

v e

m

eta

b

o

l i

te s

E B

a n

d D E

B

a r e str o n

g

l y

m

u ta

g

e n

i

c

i

n te st s

y

ste

m

s su c

h

a s

S

a

lm

o n e

l l

a,

S

a c c

h

a r o

m

y

c e s, a n

d

D

r o s o

p

h i l

a

[

1 4,

15

]

.

T h

e

m

u ta

g

e n

ic

i

t

y

o

f

a

l l

t

h

r ee

m

eta

b

o

l i

tes, E B,

E

B

D

an

d D E B

,

h

a s

b

e e n s

h

o

w

n at

b

ot

h

t

k

a n

d hp

r

t l

o c

i i

n c u

l

tu r e

d T K 6 h

u

m

a n

l

y

m

p h

o

b

l

a sto

id

c e

l ls

[

16

]

.

Si

m

i l

ar

l y

,

B D

,

E

B a n

d D E B

w

er e

m

u ta

g

e n

i

c

i

n s

p

l

e n

i

c

T

c e

l l

s

f

r o

m

ex

p

o s e

d B 6 C 3 F 1

m

i

c e

[

1 7

]

.

I

n cre a s e s

i

n s

i

ster c

h

r o

m

a t

i d

ex c

h

a n

g

e, c

h

ro

m

o s o

m

a

l

a

b

err a t

i

o n s, a n

d m

i

c r o n u c

l

e ate

d

p

e r

i

p

h

e r a

l b l

o o

d

er

y

t

h

r o c

y

te s

w

er e

f

o u n

d

a

f

te r

i

n v

i

v o e x

p

o s u r e to

B D

[

1

8

,

19

]

.

E

x

p

o s u r e o

f B D

a

l

s o c a u s e

d

c o v a

l

e n t

b i

n

d

i

n

g

to

h

e

m

o

g l

o

b i

n

[

2 0

]

a n

d

n uc

l

e a r

p

ro te

i

n s

[

2 1

,

2 2

]

a n

d p

r o te

i

n

-

D

N

A

a n

d D

N

A

-

D

N

A

cro s s

l i

n

k

s

[

2 3

]

i

n

m

i

c e.

A

s t

h

e

m

a

j

o r

m

eta

b

o

l i

te o

f B D

m

eta

b

o

l i

s

m

,

E B h

as

b

e e n s

h

o

w

n to re a ct

w

i

t

h d

e ox

y g

u a n o s

i

n e a n

d

i

n v

i

tr o

D

N

A

to

f

o r

m

t

w

o

m

a

i

n a

d d

u cts

i d

e n t

i f i

e

d

a s

7

-

(

2

-

h y d

r

ox

y

-

3

-

b

u t

e n-

l

-y

l

)

g

u an

i

n e

(

E

B-

G

ua

I

)

a n

d

7

-

(

l

-

h

y

d

ro x

y

-

3

-

b

ute n -

2

-y

D

g

u a n

i

n e

(

E B

-

G

u a

I I

) [

2 4

]

.

D E B

h

a s a

l

s o

b

e e n s

h

o

w

n

t

o r e a c

t

w

i t

h g

u a n

i

n e

t

o

g

i

v e a n a

d

d

u ct,

7

-

N

-

(

2

,

3

,

4

-t

r

i h

y d

ro x

y

b

u t

y

l

)

g

u a n

i

n e

[

23

]

.

E B

-

G

u a

I I

a n

d

7

-

N

-(

2

,

3

,4

-tr

i h y d

r o x

y b

u t

y l

)

g

u an

i

n e

w

e re

d

ete cte

d

,

b

u t n ot

q

u a nt

i

tate

d i

n t

h

e

l i

v er

D

N

A h

y

d

r o

l

y

s ate

f

r o

m

B 6 C 3 F 1

m

i

c e e x

p

o s e

d

to

5 0 0

p p

m (

l

,

4

- "

C

)

l

,

3

-

b

u ta

d

i

e n e;

h

o

w

e v er, t

h

e

y w

e r e n ot

d

ete cte

d i

n

D

N

A f

r o

m

r ats a

f

te r e x

p

o s u r e to ^

''

C

-

B D

[

2 3

]

.

T h

e r e s u

l

ts o

f

t

h

e

m

u tat

i

o n a

l

s

p

e ctru

m

i

n

d

u c e

d by

E B i

n s

p

l

e n

i

c

T

-c e

ll

s o

f

m

a

l

e

B 6 C 3 F 1

m

i

c e

h

a v e s

h

o

w

n

f

o u r

A

:

T

- >

G

:C tr a n s

i

t

i

o n s a n

d

o n e

A

:

T

- >

C

:

G

tr a n s v er s

i

o n a

m

o n

g 18 b

as e

p

a

i

r s u

b

st

i

tu t

i

o r \s

[

1 7

]

.

F

u rt

h

er

m

o r e

, n e ar

l y h

a

l f

o

f

t

h

e

p

o

i

n t

m

u tat

i

o n s

i

n

d

u c e

d b y

B

D

, E B, an

d D E B

o cc ur e

d

at

A

:

T b

as e

p

a

i

rs

[

1 7

]

.

A d d i

t

i

o na

l l

y

, t

w

o stu

d i

es o n t

h

e

f

re

q

u e n c

y

o

f

m

u tat

i

o n s

i

n t

h

e

b

o n e

m

a r r o

w

o

f

(9)

b

as e

p

a

i

rs

f

o

l l

o

w

i

n

g

B D in

h

a

l

at

i

o n e x

p

o sur e s

[

2

5

,

2 6

]

. T

h

e se

i

n v

i

v o

d

ata s u

gg

e st

t

h

at a

d

e n

i

n e

D

N

A

a

d d

u cts

m

a

y

b

e

i

n v o

l

v e

d

i

n

B D

m

u ta

g

e n e s

i

s a n

d

c a r c

in o

g

e n e s

is

.

T h

e

p

ur

p

o s e o

f

t

h

e

p

r e s e n t stu

d y i

s to c

h

ara cter

i

z e t

w

o

m

a

i

n

a

d d

u cts t

h

at a r e

f

o r

m

e

d

f

r o

m

t

h

e r e a ct

i

on o

f E B

w

i

t

h

a

d

en

i

n e, an

d

to u se

s

j

m

t

h

e s

i

ze

d

E B

-g

u a n

i

n e a n

d E B

-a

d

e n

in e

a

d d

u cts a s re

f

e r e n c e c o

m

p

o u n

d

s

t

o

d

ete ct an

d

q

u ant

i

tate t

h

e cor r es

p

o n

d i

n

g

a

d d

u ct s

p

e c

i

e s

f

or

m

e

d

i

n

D

N

A

(10)

M

A

T E R I

A

L S

A N D

M

E T

H

O D

S

C h

e

m

i

c a

l

s

E B

(

9 8 %

)

w

a s o

b

ta

i

n e

d f

r o

m

A l d

r

i

c

h

(M

i l

w

a u

k

e e,

W

I

)

.

G l

a c

i

a

l

a c et

i

c a c

i d

w

a s

b

o u

g h

t

f

r o

m

E

M

S

c

i

e n c e

(

G i b b

sto

w

n,

N

J)

.

A

d

e n

i

n e,

g

uan

i

n e,

g

u a n o s

i

n e,

c a c o

d

y

l i

c a c

i d

a n

d

c a

l f

t

h y

m

u s

D

N

A

w

er e

p

ur c

h

a s e

d f

ro

m

Si

g

m

a

(

S

t.

L

o u

i

s,

M

O

)

.

A

c eto n e,

d

i

et

h

y

l

et

h

e r,

m

et

h

a n o

l

, a n

d

H

C l

w

e r e

p

u r c

h

a s e

d

f

r o

m

M

a

l l i

n c

k

r o

d

t

(

P

a r

i

s,

K Y

)

.

A

c et

i

c a c

i d

, a

m

m

o n

i

u

m

f

o r

m

ate,

T

r

i

s,

p

o ta s s

i

u

m

d i h

y

d

ro

g

en

p

h

o s

p

h

ate, an

d

p

ota s s

i

u

m

h y d

r ox

i d

e

w

er e

b

o u

g

h

t

f

r o

m

F i

s

h

e r

Sc

i

e n t

i f i

c

(

P i

t ts

b

u r

g

,

P A

)

.

C

I

8

re v er s e

p

h

a s e r e s

i

n a n

d

p

h

o s

p

h

o r u s

p

e n ta o x

i d

e

w

e re

p

u rc

h

a s e

d f

r o

m

B

a

k

er

(

P h i l l i

p

s

b

u r

g

,

N

J)

.

H

i

g

h

Pe r

f

o r

m

an c e L

i q

u

i d C h

ro

m

a to

g

r a

p h y

(

H

P L C

)

A C 1 8

re v e rs e

p

h

a s e c o

l

u

m

n

(

d

es cr

i b

e

d

b

e

l

o

w

i

n e ac

h

s ect

i

o n o

f

m

et

h

o

d

s

)

,

a R

h

o

d y

ne

i

n

j

ecto r

(

Bax te r,

C h

a r

l

o t te,

N

C

)

e

q

u

i p p

e

d

w

i

t

h

a 2 -

m

l i

n

j

e ct

i

o n

l

o o

p

,

2

W

a te r s

5 10

H

P L C

p

u

m p

s

(M

i l l i

p

or e,

M

i l f

o r

d

,

M

A

)

, a n

d

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i

m

ar

y

s e

p

arat

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d

s e c o n

d

ar

y

v e r

i fi

c atio n o

f

t

h

e

p

u r

i

t

y

o

f

p

o o

l

e

d f

r action s o

f i

nte r e st, the

f

r a ct

i

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w

er e

d

esa

l

te

d

a n

d d

r

i

e

d

t

w

i

c e a s

d

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i b

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d

a

b

ove to c o

m

p l

ete

l y

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m

ov e sa

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I

n or

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er to

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m

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t

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w

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t o

f

t

h

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E B

-a

d

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i

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p

r o

d

u cts o

f i

n te r e st, t

h

e tu

b

e s c o n

t

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i

n

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r

y p

o o

l

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r o

d

ucts a

f

te r s

p

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p

o r atio n

w

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u t

w

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t

h p h

o s

p h

oru s

p

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w

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c

h

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b

s e

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l y

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A f

ter

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t

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e

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-a

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p

r o

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h

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y

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l

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n

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st

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ater a n

d

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m

a

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ra ct

i

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f

e a c

h

s o

l

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w

as ta

k

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g

et t

h

e a

b

so r

p

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l

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A

^

g ^ ^ ^ i

d X

^

^ ^, a n

d

t

h

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c o r r e s

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d

i

n

g

a

b

so r

p

t

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v

i

t

y

c oe

f f i

c

i

e n ts

(

or ext

i

n ct

i

o n c o e

f f i

c

i

e n ts, e

) (

p H

7

)

o

f

e a c

h

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-a

d

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n e

p

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F

o

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b

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p

t

f

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p

l

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ate r

w

i

t

h

H

C l

(

p H

1

)

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N

a

O H

(

p H

13

)

,

t

h

e a

b

s or

p

t

i

v

i

t

y

c o e

f f i

c

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f

e a c

h E B

-a

d

e n

i

n e

p

r o

d

u ct, a n

d

the a s s o c

i

ate

d X

j ^^^ ^

a n

d A

^j^n ^t

p H

^

o r

13

w

er e

d

ete r

m

i

n e

d

.

T h

e u n u s e

d p

ar t o

f

e a c

h E B

- a

d

e n

i

n e

p

r o

d

u ct

i

n

w

ate r

w

a s

d

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c c ate

d

a

g

a

i

n, a n

d

s u

b

j

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d

to

N

M

R

a n a

l y

s

i

s to

(14)

'

H

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N

M

R

S

p

e ctr a

D

ata

'

H

-

N

M

R

s

p

ectr a o

f b

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h E B

- a

d

e n

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w

a s o

b

ta

i

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d

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k

er

A

M

X

-

5 0 0

s

p

e ctr o

m

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i

n

D

M

S

O

-

d 6

. E

B

-

A d

e

I

:

5

4.

1 0

(

b

r

d

d

,

I

H

,

J

=

13

.

4 1

H

z,

J

=

8

.

3

2

H

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C

H

j-

N H

)

, 4.4 1

(

b

r

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, I

H

,

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= 1 3.4 1

H

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3

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6

9

H

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C

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j-

N H

)

,

4

.

5 1

(

b

r s,

I

H

,

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O

H

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,

5

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(

d

,

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=

1 0

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0

H

z, c

i

s

C

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2=

C

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,

5

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2 5

(

d

,

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H

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1 7

.

2 0

H

z , tr a n

C

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C

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,

5

.

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(

b

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I

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,

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)

,

5

.

9 2

(

m

,

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2=

C

H

)

,

7

.

7 0

-

8

.

0 0

(m

,

3

H

,

H

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8

,

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6-

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, a n

d N

^ -

H

)

,

8

.

2 1

an

d

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.

24

p

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p

.

m

.

(

t

w

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I

H

,

H

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2

)

.

E B

-

A

d

e

I I

:

5

3

.

8

2

(

b

r

d

d

, I

H

,

J

=

13

.

4 1

H

z,

J

=

3

.

6 9

H

z,

C

H

2-

O H

)

,

4

.

1 6

(

b

r

d

d

,

I

H

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J

=

13

.

4 1

H

z ,

J

=

8

.

3 2

H

z,

C

H

2-

O

H

)

,

5

.

2 0

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5

.

3 0

(

m

,

4

H

,

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H

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C

H

=

C

H

2, a n

d

C

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,

6

.

3 7

(

m

,

I

H

,

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H

2=

C

H

)

,

7

.

7 5

-

8

.

1 0

(

m

,

3

H

,

H

-

8

,

N

^-

H

, a n

d N

^-

H

)

,

8

.

3 5

a n

d

8

.

3 9

p

.

p

.

m

,

(

t

w

o s, I

H

,

H

-2

)

.

R

e a ct

i

o n o

f E

B

w

i

t

h C

a

l f

T

h y

m

u s

D

N

A

T

h

e

p

ro c e

d

ur e

d

esc r

i b

e

d b

e

l

o

w w

a s a

l

s o

m

o

d i f i

e

d f

r o

m

t

h

at o

f C i

t ti et a

l

.

[

2 4

]

.

C

a

l f

t

h

y

m

u s

D

N

A

(

3 6

m g

)

w

as

d i

s s o

l

ve

d i

n

1 0

m

M

T r

i

s

-

H

C l b

u

f f

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p H

7

.

2

(

7

m

l

)

b y

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l

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m

ix

i

n

g

an

d

t

h

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E B

(

3 3 4

m g

,

4

.

7 6

m m

o

l

)

a n

d

m

et

h

a n o

l

(

7

m

l

)

w

e r e a

d d

e

d

to t

h

e s o

l

ut

i

o n .

T h i

s r e act

i

o n

m

i

xtu r e

w

a s

h

eate

d

a t

3 7

°

C f

o r

1 8

h

o urs.

T h

e r e a ct

i

o n

m

i

x tu re

w

a s ex tra cte

d

t

w

i

c e

w

i

t

h 2 8

m

l d i

et

h

y

l

et

h

e r to

r e

m

o v e u nr e a cte

d E B

.

E i

ther n e utr a

l

t

h

er

m

a

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y

d

r o

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i

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d

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i

s

w

a s

d

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w

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t

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s a

m p

l

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f

ro

m

t

h

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m

a

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n

i

n

g

E B

-

D

N

A

a

q

u e o u s s o

l

u t

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I

n t

h

e

n e u tr a

l

t

h

er

m

a

l

h y d

ro

l

y

s

i

s, s a

m p

l

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l

u t

i

o n

i

n

10

m

M

c a c o

d y l

i

c a c

i

d w

a s

h y d

r o

l y

z e

d b y h

e at

i

n

g

at

9 5

°

C f

or

3 0

m

i

n ,

w

h i l

e

i

n a c

i

d

h

y

d

r o

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s

i

s, s a

m

p

l

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l

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n

0

.

1

N H

C l

w

a s

h

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d

a t

7 0

°

C f

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3 0

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i

n.

A f

te r n e utr a

l

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m

a

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r o

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s

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s, t

h

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l

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p

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w

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h

m

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h

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t

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m

a

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r o

l y

s a te

(N

T

H ) w

a s c

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d

to 4

°

C

a n

d

2

.

5

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m p

l

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w

a s

p

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d

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C

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3 0

m

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c r o c o n c e n tr ato r

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