MOLECULAR INTERACTIONS IN THE ASSESSMENT OF

THE GENOTOXICITY OF ALKYLATING AGENTS

M ic h a e l ,F . Wooder

S h e l l T o x ic o lo g y L a b o r a to r y ( T u n s t a l l ) ,

S h e l l R e s e a rc h L im i te d j -

S i t t i n g b o u r n e R e s e a rc h C e n t r e ,

S i t t i n g b o u m e ,

K e n t . .

A t h e s i s f o r t h e d e g r e e . o f

D octor- o f -Ph ilo sophy

The U n i v e r s i t y , o f <S u r re y

G u i l d f o r d , 1977.

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SUMMARY .■ ■ i

The r e c o g n i t i o n and s u b se q u e n t : d e m o n s t ra t io n * i n , v i t r o ,

o f t h e . a l k y l a t i n g : p o t e n t i a l . o f d i c h l o r v o s has l e d t o a

s u s p i c i o n t h a t , t h i s t r i e s t e r o f p h o s p h o r i c a c i d may b e a

m utagen and a c a rc in o g e n . , ■ D e s p i t e c o n s i s t e n t l y n e g a t i v e '

r e s u l t s , o b t a i n e d i n mammalian -m u ta g e n ic i ty , and c a r c i n o g e n i c i t y

t e s t s , i t was t n e v e r - t h e - 1 e s s im p o r ta n t , t o i n v e s t i g a t e t h e

a l k y l a t i n g r e a c t i v i t y i n v i v o .

I n t h e . e x p e r i m e n t s d e s c r i b e d in. t h i s t h e s i s , m ale CFE

r a t s were exposed t o a tm o sp h e re s c o n t a i n i n g 0 ,0 6 4 p g 1 ^ o f

[M e-^ C ] d i c h l o r v o s (113C i.m ol f o r 12 h o u r s . A n a ly s i s o f t h e

DNA from t h e t o t a l . s o f t t i s s u e s from 20 r a t s r e v e a l e d no

m e t h y la t i o n a t t h e .W-7 atom o f guan ine , m o i e t i e s . A c o m p a r a t iv e

s tu d y w i t h , [ m e t h y l - ^ C ] m e th a n e s u lp h o n a t e ,g a v e r i s e t o a r e a d i l y

d e t e c t a b l e e x t e n t o f m e t h y l a t i o n . o f t h e ,W-7 atom o f t h e gu an in e :

m o i e t i e s i n DNA.

The l i m i t . o f d e t e c t i o n o f m e t h y l a t i o n o f t h e DNA i n t h e

d i c h l o r v o s <s tu d y was one m e th y l g roup p e r 5 .7 x 1 0 ^ n u c l e o t i d e -

u n i t s . The p r o p o r t i o n o f t h e a d m i n i s t e r e d d o se t h a t would be

consumed i n t h i s h y p o t h e t i c a l , r e a c t i o n , would be ,0.000001%. The .,

e x p o su re p e r i o d employed i n t h i s s tu d y (12h) c o n s t i t u t e d a

s i g n i f i c a n t ; f r a c t i o n o f t h e h a l f - l i v e o f 7 -m e th y lg u a n in e m o i e t i e s

i n DNA (3 d a y s ) and. t h e c u r r e n t ; f i n d i n g s t h e r e f o r e i n d i c a t e t h a t ,

d i c h l o r v o s would n o t m e t h y l a t e . t h e DNA o f mammalian . t i s s u e s

even-w hen i t i s i n h a l e d c o n t i n u o u s ly f o r , p r o t r a c t e d p e r i o d s o f

( i i )

The a d m i n i s t r a t i o n o f - r a d i o l a b e l l e d a d e n in e , g u a n in e , •

m e th io n in e and f o rm a te t o o t h e r w i s e u n t r e a t e d r a t s , g a v e ’ r i s e

t o t h e e x c r e t i o n o f r a d i o l a b e l l e d m e th y la t e d p u r in e s i n

u r in e o T h is f i n d i n g i n d i c a t e s t h a t , t h e d e t e c t i o n o f r a d i o - .

l a b e l l e d m e th y la t e d p u r i n e s , p e r s e , i n t h e u r i n e . o f a n im a ls

exposed to m e t h y l - l a b e l l e d m e t h y l a t i n g a g e n ts does n o t con-i

s t i t u t e e v id e n c e for? th e . s p o n ta n e o u s m e t h y l a t i o n o f t h e p u r i n e

m o i e t i e s o f c n u c l e o s i d e s a n d . n u c l e i c a c id s by m e t h y l a t i n g

a g e n t s , i n v i v o *,

( i i i )

ACKNOWLEDGEMENTS

I am v e ry g r a t e f u l ' t o D r . A. S , W rig h t who a c t e d as my

i n d u s t r i a l s u p e r v i s o r , p r o v i d i n g a t a l l t im e s v a l u a b l e a d v ice ,

and e n c o u ra g e m e n t . I am a l s o g r a t e f u l t o D r . L, .J . K ing o f

The U n i v e r s i t y o f S u r r e y , who a c t e d as. my U n i v e r s i t y

s u p e r v i s o r , d u r in g t h e c o u r s e o f t h i s w ork , and p r o v id e d

e x c e l l e n t c o u n c i l and p e r s p e c t i v e .

I would l i k e t o th a n k th e Management o f S i t t i n g b o u r n e ,

R esea rch , C e n t re and to D r . D. E . S te v e n s o n , D i r e c t o r o f t h i si *

L a b o r a t o r y , jfor a l l o w in g me t o r e s e a r c h t h i s p r o j e c t .

My th a n k s i s a l s o due to D r . D. H. H u tson f o r v a l u a b l e

d i s c u s s i o n s and f o r t h e g i f t s o f d im e th y ln a p h th y lp h o s p h a te

and d e u t e r a t e d d i c h l o r v o s , Mr. P . A. H a r th o o m f o r s y n t h e s i s i n g

th e r a d i o a c t i v e l y l a b e l l e d d i c h l o r v o s and Mr, D, B l a i r and

D r. H. R o d e r ic k f o r a s s i s t a n c e w i t h vt h e i n h a l a t i o n e x p e r i m e n t s ,

The mass s p e c t r a f o r t h i s t h e s i s w ere o b t a i n e d w i t h t h e

a s s i s t a n c e o f D r s . M, K. Baldw in and I , Howe. A s s i s t a n c e

w i t h th e s t a t i s t i c a l a n a l y s i s was g iv e n by Mr;, W, V, M cC arthy .

I would a l s o l i k e to th a n k th e members o f t h e G e n e ra l

S e r v ic e s D i v i s i o n , and i n p a r t i c u l a r M rs . R. G reen , Mr, D, H a l l

and Mr. K. Attwood w i t h o u t whose e f f o r t s th e p r e s e n t a t i o n

o f t h i s t h e s i s would n o t have b een p o s s i b l e .

To p e r s o n s n o t m en t io n e d a bove , who a s s i s t e d i n t h e work

c o n ta i n e d i n t h i s t h e s i s , I g iv e my g r a t e f u l t h a n k s ,

( iv )

CO N T E N T S

CHAPTER 1 - I n t r o d u c t io n ^

Page No,

1 .

1 o.l I n , v iv o r e a c t i o n s o f a l k y l a t i n g a g e n ts

with,DNA

1 . 2 . N a t u r a l l y o c c u r r i n g m e th y la t e d b a s e s

103 A l k y l a t i n g a c t i v i t y , o f p h o sp h o ru s e s t e r s

1 04 Aims o f ■ th e p r e s e n t ; s tu d y

17

26

36

CHAPTER 2 - M a t e r i a l s and S y n t h e s e s • 37

2.1... M a te r i a l s .

2 02 R adio c h e m ic a ls

14 n .[Me-. C ] D l ch l o r v o s

Mass s p e c t r o m e t r y

S - [ M e th y l - ̂ C ] g l u t a t h i o n e

2 .3 S y n th e s i s o f r e f e r e n c e compounds

3 -M ethy l a d e n in e

0 -M e th y lg u a n in e

2 04 . Anim als

37

38

38

4°

44

44

44

46

46

CHAPTER 3 - D i r e c t A l k y l a t i o n S tu d ie s -

3 .1 I n t r o d u c t i o n

48

48

(v )-

3 02 Methods 49

A l k y l a t i o n •o f DNA. i n . v i t r o 49

H y d r o ly s i s o f DNA . 49

iF . ra c t io n a t io n o f DNA h y d r o l y s a t e s 50

M easurementi o f ■r a d i o a c t i v i t y 51

Animal- e x p e r im e n ta t i o n . 51

I n v iv o a l k y l a t io n s t u d i e s w i t h ' f me 't hy 1-14 G ] me t h an e s u 1 ph on a t e

In . v iv o a l k y l a t i o n s tu d y w i t h ,d i [ m e th y 1 -1 4 CJ s u l p h a t e .

L a b e l l i n g . o f n e o n a t a l r a t DNA w i t h . sodium L14C] fo rm a te :

L a b e l l i n g l i v e r DNA f o l l o w i n g p a r t i a lh e p a te c to m y

I s o l a t i o n v o f DNx4 5 6 '

E s t im a t i o n o f DNA 58

R ecovery o f 7 - m e t h y lg u a n in e i f r o m - t h e , 58s c i n t i l l a t i o n m ix tu re .

P a p e r c h ro m a to g ra p h y 59

3o 3 R e s u l t s 59

In . v i t r o s t u d i e s 59

I n v iv o s t u d i e s 67

CHAPTER 4 - M e th y la te d P u r in e s i n U rine; 97

4 o1 I n t r o d u c t i o n . 97 ,

4 o 2 M ethods' 98

A n im a l . e x p e r i m e n t s . 98 '

( v i )

C o l l e c t i o n < o f <u r i n e and e x t r a c t i o n o f ■ u r i n a r y p u r in e s i

F r a c t i o n a t i o n ^ o f u r i n a r y p u r in e s

D e te r m in a t io n o f <r a d i o a c t i v i t y .

P a p e r ch ro m a to g rap h y

T h i n - l a y e r ch ro m a to g rap h y

Page No

99

100

101

101

101

4o3, R e s u l t s 102

CHAPTER 5 r . DNA S t r a n d , Damage 130

5..1 I n t r o d u c t i o n 130

5e2 Methods 131

L a b e l l i n g o f l i v e r DNA 131

L a b e l l i n g o f l i v e r p r o t e i n , a n d RNA 131

T r e a t m e n t : ofi ■ a n im a ls 132

P r e p a r a t i o n o f l i v e r c e l l s u s p e n s i o n < 132

S e d im e n ta t io n v a n a l y s i s 133

D e te r m in a t io n o f r a d i o a c t i v i t y 138

I n v e s t i g a t i o n . o f ' the, . r a p i d l y s e d im e n t in g . . ^m a t e r i a l ,

R e p a i r of. s i n g l e - s t r a n d damage 139

5o3 R e s u l t s 139

CHAPTER 6. - D i s c u s s io n . 155

6 o l . D i r e c t ; I n t e r a c t i o n w i t h DNA 155

6o2 O r ig in s of-. U r in a r y M e th y la te d P u r in e s 168

L i t e r a t u r e C i t e d 175

CHAPTER 1

I n t r o d u c t i o n

■*-°l I n v iv o r e a c t i o n s o f a l k y l a t i n g a g e n ts w i t h DNA

The b i o l o g i c a l l y a c t i v e a l k y l a t i n g a g e n ts a r e a c h e m ic a l ly

d i v e r s e group o f s y n t h e t i c and n a t u r a l l y o c c u r r i n g compounds

whose o n ly common f e a t u r e a p p e a r s t o be t h e i r e l e c t r o p h i l i c

r e a c t i v i t y . I n some m o le c u le s t h i s r e a c t i v i t y i s i n h e r e n t e . g .

MMS and DMS ( F ig . 1) b u t i n many o t h e r m o le c u le s t h i s r e a c t i v i t y

i s o n ly m a n i f e s t e d a f t e r m e t a b o l i c a c t i v a t i o n e . g . DMN and MAM

( F ig . 1 ) . S e v e r a l e x c e l l e n t r e v ie w s d e a l i n g w i t h t h e s u b j e c t

o f t h e i r 'm e t a b o l i c a c t i v a t i o n have b een p u b l i s h e d ( e . g . M i l l e r

and M i l l e r , 1969a; 1969b; 1 9 7 1 ) . Two mechanisms a r e g e n e r a l l y

a c c e p te d f o r i n v iv o a l k y l a t i o n r e a c t i o n s . By d e f i n i t i o n ,

a l k y l a t i o n r e a c t i o n s i n v o lv e n u c l e o p h i l i c s u b s t i t u t i o n a t

t h e e l e c t r o p h i l i c ca rb o n atom o f t h e a l k y l a t i n g a g e n t . Two

r e a c t i o n mechanisms a r e n o r m a l ly a v a i l a b l e f o r su c h n u c l e o p h i l i c

s u b s t i t u t i o n s (Sn ) . One o f them c o n ta i n s o n ly one s t a g e and

p ro c e e d s v i a an e l e c t r o n t r a n s f e r from t h e s u b s t i t u t i n g a g e n t

t o t h e s e a t o f s u b s t i t u t i o n , and from t h e l a t t e r t o t h e d i s ­

p l a c e d group (E q u a t io n 1 ) . I n t h i s m echanism , two m q le c u le s

s im u l t a n e o u s l y unde rgo c o v a le n c y change and t h e m echanism

i s t h e r e f o r e b i m o l e c u l a r (S^2)

N-Alk + P (Sn2)N + A ik - X •>

( n u c l e o p h i l e )

E q u a t io n 1

CH —S—OCH, Methyl methanesulphonate3 it 3

(MMS)

CHg—O v y/0 Dimethyl sulphate

C H , - 0 ' ' % (DMS)

CHg—N v Dimethyl nitrosamine>N=0 (DMN)C H g - N " v u i v i i m /

CHg—N=N CH20H Methyl azoxymethanol♦ (MAM)

C H3—N —C—N H2 N—Methyl —N —n itrosourea

NO 0 (MNU)

CHg—N - C —N—N 02 N -M eth y l-N '-N itro —N1—Nitrosoguanidine

NO NHH ,MNNG>

1,2 dimethyl hydrazine CHg— N —N—CH, (DMH)

i i 3H H

Fig 1 Some biologically active alkylating agents

- 3 -

The seco n d mechanism in v o lv e s two s t a g e s : a s low h e t e r o l y s i s

o f t h e a l k y l a t i n g a g e n t to y i e l d a ca rbon ium io n fo l lo w e d

by a r a p i d c o - o r d i n a t i o n o f th e ca rbon ium io n and th e n u c le o ­

p h i l e ( E q u a t io n 2 ) 0 B ecause o n ly one m o le c u le i s u n d e rg o in g

a c o v a le n c y change d u r in g th e r a t e - d e t e r m i n i n g s t a g e , i , e „

t h e g e n e r a t i o n o f t h e ca rbon ium i o n , t h e mechanism i s u n i -

m o le c u la r (Sjql) o

T h is d i v i s i o n on t h e b a s i s o f m echanism i s r e f l e c t e d i n t h e

n a t u r e o f t h e i n v iv o r e a c t i o n p r o d u c t s w i t h n u c l e i c a c i d s .

W ith a g e n ts r e a c t i n g b y th e SN2 m echanism t h e r e a c t i o n te n d s

t o be d i r e c t e d tow ards t h e p u r i n e and p y r im id in e r i n g n i t r o g e n

l e s s s e l e c t i v e , r e a c t i n g w i t h b o th oxygen and n i t r o g e n a to m s .

I t i s becom ing e v id e n t t h a t a l l o f t h e b a s e s i n DNA and

i n some i n s t a n c e s t h e d e o x y r ib o s e p h o s p h o d ie s t e r b a c k b o n e a r e

i n v iv o t a r g e t s f o r a l k y l a t i n g a g e n t s . However, i t i s c l e a r

t h a t i n v iv o c e r t a i n segm ents i n DNA a r e more s u s c e p t i b l e

t o a l k y l a t i o n t h a n a r e o t h e r s ; i . e . t h e p a t t e r n o f a l k y l a t i o n

i s n o t hom ogeneous. Thus, i t h a s b e e n shown t h a t t h o s e a r e a s

o f c h ro m a tin t h a t a r e a c c e s s i b l e t o n u c l e a s e s , i . e . r e g i o n s

o f DNA t h a t a r e n o t p r o t e c t e d by h i s t o n e s , a r e t h e m ost p ro n e

s low

f a s t■> N A ik( n u c l e o p h i l e )

!E q u a t io n I I

a tom s, w h i l e a g e n ts r e a c t i n g by th e S ^ l m echanism te n d to be

- 4 -

to a l k y l a t i o n (Cooper e t a l , 1975; Cooper and I t z a k i , 1975;

I t z a k i e t a l , 1976; Ramanathan e t a l , 1 9 7 6 ) . These r e a c t i o n s

have b e e n th e s u b j e c t o f many e x c e l l e n t rev ie w s (Law ley , 1966;

1 9 7 2 a ,b ; 1974; Sarma e t a l , 1975; S i n g e r , 1975) and t h e r e f o r e

d i s c u s s i o n o f them h e r e w i l l n o t be e x t e n s i v e . S u f f i c e i t t o

s a y t h a t w i th a v a r i e t y o f m e th y l-a t in g a g e n ts atoms a t t h e

f o l l o w i n g n u c l e o p h i l i c c e n t r e s have been shown t o be r e a c t i v e

tow ards a v a r i e t y o f m e t h y l a t i n g a g e n t s : - t h e W-l, W-3 and

W-7 atoms o f a d e n in e , t h e W-3, 0 and W-7 atoms o f g u a n in e

and t h e W-3 atom o f c y t o s i n e . I n a d d i t i o n , i n v i t r o s t u d i e s

have shown t h a t t h e oxygen atoms o f DNA b a s e s a r e r e a c t i v e

tow ards n i t r o s o compounds i n v i t r o (Lawley e t a l , 1973; S i n g e r ,

1 9 76a ,b ) o The o r d e r o f r e a c t i v i t y i n d o u b le s t r a n d e d DNA i s

02 o f T = 06 o f G > 04 o f T » 02 o f C ( S in g e r , 1 9 7 6 b ) .

F ig u r e 2 shows th e l o c a t i o n o f t h e s e s i t e s i n t h e i r r e s p e c t i v e

b a s e s t o g e t h e r w i t h an i n d i c a t i o n o f t h e a g e n ts t h a t r e a c t

w i t h them . Q u a n t i t a t i v e l y t h e m ost r e a c t i v e s i t e i s th e W-7

atom o f t h e g u a n in e r i n g , a c c o u n t in g f o r more th a n 60% o f t h e

t o t a l b a s e a l k y l a t i o n . T h is r e a c t i v i t y has been a t t r i b u t e d

t o i t s p e r i p h e r a l p o s i t i o n i n th e w ide g roove o f t h e W atson-

C r ic k model o f t h e d o u b le h e l i x (R e in e r and Zamenhof, 1957;

Luck and Zimmer, 1972). I n a d d i t i o n i t has b e e n p r o p o se d t h a t

t h e h ig h n u c l e o p h i l i c i t y o f t h e W-7 i s r e l a t e d t o th e r o l e

o f g u a n in e as a h y d ro g en bond d o n o r . I n t h e W a ts o n -C r ic k model

o f t h e DNA h e l i x , g u a n in e d o n a te s two h y d ro g en bonds b u t a c c e p t s

o n ly o n e . T h i s , i t i s s u g g e s t e d , r e s u l t s i n an i n c r e a s e d e l e c t r o n

d e n s i t y o v e r t h e g u a n in e r i n g w h ich may be r e f l e c t e d by th e

MNU (f)MMS (c)(d)(g) DMS (i) MNNG (b)

DIMAchain.

DMN (d)(g)(k) MNU (e)(f) MMS (c)(d)(g) DMS (i) MNNG (b)

H ( MNNG (b)< MNU (c)(f)

f (DMN (d)(g)(k)

DNA chain

MNNG (b) MNU (c) DMS (i) MMS(g)(h) DMN(g)

a) Shank & Magee (1967)b) Lawley & Thatcher (1970)c) Kleihues etal (1974)d) O 'C onnorif al (1973)e) Margison & Kleihues (1975)f) Maitra & Frei (1975)g) Capps etal (1973)h) O'Connor et al (1972)i) Lawley (1976)j) Lawley etal (1973) k) Margison etal (1976)

N I

DNA chain

( MNU (e){ MMS (d)(g)( DMN (d)(g)(k)

Fig 2 Sites of interaction of methylating agents with nucleic acids in vivo and in vitro

I MMS (c)(g) MAM (a) DMN (g)(k) MNU (e)(f) MNNG (b) DMS (i)

- 6 -

i n c r e a s e d n u c l e o p h i l i c i t y o f t h e W-7 atom (Sarma e t a l , 1975).

M u ta g e n e s is and c a r c i n o g e n e s i s , t h e b i o l o g i c a l e f f e c t s

o f m ost c o n c e rn i n r e l a t i o n t o t h e a l k y l a t i n g a g e n t s , have

b o th b e e n a t t r i b u t e d t o changes i n b a s e s t r u c t u r e w i t h sub ­

s e q u e n t m i s r e p a i r o r m is c o d in g . C o l l e c t i v e l y t h e s e e f f e c t s

have b een g rouped u n d e r t h e g e n e r a l h e a d in g o f g e n o t o x i c i t y .

Thus a g e n o to x ic compound i s d e f in e d as "an y a g e n t w h ic h ,

by v i r t u e o f i t s p h y s i c a l o r c h e m ica l p r o p e r t i e s , can in d u c e

o r p ro d u c e h e r i t a b l e changes i n t h o s e p a r t s o f t h e g e n e t i c

a p p a r a tu s o f s o m a t ic c e l l s , r e s p o n s i b l e f o r th e h o m e o s t a t i c

c o n t r o l , and th e r e b y d e te r m in in g them t o m a l ig n a n t t r a n s ­

fo rm a t io n " (D ru c k re y , 1973; 1 9 7 5 ) . L a r g e ly on t h e b a s i s o f

i t s q u a n t i t a t i v e i m p o r ta n c e * a l k y l a t i o n a t th e W-7 atom o f

g u a n in e was presum ed to be c a u s a l l y r e l a t e d to t h e s e e f f e c t s .

However, i n v i t r o s t u d i e s w i t h s y n t h e t i c po lym ers c o n t a i n i n g

v a r y i n g p r o p o r t i o n s o f 7 -m e th y lg u a n in e have d e m o n s t ra te d t h a t

th e p r e s e n c e o f t h i s b a s e i s n o t " m u ta g e n ic " p e r s e a t t h e

l e v e l o f t r a n s c r i p t i o n (Ludlum, 1970) o r a t th e l e v e l o f t r a n s ­

l a t i o n (W ilhelm and Ludlum, 196 6 ) . F u r th e r m o r e , H e n d le r

e t a l . (1970) have shown t h a t i n an i n v i t r o DNA p o ly m e ra s e

s y s te m , 7 - m e t h y l - 2 ’-d e o x y g u a n o s in e t r i p h o s p h a t e can s u b s t i t u t e ,

t o a l i m i t e d d e g r e e , f o r 2 ’- d e o x y g u a n o s in e t r i p h o s p h a t e . F u r t h e r

d o u b t h a s b een c a s t on th e r e l e v a n c e o f t h i s b a s e t o m uta­

g e n e s i s by s t u d i e s on b a c t e r i o p h a g e . I n t h i s sy s te m b o th IMS

and EMS r e a d i l y form 7 - a l k y l g u a n i n e , b u t EMS i s more p o t e n t

as a m utagen th a n MMS ( L o v e le s s , 1 9 5 9 ) . T h is a p p a r e n t d i s c r e p a n c y

can n o t be e x p la i n e d on th e b a s i s o f th e e t h y l group b e i n g u n iq u e

- 7 -

as MNU p ro v ed to be an e f f e c t i v e m u tagen . M e c h a n i s t i c a l l y EMS

b r i d g e s t h e gap be tw een S^2 a g e n ts (e,»g. MMS)' and S^.1 a g e n ts£

( e . g . MNU) g i v in g a m e a s u ra b le e x t e n t o f 0 - a l k y l a t i o n . As

th e b i o l o g i c a l d a t a was: amassed i t became e v i d e n t t h a t t h e r e

was o n ly a p o o r c o r r e l a t i o n be tw een th e l e v e l s o f 7- m e th y l ­

g u a n in e and b i o l o g i c a l e f f e c t ( i n m ost c a s e s tumour p r o d u c t i o n ) .•

T h is has b e e n d e m o n s t r a te d w i t h many d i f f e r e n t a l k y l a t i n g . a g e n t s ,

DMN (C raddock , 1973)', MNU, : DMN,. DMS and MMS (Swann and Magee,

1 9 6 8 ) , DEN, ENU and EMS (Swann and Magee, 1 9 7 1 ) , ENU and MNU

( L i j i n s k y e t a l , 1 9 7 2 ) , MNU and MMS ( K le ih u e s and Magee,. 1973)

and DEN (Ross e t a l , 1 9 7 1 ) . A t t e n t i o n was t h e r e f o r e fo c u s e d

on th e m ino r b a s e s , and i n p a r t i c u l a r 0 - m e t h y l g u a n i n e , . th e s i t e

of i n t e r a c t i o n s u g g e s t e d by L o v e le s s (1969) as b e i n g - r e s p o n ­

s i b l e f o r m u ta g e n e s i s i n th e b a c t e r i o p h a g e . I n v i t r o s t u d i e s

6w i t h copo lym ers o f - c y t i d y l i e and 0 - m e t h y lg u a n y l i c a c id s u se d

as te m p la te s , f o r RNA p o ly m e rase d e m o n s t r a te m i s - i n c o r p o r a t i b n

o f UMP o r AMP i n t o th e p r o d u c t copo lym er-(G erchm an and Ludlum,

619.73) , The s t u d i e s w i t h p o ly 0 - m e t h y lg u a n y l i c a c id and p o ly ,

60 - e t h y l g u a n y l i c a c id homopolymers (Mehta and Ludlum, ,1976)

have shown t h a t , t h e y do no t, form h e l i c e s w i th p o ly (.C) n o r

f o r t h a t m a t t e r w i t h , p o l y (U) , Thus 0 - m e th y lg u a n in e d i s r u p t s

b a s e p a i r i n g and a f f e c t s t h e s e c o n d a ry s t r u c t u r e and m ust be

r e g a r d e d Mp r o - m u ta g e n ic M, In v iv o s t u d i e s , (K le ih u e s and Mageej

1973) d e m o n s t r a te d a r e l a t i v e l y high: l e v e l o f m e t h y l a t i o n a t

t h e 0 - p o s i t i o n o f g u a n in e i n th e DNA o f r a t b r a i n f o l l o w i n g

ad m in istra tion o f MNU, Ktumourigenic in b r a in ) . ,The le v e l o f

60 -guanine methylation was 12% of that found at the W-7

p o s i t i o n and was n o t 'o b s e r v e d w i t h MMS. A s i m i l a r s i t u a t i o n £

was found f o r 0 - g u a n in e a l k y l a t i o n i n l i v e r DNA f o l lo w in g

e x p o s u re to DMN (a p p ro x . . 12% o f t h a t a t W-7; C raddock , 1973).,

M inor b a s e s w ere found i n b o t h s t u d i e s , b u t t h e i r g e n e r a t i o n

d id n o t c o r r e l a t e w i t h tum our p r o d u c t i o n , e . g . K le ih u e s and

Magee (1973) found t h a t th e amounts o f 3 - m e th y la d e n in e i n b r a i n

DNA w ere h i g h e r by a f a c t o r o f two f o l lo w in g t r e a t m e n t w i t h MMS.

Thus i t seem ed, on t h e b a s i s o f t h e s e s t u d i e s t h a t t h e 0 - a l k y l -

g u a n in e p ro d u ced i n t h e t a r g e t o rg an s was c a u s a l l y r e l a t e d t o

tum our p r o d u c t i o n . , However, i t was l a t e r shown (Goth and R ajew sky ,

1974a) . t h a t th e i n i t i a l e x t e n t o f a l k y l a t i o n p ro d u ce d a t th e £

0 p o s i t i o n o f g u a n in e by ENU was t h e same: f o r b o t h b r a i n

( t a r g e t o rgan ) and l i v e r ( n o n - t a r g e t o r g a n ) , However, th eg

p e r s i s t e n c e o f 0 - e t h y l g u a n i n e i n t h e b r a i n was much g r e a t e r

( h a l f l i f e a p p ro x . 220h) th a n i n l i v e r ( h a l f l i f e . a p p r o x . 3 0 h ) ,

The r e s u l t s o f a se co n d s tu d y (Goth and R ajew sky , 1974b)

showed that while the molar fractions of W-7 ethylguanine,60 - e t h y l g u a n i n e , W-3 e t h y l a d e n i n e and W-7 e t h y l a d e n i n e w ere

i n i t i a l l y s i m i l a r i n b o th t a r g e t ( f o e t a l o r 10 day o ld r a t

b r a i n ) a n d n o n - t a r g e t ( l i v e r ) t i s s u e s , t h e r a t e o f rem ova l of-

0 - e t h y l g u a n i n e was s lo w e r i n t a r g e t o r g a n s . The p e r s i s t e n c e o f

th e l e s i o n i n t a r g e t t i s s u e was l a t e r c o n f i rm e d u s in g MNU and

i n t e r e s t i n g l y , t h e p e r s i s t e n c e i n th e DNA o f k id n e y (an

o c c a s i o n a l t a r g e t o rg an ) was i n t e r m e d i a t e be tw een t h a t i n l i v e r

and i n b r a i n (K le ih u e s and M a rg iso n , 1974). R e p e a te d a p p l i c a t i o n

o f MNU to r a t s r e s u l t s i n a s e l e c t i v e a c c u m u la t io n o f 0 -

m e th y lg u a n in e i n b r a i n as compared w i t h k id n e y , s p l e e n and

- 9 -

6l i v e r . F o r c om par ison th e t e r m in a l l e v e l s o f 0 -m e th y lg u a n in e

i n l i v e r r e p r e s e n t e d l e s s th a n 1% o f t h a t in b r a i n (M arg ison

and K le ih u e s , 1 9 7 5 ) . I n a t l e a s t one c a se how ever t h e r a t e

o f removal, o f 0 - m e th y lg u a n in e m o ie t i e s and tu m o u r ig e n e s i s

w ere a n t i - p a r a l l e l (Den E n g le s e , 1974),.

The rem oval o f a l k y l a t e d (damaged) b a s e s from DNA i s n o t £

r e s t r i c t e d t o 0 - m e t h y lg u a n in e . Most a l k y l a t e d p u r in e s have

b e e n shown to be d e p u r i n a t e d from DNA a t a r a t e g r e a t e r th a n

t h a t m easu red f o r n o rm al b a s e s ( L in d h a l and N yberg , 1972) .

In some c a s e s t h e r a t e o f d e p u r i n a t i o n i n v iv o i s s i g n i f i c a n t l y

g r e a t e r th a n t h a t found i n v i t r o (T a b le 1 ) , i n d i c a t i n g th e

p o s s i b l e in v o lv e m e n t o f a " r e p a i r enzym e" . K i r t i k a r and

G o ld th w a i t (1974) and K i r t i k a r e t a l (1976) have d e s c r i b e d such

an enzyme, e n d o n u c le a s e I I , i n E a c o l i . The enzyme h a s b o th

p h o s p h o d ie s t e r a s e and W -g ly c o s id a s e a c t i v i t y , t h e l a t t e r

b e in g d i r e c t e d tow ards a l i m i t e d number o f p u r i n e s » Thus i t

6w i l l r e l e a s e 0 - m e th y lg u a n in e , 3 - m e th y la d e n in e and s m a l l amounts

of- 1 - . and 7- m e th y la d e n in e s from m e th y la t e d DNA. 7 -M e th y lg u a n in e

i s n o t r e l e a s e d by t h i s enzyme, and i t i s l i k e l y t h a t t h e r e l e a s e

o f m ost o f t h i s b a s e i n v iv o i s v i a c h e m ica l h y d r o l y s i s o f

th e c a r b o n - n i t r o g e n bond l i n k i n g th e W-9 o f t h e p u r i n e t o th e

G-l o f th e d e o x y r ib o s e m o ie ty ( F ig . 3 ) , The p r o b a b l e s t e p s i n

t h i s e x c i s i o n p r o c e s s , d e r i v e d from s t u d i e s i n b a c t e r i a , a r e

shown i n F i g . 4 . Damaged a r e a s a r e r e c o g n i s e d p r o b a b ly on th e

b a s i s o f d i s t o r t i o n o f th e DNA by th e e n d o n u c le a s e (van L a n k e r

and Tomura, 1972) . A reas o f " s p o n ta n e o u s " d e p u r i n a t i o n , can

a l s o b e r e c o g n i s e d and V e r ly e t a l 1973 and V e r ly and P a q u e t t e

(1973) have d e s c r i b e d an enzyme from r a t l i v e r w h ich a c t s on

- 10 -

T a b le 1 - S t a b i l i t y o f A lk y l a t e d B ases i n DNA

M e th y la te d b a s e System PH Temp. t j (h) R e f .

In v i t r o 5 .0 37° 79 .3 (1)7 -M e th y lg u a n in e In v i t r o

I n v i t r o7 .07 .2

37°37°

144105

(1)(2)

I n v iv o — - 72 (1)

7 - E th y lg u a n in e In v iv o ( b r a i n ) In v iv o ( l i v e r )

- - ^ 89 64

(3)(3)

In v i t r o 5 .0 37° 1 1 .5 (4)3 -M e th y la d e n in e In v i t r o

In v i t r o7 .07 .2

37°37°

2438

(4)(2)

I n v iv o - - 3 (4)

In v i t r o _ 33 (3)3 - E th y la d e n in e In v iv o ( b r a i n ) - - 16 (3)

In v iv o ( l i v e r ) — — 12 (3)

7 -M e th y la d e n in e In v i t r o I n v iv o

7 .2 37° 2 .82 .6

(2)(2)

3 -M e th y lg u a n in eI n v i t r o I n v iv o

7 .2 37° >1053 .6

(2)(2)

Q6 -M e th y lg u a n in e In v i t r o I n v iv o

—13 (5)

In v iv o ( b r a i n )- - >229 (3)

0 - E th y l g u a n in e I n v iv o ( l i v e r )- - * 36 (3)

Normal p u r i n e s In v iv o 7 .4 37° 6 .4 x 106 (6)

(1) M arg ison e t a l , 1973(2) Lawley and W arren , 1976(3) Rajewsky and Goth , 1976(4) M arg ison and O’ Connor, 1973(5) 0 ’ Connor e t a l , 1973(6) L in d a h l and N yberg, 1972

o —z

+

+X

0II. 0 . - 0 c1

o — z

a.—0 —0 '

Fig

3 Sp

onta

neou

s de

purin

atio

n of

7—M

ethy

lgua

nine

in

DN

A

1. Recognition endonuclease

i2. Incision X

endonuclease _____________________________

3. Removal of lesion Xexonuclease

4. Repair replication DNA polymerase

5. Rejoining polynucleotide ligase

A A A AAV V V V '

“ l

A AAAA1

V V V V

Fig 4 Probable steps in the removal of DIMA lesion and repair of the damage

- 13 -

d e p u r i n a t e d r a t h e r th a n a l k y l a t e d DNA, The r e l a t i o n s h i p o f

t h e s e two enzyme sy s te m s t o each o t h e r i s n o t known. A c t io n

o f t h e e n d o n u c le a s e p ro d u c e s a t r a n s i e n t b r e a k i n t h e DNA

c h a i n . These b r e a k s , p ro d u ced e i t h e r p a r t l y o r e n t i r e l y by

enzyme a c t i v i t y , have b e e n m easu red i n v i v o , l a r g e l y in l i v e r

DNA, f o l l o w i n g th e a p p l i c a t i o n o f s e v e r a l a l k y l a t i n g a g e n ts

(T a b le 2 ) g, (Cox e t a l , 1 9 7 3 a ,b ; Sarma e t a l , 1973a; DamjanoV

e t a l , 1973; S t e w a r t e t a l , 1973; S t e w a r t and F a r b e r , 1973;

R a ja la k s h m i and Sarma, 1973; Goodman and P o t t e r , 1 9 7 2 ) .

Removal o f th e damaged r e g i o n o c c u rs th ro u g h th e a c t i o n

o f an e n d o n u c le a s e ( F i g , -4 ) and i s f o l lo w e d by r e p a i r s y n t h e s i s

(u n sc h e d u le d DNA s y n t h e s i s ) p re su m a b ly u s in g th e undamaged

s t r a n d as t h e t e m p la t e . I n c o r p o r a t i o n o f n u c l e o t i d e s , predom­

i n a n t l y t h y m i d i n e , i n t o t h e DNA o f c u l t u r e d c e l l s has been

d e m o n s t ra te d f o l l o w i n g t r e a tm e n t w i th c a rc in o g e n s (S e l to w and

Regan, 1972; S t i c h e t a l , 1972; L e iberm an and F o r b e s , 1 9 7 3 ) .

The. gap i s f i n a l l y s e a l e d by a p o l y n u c l e o t i d e l i g a s e , The

f i d e l i t y o f s o - c a l l e d e x c i s i o n r e p a i r i s good, as ju d g e d from

s t u d i e s i n b a c t e r i a .

F a i l u r e o r i n a b i l i t y to e x c i s e , damage and r e g i o n s from

th e DNA does n o t n e c e s s a r i l y b lo c k DNA s y n t h e s i s , M ost, i f n o t

a l l , c e l l s have th e a b i l i t y to " b y p a s s " su c h l e s i o n s by a p r o c e s s

d e s c r i b e d as ' p o s t - r e p l i c a t i o n r e p a i r ' . The o p e r a t i o n o f such

a sy s te m h a s b e e n r e p o r t e d i n r a t l i v e r DNA i n v iv o f o l l o w i n g

e x p o s u re to DMN (R a ja la k sh m i and F a r b e r , 1 9 7 3 ) . T h is sy s te m

a p p e a rs s i m i l a r t o t h a t : d e s c r i b e d i n r e p a i r d e f i c i e n t E. c o l i

(Rupp and H o w a rd -F la n d e rs , 1968) and mouse L5178Y c e l l s

- 14 -

T a b le 2 - A l k y l a t i n g A gen ts C ausing DNA S t ra n d

B reaks i n L i v e r DNA i n v iv o

S i n g le s t r a n d b r e a k s ( r a p i d l y r e p a i r e d <72h)

S in g le s t r a n d b r e a k s ( s lo w ly r e p a i r e d >72h)

M ethyl m e th a n e s u lp h o n a te

N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e

N - n i t r o s o - N - m e th y lu r e th a n e

H ycanthone m e th a n e s u lp h o n a te

Me t h y 1azo xym ethano l..........

D ime t h y I n i t r o s am ine.....

E t h y l m e th a n e s u lp h o n a te

N - n i t r o s o m e t h y lu r e a

- 15 -

(Lehmann, 1 9 7 2 ) . . F in d in g s t h a t m ic ro -o rg a n is m s d e f i c i e n t i n

p o s t - r e p l i c a t i o n : r e p a i r a r e r e l a t i v e l y r e s i s t a n t tow ards t h e

m u ta g e n ic a c t i o n o f a number o f a l k y l a t i n g a g e n ts h as s u g g e s te d

t h a t t h i s p r o c e s s i s " e r r o r - p r o n e " ( S t r a u s s , 1974)«

R e p l i c a t i o n o f DNA c o n t a i n i n g damaged r e g io n s may be d e l ­

e t e r i o u s P r o -m u ta g e n ic l e s i o n s such as 3 - m e th y la d e n in e , 3 - m e th y l -

6 4g u a n in e , 0 - m e th y lg u a n in e o r 0 -m e th y l th y m in e i n t h e p a r e n t

s t r a n d may i n t r o d u c e GC ■-* AT o r AT -> GC t r a n s i t i o n s . S u g g e s te d

anomalous p a i r i n g s a r e shown i n F i g . 5« O th e r l e s i o n s such

as 3 - m e t h y lc y to s i n e , 3 -m e th y l th y m in e and 1 - m e th y la d e n in e a r e

p r i m a r i l y i n a c t i v a t i n g (L aw ley , 1 9 7 4 ) .

I t would a p p e a r t h e r e f o r e t h a t th e g e n o to x ic e f f e c t s o f

th e a l k y l a t i n g a g e n ts can b e e x p la in e d by a d e f i c i e n c y i n t h e

ex c is io n ^ r e p a i r p r o c e s s . T h is h y p o t h e s i s i s s u p p o r t e d i n p a r t

by th e e x p e r im e n ts on th e p e r s i s t e n c e o f 0 - a l k y l g u a n i n e i n

t a r g e t o rg a n DNA f o l lo w in g e x p o s u re t o n i t r o u r e a s (K le ih u e s

and M a rg iso n , 1974; M arg ison and K l e ih u e s , 1975; Goth and

R ajew sky, 1 9 7 4 a ,b ) and from th e s t u d i e s on x e ro d e rm a p igm entosum

c e l l s w h ich l a c k t h e a b i l i t y to e x c i s e p y r im id i n e d i m e r s . I n

t h i s c o n te x t m easurem ent o f ' a l k y l a t i o n r e a c t i o n a t th e W-7

atom o f g u a n in e would a p p e a r to have l i t t l e r e l e v a n c e t o th e

g e n e to x i c e v e n t s . However, t h e W-7 atom rem a in s t h e m ost

a v a i l a b l e and r e a c t i v e s i t e i n DNA and t h e r e f o r e t h e m ost

s e n s i t i v e m easu re of: a l k y l a t i o n r e a c t i o n s .

To

OCH

N‘IH

Thym ine: O6—Methylguanine

CH

CH

Thymine : 7—Methylguanine

CH

OCH.

f Y " ' ' ' - a ' ' 'V ssA-C H 3rY

N N

CH„

SN 1STI I

H CH,

0 4—M ethylthymine: Guanine

Cytosine : 3—Methyladenine

Thymine : 3—Methylguanine

Fig 5 Suggested anomalous paring of alkylated bases

(Lawley 1974)

- 17 -

1 o 2 N a t u r a l l y o c c u r r i n g m e th y la te d ' b a s e s

14I n v iv o s t u d i e s w i t h C - l a b e l l e d a l k y l a t i n g a g e n ts have

shown, t h a t i n a d d i t i o n to th e p a t t e r n o f a l k y l a t i o n p r e d i c t e d

from t h e i n v i t r o e x p e r im e n t s , a g e n e r a l l a b e l l i n g o f t h e b a s e s

o c c u rso A lth o u g h th e r a d i o l a b e l i s d e r i v e d from th e a l k y l a t i n g

a g e n t s , i t s p r e s e n c e i n th e b a s e s ( e . g . a d e n in e and g u a n in e

w hich have no a t t a c h e d m ethy l g ro u p s) i s c l e a r l y n o t t h e r e s u l t

o f a d i r e c t a l k y l a t i o n r e a c t i o n , , Such l a b e l l i n g h a s f o r exam p le ,

b e e n shown t o o c c u r i n r a t l i v e r r-RNA f o l lo w in g e x p o s u re t o

[^C]MMS (McElhone e t a l , 1 9 7 1 ) , i n r a t s p l e e n RNA (Lee e t a l ,

1964) and r a t l i v e r DNA (Lawley e t a l , 1968; C raddock 1969;

Capps e t a l , 1973) f o l l o w i n g e x p o s u re t o [^C]DMN; i n t h e

n u c l e i c a c id s o f mouse c o lo n (Hawks e t a l , 1972) and th e DNA

o f mouse c o lo n (Hawks and Magee, 1974;) f o l l o w i n g e x p o s u re

rl4 .t o [ C ]1 , 2 - d i m e t h y l h y d r a z m e 0 The RNA and DNA o f r a p i d l y

d i v i d i n g t i s s u e s ( e . g . i n t e s t i n a l :e p i t h e l i u m ) a r e t h o s e m ost

e f f e c t i v e l y l a b e l l e d u n d e r t h i s c o n d i t i o n . The o b s e rv e d p a t t e r n

o f l a b e l l i n g c l o s e l y re s e m b le d t h a t o b t a i n e d w i t h [ ^ C - m e th y l ]

m e th io n in e ( E a rb e r e t a l , 1967; Craddock e t a l , 1968; C raddock ,

rl4 ■, .1972) and w i t h sod ium [ C ] fo rm a te ( S i b a t o n i , 1975; S m e l l ie

e t a l , 1958; P e r r e t t a , 1967, C raddock , 1 9 6 9 /7 0 ) . I t i s assumed

t h e r e f o r e t h a t 1-C f r a g m e n t s , r e l e a s e d d u r in g th e m e t a b o l i c

d e g r a d a t i o n o f th e a l k y l a t i n g a g e n t s , e n t e r t h e 1-C p o o l s and

a r e th u s i n c o r p o r a t e d d u r in g d e n o v o b a s e s y n t h e s i s .

B ecause b i o m e t h y l a t i o n i s a fu n d a m e n ta l n a t u r a l p r o c e s s

i t . i s im p o r ta n t t o b e a b l e t o d i s t i n g u i s h b e tw e en m e t h y la t e d

b a s e s p roduced by n a t u r a l m e th y la s e a c t i v i t y and t h o s e p ro d u c e d

- 18 -

by d i r e c t , non-enzym e m e d ia te d a l k y l a t i o n . In v iew o f t h e con­

t r i b u t i o n made b y . t h e a l k y l a t i n g a g e n ts t o th e 1-C p o o l s ,

a d e t a i l e d know ledge o f th e no rm al o c c u r r e n c e o f m e th y la t e d

b a s e s i s e s s e n t i a l and many co m p reh en s iv e re v ie w s have b e e n

w r i t t e n on th e s u b j e c t o f n u c l e i c a c id m e t h y la t i o n (Borek an4

S r i n e v a s a n , ,1966 ; S r in e v a s a n and B orek , 1964, 1967; S t a r r

and S e l l s , 1 9 6 9 ) . I n a d d i t i o n t o m e th y la t e d r i b o s e , m e th y la t e d

d e r i v a t i v e s o f a l l f o u r b a s e s have b e e n d e s c r i b e d i n t-RNA

and r-RNAe , T h e s e . a r e ( F ig . 6) 1 - m e th y la d e n in e , 2 - m e th y la d e n in e ,

6 6 6 W - m e t h y la d e n in e , N ,W - d im e th y l a d e n i n e , 1 - m e th y lg u a n in e ,2 . 2 2

M - m e th y lg u a n in e , M ,W - d im e th y l g u a n i n e , 7 - m e th y lg u a n in e ,

3 - m e t h y lc y to s i n e , W ^ -m e th y lc y to s in e , 5 - m e t h y l c y t o s i n e , 3 - m e th y l -

u r a c i l and 5 - m e t h y l u r a c i l ( H a l l , 1965; Munns e t a l , 1 9 7 4 ) .

A r e c e n t p a p e r by A lb a n i e t a l (1976) d e s c r i b e d th e p r e s e n c e

o f 5 - m e th o x y u r a c i l ( F ig . 6) i n t-RNA o f B. s u b t i l i s , b u t as

y e t no e v id e n c e e x i s t s f o r i t s o c c u r r e n c e i n mammalian c e l l

t-RNA. These m o d i f i c a t i o n s have b e e n shown to o c c u r p o s t -

s y n t h e t i c a l l y i . e . a t th e p o l y n u c l e o t i d e l e v e l (Mandel and

B o rek , 1961; B o r e k , ,1963 ; F l e i s s n e r and B o rek , 1963) by t h e

a c t i o n o f ' s p e c i f i c , m e th y la s e s w i t h 5 - a d e n o s y l m e th io n in e

a c t i n g as th e m e th y l dono r (Mandel and B orek , 1961, 1 9 6 3 ) . .

U n t i l c o m p a r a t iv e ly r e c e n t l y t h e m-RNA-molecule was b e l i e v e d

to b e u n m e th y la te d . However, w i t h t h e a p p l i c a t i o n o f a f f i n i t y

ch rom a tog raphy h i g h l y p u r i f i e d m-RNA w a s .p ro d u c e d . I t was t h e n

shown q u i t e c l e a r l y that,m-RNA from mouse L - c e l l s ( P e r r y and

K e l l e y , 1974) and from N ovakoff hepa tom a c e l l s ( D e s r o s i e r s

e t a l , 1974) c o n ta in e d m e th y la t e d b a s e s . The s u g g e s t e d l o c a t i o n

I —Methyladenine N6— Methyladenine NG, N6—Dimethyladenine

NH

HN

CH

2—Methyladenine

CH„—N

1—Methylguanine

CH

HIM

H2

7—Methylguanine

HN

CH

N2 —Methylguanine

CH, Hf V \ h

d H

N2 ,N* — D imethy Igu ani ne

NH NH

CH3 CH-NHN

HH H

CH3HN

HH H

5—Methylcytosine A/3—Methylcytosine 5—Methyluracil(thymine)

OCHHN

H5—Methoxyuracil

HNCH,

OH

3 —Methyluracil N4— Methylcytosine

Fig 6 Methylated bases present in RNA molecules

- 20 -

( a t th e 5* te r m in u s ) was s u b s e q u e n t ly co n f i rm e d and t h e s e

m e th y l g roups w ere shown to b e c o n ta in e d i n a s t r u c t u r e con­

t a i n i n g 7 - m e th y lg u a n in e i n a 5 ’- 5 ’p y ro p h o s p h a te l i n k e d w i t h

a 2 ' - 0 - m e t h y la t e d n u c l e o t i d e . Two such s t r u c t u r e s have b een

d e s c r i b e d i n RNA v i r u s e s , c y to p la s m ic p o l y h e d r o s i s v i r u s

( F u r u i c h i , 1974; F u r u i c h i and M u ira , 1975) and r e o v i r u s

( F u r u i c h i e t a l , 1975a) as 7mGpppAmp and 7mGpppGmp. These ’ c a p ’

s t r u c t u r e s have a w ide d i s t r i b u t i o n and have b e e n d e s c r i b e d

i n m-RNA d e r i v e d from H e la c e l l s (Nuss e t a l , 1975; G roner

and H u rw i tz , 1975; E n z in g e r and M oss, 1976; Wei e t a l , 1 9 7 5 ,1 9 7 6 ) ,

mouse myeloma c e l l s (Cory and Adams, 1975a, 1975b; Adams and

C ory , 1 9 7 6 ) , E h r l i c h s A s c i t e s c e l l s ( B a j s z a r e t a l , 1976),

i n g lo b in m-RNA from duck ( P e r r y and S c h e r r e r , 1975) and from

r a b b i t (M u lh u k r ish an e t a l , 1975; Hunt and O akes, 197 6 ) .

These s t r u c t u r e s a r e a l s o found i n t h e m-RNA from s e v e r a l mam­

m a l ia n v i r u s e s e . g . r e o v i r u s ( S h a tk i n , 1974; F u r u i c h i e t a l ,

1 9 7 5 ) , v a c c i n i a v i r u s (Wei and M oss, 1 9 7 4 ) , SV40 v i r u s ( A lo n i ,

1975a) and Rous sa rcom a v i r u s ( K e i th and F r a n k e l - C o n r a t , 1 9 7 5 ) .

I n a d d i t i o n ' t o t h e capped e n d s , m-RNA c o n ta i n s 6-m e th y la m in o

6 6p u r in e l o c a t e d i n t h e s p e c i f i c s e q u e n c e s Gpm ApC and Apm ApC

and n o t i n t h e p o ly A segm ent a t t h e 3 ’ end ( D e s r o s i e r s e t a l ,

T 9 7 4 ; P e r r y e t ^ a T , 1975; D e s r o s i e r s e t a l 7~1975 )t ^

The s i t u a t i o n w i th r e g a r d t o mammalian DNA i s l e s s complex

th a n t h a t o f RNA. 5 - M e t h y lc y t o s in e , f i r s t i d e n t i f i e d i n t h e

DNA from c a l f thym us, b e e f s p l e e n and ram sperm (Wyatt, 1950,

1951a , 1951b) i s th e o n ly m e th y la t e d b a s e found i n mammalian

DNA, a l th o u g h 6- in e th y la m in o p u r in e . i s found i n t h e DNA o f b a c t e r i a l

- 21 -

c e l l s o S u b se q u e n t s t u d i e s i n r a t l i v e r and s p l e e n (S h e id

e t a l , 1 9 6 8 ) , c h ic k embryo (K a p p le r 1 9 7 1 ) , c u l t u r e d mouse

f i b r o b l a s t s (Burdon and Adams, 1 9 6 9 ) , H e la c e l l s (G e ra c i

e t a l , 1974) and v a r i o u s o rg a n s o f th e r a t (V anyush in e t a l ,

1973) have a l l c o n f i rm e d t h e p r e s e n c e o f 5 - m e t h y lc y to s i n e i n

mammalian c e l l DNA and h a v e d e m o n s t ra te d b o th an i n t e r - and

i n t r a - s p e c i e s v a r i a t i o n i n i t s d i s t r i b u t i o n . The m e th y l g ro u p s o f

t h e s e r e s i d u e s a r e i n t r o d u c e d i n t o t h e DNA a t th e po lym er l e v e l .

The m e th y l group i s d o n a te d by S - a d e n o s y lm e th io n in e and i t s

t r a n s f e r t o c y t o s i n e i s e f f e c t e d by a s p e c i f i c m e th y l t r a n s f e r a s e .

The f a c t t h a t 5 - m e t h y lc y to s i n e i s s y n t h e s i s e d a t t h e p o ly ­

n u c l e o t i d e l e v e l c l e a r l y d i s t i n g u i s h e s i t from thym ine ( 5 - m e th y l -

u r a c i l ) , t h e m eth y l group o f w h ich i s i n c o r p o r a t e d a t th e monomer

5 10l e v e l w i t h t h e m e th y l c a rb o n o r i g i n a t i n g from W ,W - m e t h y le n e -

t e t r a h y d r o f o l a t e (FH ^)„

I n c u l t u r e d mouse f i b r o b l a s t s , t h e m e t h y l a t i o n o f c y t o s i n e

o c c u rs w i t h i n 1 h o f DNA s y n t h e s i s , d u r in g th e S -p h a s e o f t h e

c e l l c y c le (Burdon and Adams, 1969 ) ; th e same i s t r u e f o r DNA

s y n t h e s i s by H e la c e l l n u c l e i i n v i t r o (G e ra c i e t a l , 1 9 7 4 ) .

The d i s t r i b u t i o n o f 5 - m e t h y lc y to s i n e i n t h e DNA m o le c u le a p p e a r s

t o be non-random ( S h a p i r o and C h a r g a f f , 1960; Sm ith and Markham,

1 9 5 2 ) . I n N o v ik o f f hepa tom a c e l l s th e 5 - m e t h y lc y to s i n e i s a s s o c ­

i a t e d w i t h p y r im id i n e i s o s t i c h s ( S n e id e r , 1971) and v i r t u a l l y

a l l i s a s s o c i a t e d w i t h th e d o u b l e t 5MeCpG ( S in s h e im e r , 1 9 5 5 ) .

Use o f a n t i s e r u m to 5 - m e t h y lc y to s i n e h a s shown t h a t i t i s

a s s o c i a t e d w i t h th e C-band r e g io n s o f c e n t r o m e r ic h e t e r o c h r o m a t i n ,

and t h a t mouse s a t e l l i t e DNA c o n t a i n s more 5 - m e t h y l c y t o s i n e

t h a n m ain band DNA ( M i l l e r e t a l , 1974) .

- 22 -

P r o b a b ly th e m ost s i g n i f i c a n t f i n d i n g , in t h e p r e s e n t c o n t e x t ,

i s t h a t t h e m e th y la t e d b a s e s p ro d u ce d by d i r e c t c h e m ic a l a l k y -

l a t i o n a r e n o rm a l ly a b s e n t from mammalian DNA. However, r e p o r t s

o f ' t h e p r e s e n c e o f m ino r m e th y la t e d b a s e s o t h e r t h a n 5 - m e th y l ­

c y t o s i n e have b e e n made, f o r exam ple Culp e t a l (1970) c la im e d

th e p r e s e n c e o f A/-3 m e t h y lc y t o s in e i n human em bryonic lu n g

c e l l s and W-3 m e t h y l c y t o s i n e , W-l m e th y lg u a n in e , W-7 m e th y lg u a n in e ,

2 . 2 2 .W - m e th y lg u a n in e and W ,W - d im e th y lg u a n in e i n H e la c e l l DNA.

These e x p e r im e n ts i n H e la c e l l s have s i n c e been r e p e a t e d by

Lawley e t a l (1972) who found t h a t no m e th y la t e d b a s e o t h e r

th a n 5 - m e th y c y to s in e was p r e s e n t i n such DNA. In v iv o s t u d i e s

i n t h e r a t (C raddock e t a l , 1968) f a i l e d t o d e t e c t 7 - m e th y lg u a n in e

i n l i v e r DNA f o l l o w i n g [ ^ C - m e th y l ] m e th i o n i n e w h i l s t i t s p r e s e n c e

was c l e a r l y d e m o n s t ra b le i n th e RNA f r a c t i o n . These f i n d i n g s

w ere l a t e r c o n f i rm e d i n a s tu d y u s in g s e le n o m e t h io n i n e , w h ich

as s e l e n o a d e n o s y l s e l e n o m e th io n in e ( a c t i v e s e le n o m e th io n in e )

can a p p a r e n t l y a c t as a m e th y l dono r i n p l a c e o f S - a d e n o s y l

m e th io n in e (C raddock , 1972)„

E v id en ce f o r t h e u r i n a r y e x ic r e t io n o f m e th y la t e d b a s e s

i n t h e human d a t e s back to 1898 when t h e p r e s e n c e o f 7 - m e th y l -

g u a n in e was docum ented by K ru g e r and Saloman (1898a; 1 8 9 8 b ) .

However, i t was n o t u n t i l t h e work o f Weissmann e t a l (1957a;

1957b) t h a t th e f u l l r a n g e o f m e th y la t e d b a s e e x c r e t i o n became

a p p a r e n t . Chheda (1975) h a s r ev ie w e d th e c u r r e n t s i t u a t i o n w h ic h

i s sum m arised i n T a b le 3„ The o r i g i n o f t h e s e b a s e s would a p p e a r

t o b e th e m e th y la t e d b a s e s p r e s e n t i n t h e v a r i o u s R N A -sp ec ie s ,

r e l e a s e d from th e RNA d u r in g i t s m e t a b o l i c d e g r a d a t i o n . As t h e y

- 2 3 -

T a b le 3 - P u r in e and P y r im id in e D e r i v a t i v e s E x c r e t e d

i n Normal Human U r in e ( a f t e r Chheda, 1975)

Compound mg/day

A denine 1 .41- m e th y la d e n in e 0 . 3 - 0 . 4A/6- m e th y la d e n in e 0 .3 7A denos ine T race1- m e th y la d e n o s in e Not q u a n t i f i e dM ^-m ethy ladenos i n e 10-14

S -a d en o sy lm e t h io n in e 23 .8 ■-

C y to s in e 0 . 5 - 0 .63 - m e t h y lc y to s i n e 1.1C y t i d in e T ra c e

Guanine 0 . 3 - 1 . 31-m e th y lg u a n in e 0 . 6A/2-me t hy 1 guan in e 0 . 3 - 0 .6A/2A/2—d ime t hy 1 guan in e T ra ce7 -m e th y lg u a n in e 2 . 7 - 7 . 88- h y d r o x y - 7 -m e th y lg u a n in e 1 .6G uanosine T ra c eA/2A/2- .d imeth y l g u a n o s i n e : 1 . 1- 2 . 2A/2-m et hy 1 guano s i n e 0 . 2- 0 .31-me th y lg u a n o s in e 0 .4 - 0 .6 "

U r a c i l 3 . 7 - 1 3 .75 - m e t h y l u r a c i l 3 .55 - h y d r o x y m e th y lu r a c i l T ra c e

X a n th in e 2 . 8- 8 .71- m e t h y lx a n th i n e ( 1) Not q u a n t i f i e d3 - m e th y lx a n th in e (2) 3 .07-me t h y 1x a n th i n e 0 . 7 - 7 . 0 '

H ypoxan th ine 6 .81-me th y lh y p o x a n th in e 0 . 4 - 0 . 9I n o s i n e 0 .1 81- m e t h y l i n o s i n e 2 . 1- 2 .9

1- m e t h y ln i c o t i n a m id e 2 .8

(1) Young e t a l , 1971(2) B u t t s e t a l , 1971

- 24 -

r e p r e s e n t p o s t - s y n t h e t i c m o d i f i c a t i o n s t o t h e RNA th e y would

n o t b e r e - u t i l i s e d , and t h e r e would t h e r e f o r e seem l i t t l e a d v a n t ­

age t o be g a in e d by t h e i r s a l v a g e . C o n s e q u e n t ly t h e y a r e e x c r e t e d

unchanged i n th e u r i n e . T h is c o n c lu s io n may be s u p p o r t e d by

th e f a c t t h a t no m e th y la t e d n u c l e o t i d e o r n u c l e o s i d e p r e c u r s o r

p o o ls have b e e n d e t e c t e d i n t i s s u e e x t r a c t s o r b a c t e r i a l c e l l s

(B o rek , 1 9 6 3 ) . A ls o , i f c h e m ic a l ly p r e p a r e d m e th y la t e d b a s e s

a r e o f f e r e d to th e i n t a c t o rg a n is m th e y a r e n o t i n c o r p o r a t e d

and i n th e c a se o f 7 - m e th y lg u a n in e , a r e e x c r e t e d l a r g e l y U n c h a n g e d

i n th e u r i n e (B orek , 1963; Craddock e t a l , 196 8 b ) . W ith t h e

e x c e p t io n o f x a n t h i n e , 1 - m e th y lx a n th in e , 3 - m e th y lx a n th in e and

7 - m e th y lx a n th in e (Young e t a l , 1971; B u t t s e t a l , 1971; van

Gennip e t a l , 1973) t h e c o n c e n t r a t i o n s o f m e th y la t e d b a s e s

i n u r i n e a r e in d e p e n d e n t o f d i e t . The f a c t t h a t a d m i n i s t r a t i o n

_14 n . .o f [ C -m e th y l ]m e th io n in e to t h e i n t a c t an im a l r e s u l t s m t h e

l a b e l l i n g o f t h e u r i n a r y p u r in e s (Mandel e t a l , 1966; Ahonen

e t a l , 1972) a rg u e s v e ry s t r o n g l y f o r t h e i r endogenous o r i g i n .

The d a i l y e x c r e t i o n o f 7 - m e th y lg u a n in e has b een d e te r m in e d

i n t h e r a t . C raddock and Magee (1967) r e p o r t e d a d a i l y e x c r e t i o n

o f 65 yg.^ Chu and Lawley (1975a) have r e p o r t e d an e x c r e t i o n

r a t e o f 90 y g /d a y . A d m i n i s t r a t i o n o f DMN (30 m g.kg ^) c a u se d

a 2 - f o l d i n c r e a s e i n th e u r i n a r y e x c r e t i o n o f 7 - m e th y lg u a n in e

(Chu and Law ley , 1 9 7 5 a ) . T ha t a t l e a s t p a r t o f t h i s was d e r i v e d

from DNA was d e m o n s t ra te d u s in g r a t s w i t h DNA p r e - l a b e l l e d w i t h

sodium [ ^ C ] f o rm a te (Magee e t a l , 1967; C raddock and Magee,

1 9 6 7 ) . However, a f t e r t r e a tm e n t w i t h MMS (lOOmg.kg ^) u r i n a r y

l e v e l s o f 7 -m e th y lg u a n in e rem a in e d a t t h e c o n t r o l l e v e l . W h i l s t

- 25 -

i t can b e c l e a r l y d e m o n s t ra te d t h a t r a d i o a c t i v i t y d e r i v e d from14

th e m e th y l group o f I CJMMS can be t r a n s f e r r e d t o u r i n a r y 7 -

m e th y lg u a n in e (Chu and L aw ley , 1 9 7 4 ) , th e p o s i t i o n o f t h i s l a b e l

i n t h e J - m e th y l g u a n in e m o le c u le i s unknown.

A lthough th e u r i n a r y e x c r e t i o n o f m e th y la te d x a n t h i n e s

p r o b a b ly r e f l e c t s d i e t a r y i n t a k e i t has b een s u g g e s te d t h a t

3 - m e th y lx a n th in e may be d e r i v e d from 3 - m e th y la d e n in e . T h is l a t t e r

b a s e , th e m ost a b u n d a n t o f th e m ino r p r o d u c t s g e n e ra te d , by

t h e i n t e r a c t i o n o f m e t h y l a t i n g a g e n ts and DNA, i s n o t n o r m a l ly

r e c o g n i s e d as b e in g p r e s e n t i n RNA. Howeverj i t h a s b een r e p o r t e d

as a p r o d u c t i n E. c o l i and y e a s t t-RNA f o l lo w in g h y p e r m e th y l a t i o n

w i th t-RNA me t h y l a s e d e r i v e d from v a r i o u s h a m s te r t i s s u e s

(M cFarlane and Lee,, 1970; M cF arlane , 1 9 7 2 ) , These f i n d i n g s w ere

n o t c o n f i rm e d by J a c k s o n and Pegg (1 9 7 5 ) , However, t h i s i s s u e

m ust rem a in open i n v iew o f t h e d e s c r i p t i o n (A x e lro d and D a ly ,

1962) o f a m e th y la s e t h a t i s c a p a b le o f m e t h y l a t i n g a d e n in e

a t t h e S I -3 p o s i t i o n .

I t i s t h e r e f o r e a p p a r e n t t h a t th e m easurem ent o f r a d i o a c t i v i t y

c o n ta i n e d i n u r i n a r y p u r in e s c a n n o t b e u t i l i s e d as a m ethod

f o r q u a n t i t a t i v e l y m e a s u r in g i n v iv o a l k y l a t i n g r e a c t i v i t y

and i t s u t i l i t y i n t h e q u a l i t a t i v e s e n s e i s a l s o s e v e r e l y l i m i t e d .

D e m o n s t r a t io n o f a l k y l a t i n g r e a c t i v i t y i n v iv o m ust t h e r e f o r e

r e s t on th e i s o l a t i o n o f m e th y la t e d b a s e s from c e l l u l a r n u c l e i c

a c i d s , and i n t h i s c o n te x t i t w ould a p p e a r t h a t DNA w i t h i t s

v e ry low b ack g ro u n d o f n a t u r a l m e t h y la t i o n s i s t h e b e s t r e c e p t o r

m o le c u le f o r such s t u d i e s ,

r

- 26 -

1«3 A l k y l a t i n g a c t i v i t y o f p h o spho rus e s t e r s

E s t e r s o f p e n t a v a l e n t p h ospho rus c o n s t i t u t e th e m ost im p o r t ­

a n t c l a s s o f b i o l o g i c a l l y a c t i v e o rg an o p h o sp h o ru s compounds.

They a r e employed e x t e n s i v e l y as i n s e c t i c i d e s i n b o th com m erc ia l

and d o m e s t ic s i t u a t i o n s , i n many c a se s r e p l a c i n g th e more e n v i r o n -i

m e n t a l l y and b i o l o g i c a l l y p e r s i s t e n t o r g a n o c h l o r i n e compounds.

W ith few e x c e p t io n s t h e s e o rg a n o p h o s p h a te p e s t i c i d e s a r e n e u t r a l

e s t e r s o f p h o s p h o r i c a c id o r p h o s p h o r o t h i o n ic a c i d ( i t s s u lp h u r

a n a l o g u e ) I n su ch e s t e r s th e p h o spho rus atom te n d s to b e e l e c t r o n

d e f i c i e n t due t o th e p o l a r i s e d p h o s p h o ry l g r o u p s . Thus t h e r e

i s a n e t p o s i t i v e c h a rg e on th e p h ospho rus atom , w h ich as a

c onsequence d i s p l a y s e l e c t r o p h i l i c c h a r a c t e r i s t i c s . N u c l e o p h i l i c

_ 2 -

a t t a c k on th e o r g a n o p h o s p h a te s by h a rd n u c l e o p h i l e s (OH , HPO^ )

i s c e n t r e d a t p h o s p h o ru s , as shown i n E q u a t io n I I I .

n u c l e o p h i l e . I t i s by t h i s r e a c t i o n t h a t th e o rg a n o p h o sp h o ru s

i n s e c t i c i d e s a r e th o u g h t to e x e r t t h e i r b i o l o g i c a l e f f e c t .

Thus a t t a c k o f t h e p hospho rus atom by th e s e r i n e h y d ro x y l

s i t u a t e d a t th e a c t i v e e s t e r a t i c s i t e o f a c e t y l c h o l i n e s t e r a s e , =

a c t i v a t e d as a n u c l e o p h i l e by th e p r e s e n c e o f t h e i m id a z o le

group o f an a d j a c e n t h i s t i d i n e r e s i d u e , r e s u l t s i n p h o s p h o r y l a t i o n

Equation^ I I I

The n e t r e s u l t o f such an a t t a c k i s p h o s p h b r y l a t i b n o f t h e

- 27 -

o f t h e s e r i n e h y d ro x y l and i n a c t i v a t i o n o f t h e enzyme,

N u c l e o p h i l i c . a t t a c k o f t h e o rg a n o p h o s p h a te s by s o f t n u c l e o -

o f th e e s t e r g ro u p in g r a t h e r th a n th e phospho rus a tom . Th is

r e s u l t s , as shown i n E q u a t io n IV i n a l k y l a t i o n and n o t p h o sp h o ry ­

l a t i o n o f t h e n u c l e o p h i l e .

The a l k y l a t i n g r e a c t i v i t y o f t h e n o n - i n s e c t i c i d q l l y a c t i v e p h o s ­

p h o ru s e s t e r s has b e e n e x p l o i t e d f o r many y e a r s i n s y n t h e t i c

c h e m i s t r y . T r i a l k y l p h o s p h a te s , f o r ex am p le , have b een u se d

to a l k y l a t e , amines (B i l lm a n e t a l , 1942; Thomas e t a l , 1 9 4 6 ) ,

a l i p h a t i c a l c o h o l s (Toy, 1 9 4 4 ) , t h i o u r e a ( P a r k e r and S m ith , 1961)

a ry la m in e s ( Jo n e s e t a l , 1966) and i m id a z o le (Yamanchi and

K i r o s h i t a , 1973a) . The a l k y l a t i o n o f m o le c u le s o f g r e a t e r b i o l o g i c a l

s i g n i f i c a n c e , have been r e c e n t l y r e p o r t e d t o o c c u r u n d e r n o n -

p h y s i o l o g i c a l c o n d i t i o n s . Yamanchi and K i r o s h i t a (1973b) demon­

s t r a t e d t h e a l k y l a t i o n o f th e p y r im id in e s thym ine and u r a c i l

w i th t r i m e t h y l p h o s p h a te . I n s u b s e q u e n t s t u d i e s th e same a u th o r s

d e m o n s t ra te d th e a l k y l a t i o n o f a r a n g e o f p u r i n e s , nam ely x a n t h i n e , ...

t h e o p h y l l i n e , th eo b ro m in e and a d e n in e by t r i a l k y l p h o s p h a te s

(Yamanchi and K i r o s h i t a , 1 9 7 5 ) .

2 -p h i l e s (RSH, I , $2 ^ 3 ) i s u s u a l l y d i r e c t e d tow ards th e a - c a r b o n

CH--0 y/0^ r " x c h 3 +

CH — 03 w\ p /

E q u a t io n IV

- 28 -

A lthough such e x p e r im e n ta t i o n , h a s d e m o n s t ra te d t h a t o rg a n o -

p h o s p h a te s can a c t as a l k y l a t i n g a g e n t s , th e c o n d i t i o n s employed

a r e to o v ig o ro u s f o r any m e a n in g fu l e x t r a p o l a t i o n t o i n v i v o .

T h is was r e c o g n i s e d by Yamanchi e t a l (1976) who have s t u d i e d

th e a l k y l a t i n g r e a c t i v i t y o f t r i m e t h y lp h o s p h a te to w ard s t h e

f r e e b a s e s c y t o s i n e , th y m in e , u r a c i l , a d e n in e and g u a n in e u n d e r

much m i l d e r c o n d i t i o n s (aqueous s o l u t i o n , pH 9-12 a t 3 5 - 6 0 ° ) .

Em ploying a t l e a s t a 3 - f o l d m o la r e x c e s s o f t r i m e t h y l p h o s p h a t e

m e t h y la t i o n o f a l l b a s e s was d e m o n s t ra te d as f o l l o w s : c y t o s i n e

W-l > W-3, thymine W-T = W-3, uracil W-l = W-3, adenine W-9

= W - 3 > W-7 > W-l and guanine W-l ^ W-7 > W-3 > W-9 > 0 ^ .

Two i n t e r e s t i n g p o i n t s emerged from t h e i r s t u d y , f i r s t l y th e

W-7 atom of guanine was not the major reaction site and secondly6t h a t r e a c t i o n a t t h e 0 atom o f g u a n in e was d e t e c t e d , n o t a lo n e

6b u t i n a t r i m e t h y l a t e d d e r i v a t i v e (0 , 3 , 7 - t r i m e t h y l g u a n i n e ) .

A lth o u g h p o o r ly docum ented a l k y l a t i o n o f t h e s e n u c l e o p h i l i c

c e n t r e s has b e e n d e m o n s t r a te d i n DNA0 Kononova and Gumarou (1971)

d e m o n s t ra te d th e i n v i t r o a l k y l a t i o n by t r i m e t h y lp h o s p h a te

o f t h e DNA o f b a c t e r i o p h a g e T4B and w ere a b l e t o i d e n t i f y 7 -m e th y l—

g u a n in e and 1 - m e th y la d e n in e as t h e r e a c t i o n p r o d u c t s * R o senk ranz

and R osenkranz (1972) a l s o d e m o n s t ra te d an i n t e r a c t i o n be tw een

DNA and t r i m e t h y lp h o s p h a te i n v i t r o „ W h i l s t t h e s e w o rk e rs d i d

no t . i d e n t i f y th e p r o d u c t s o f t h e r e a c t i o n w i t h DNA, th e y found

a r e d u c t i o n i n t h e s e d im e n ta t i o n c o e f f i c i e n t o f DNA f o l l o w i n g

t r e a tm e n t w i t h o rg a n o p h o s p h a te s ...

I n f o r m a t io n on th e a l k y l a t i n g r e a c t i v i t y o f t h e i n s e c t i c i d a l

o rgan o p h o sp h o ru s e s t e r s i s s p a r s e i n c om par ison w i t h t h e i r

- 29 -

p h o s p h o r y l a t i n g r e a c t i v i t y . The p r e f e r e n c e o f s o f t n u c l e o p h i l e s

f o r th e a - c a r b o n o f t h e e s t e r h a s p r o v id e d a c o n v e n ie n t r o u t e

t o th e p r e p a r a t i o n o f d e s - a l k y l d e r i v a t i v e s ( H i lg e t a g and

Teichm ann, 1 9 6 5 ) . I n a d d i t i o n many unw anted m o d i f i c a t i o n s t h a t

o c c u r d u r in g th e m a n u fa c tu re and s t o r a g e o f o rg a n o p h o s p h a te s

a r e a t t r i b u t a b l e t o a l k y l a t i n g r e a c t i o n s . A good exam ple i s

p r o v id e d by dem eton-S -m ethyT w hich , d u r in g s t o r a g e , i s c o n v e r te d

i n t o a su lp h o n iu m compound o f much g r e a t e r t o x i c i t y . T h is was

shown by H eath and V andekar (1957) t o be due to one m o le c u le

a l k y l a t i n g th e s u lp h i d e group o f a n o th e r (E q u a t io n V ) .

E to and h i s c o -w o rk e rs have s t u d i e d th e a l k y l a t i n g r e a c t i v i t y

o f t h e s a l i g e n i n c y c l i c p h o spho rus e s t e r s and have shown t h a t

th e y w i l l a l k y l a t e m e rc a p ta n s su ch as g l u t a t h i o n e (E to e t a l ,

1968) and c y s t e i n e (Ohkawa and E to , 1 9 6 9 ) . They h av e a l s o shown

t h a t t h e s e compounds w i l l r e a c t w i t h and i n h i b i t t h e fSH e n zy m es1

such as p a p a in and y e a s t a l c o h o l d e h y d ro g e n a se (Ohkawa and E to ,

1 9 6 9 ) . D e s p i t e t h e p o s s i b l e i m p l i c a t i o n s o f t h e s e f i n d i n g s t o

t h e t o x i c i t y o f th e o rg an o p h o sp h o ru s p e s t i c i d e s o n ly one su ch

compound 2 , 2 - d i c h l o r o e t h e n y l d im e th y l p h o s p h a te ( F ig . 7 a , d i c h l o r v o s )

h as b een s t u d i e d i n any d e t a i l . L o f r o th e t a l (1969) r e p o r t e d

t h e i n v i t r o a l k y l a t i n g r e a c t i v i t y o f t h i s compound tow ards

a number o f n u c l e o p h i l e s i n c l u d i n g w a t e r , c h l o r i d e i o n s , s e v e r a l

m e rc a p ta n s and 4 - ( 4 - n i t r o b e n z y l ) p y r i d i n e . T h is l i s t o f n u c l e o p h i l e s

2(CH„0)of>SCHoCHoSCoH_ > (CH-O) JsCH.CH.S-C.H,. + 0-PSCHoCHoSCoH1

E q u a t io n V

- 30 -

CHo0 ^ 03

ch30 k0CH = CC1,

(a ) 2 , 2- d i c h l o r o e t h e n y l d im e th y l p h o s p h a te ( d i c h l o r v o s )

CHo0 .0

ch3o/ CH - CC10

r 3OH

(b) D im ethy l 1- h y d r o x y - 2 , 2 , 2 - t r i c h l o r o e t h y l p h o s p h o n a te ( t r i c h l o r p h o n )

ch3o.

“ 3°CH CC1,

oc- c3h 7

(c ) D im ethy l 1 - n - b u t y r y l o x y 2 , 2 , 2 - t r i c h l o r e t h y l p h o s p h o n a te ( b u to n a t e )

F i g . 7 O rganophosphorus p e s t i c i d e s exam ined f o r i n v iv o

a l k y l a t i n g r e a c t i v i t y

- 31 -

has s i n c e b een e x te n d e d t o i n c l u d e g u a n o s in e ( L o f r o t h , 1970;

R osenk ranz and R o se n k ra n z , 1 9 7 2 ) , i s o l a t e d DNA ( L S f r o th , 1970;

R osenkranz and R o se n k ran z , 1972; W ennerberg , 1973; W ennerberg

and L o f r o t h , .1974; Lawley e t a l , 1 9 7 4 ) . A d e t a i l e d c o m p a ra t iv e

s tu d y o f t h e r e a c t i o n o f o r g a n o p h o s p h a te s i n c l u d i n g d i c h l o r v o s

and e s t a b l i s h e d a l k y l a t i n g a g e n ts w i t h 4 ( 4 - n i t r o b e n z y l ) p y r i d i n e

by B ed fo rd and R ob inson (1972) showed d i c h l o r v o s to b e l e s s

r e a c t i v e th a n b o th MMS and DMS, b u t more r e a c t i v e th a n t r i m e t h y l ­

p h o s p h a te , w hich gave o n ly a v e ry weak p o s i t i v e r e a c t i o n , , S t u d ie s

w i t h c e l l s i n c u l t u r e have a l s o shown t h a t d i c h l o r v o s i s c a p a b le

o f a l k y l a t i n g t h e n u c l e i c a c id s o f b o th b a c t e r i a l c e l l s (W ennerberg ,

1973; Lawley e t a l , 1974; W ennerberg and L o f r o t h , 1974) and

H e la c e l l s (Lawley e t ' a l , 1 9 74 ) .

T hese e s s e n t i a l l y i n v i t r o s t u d i e s d e m o n s t r a te t h a t p h o s p h o r i c

a c i d t r i e s t e r s su ch as d i c h l o r v o s p o s s e s s a l k y l a t i n g p r o p e r t i e s .

D ic h lo rv o s m e t h y la t e s DNA v i a an S^2 m echanism . I n t h i s r e s p e c t

d i c h l o r v o s r e s e m b le s MMS b u t o f t h e tw o, t h e o rg a n o p h o s p h a te

i s much t h e w e a k e r a l k y l a t i n g a g e n t . However, t h e r e s u l t s

o b t a i n e d i n t h e s e model sy s tem s c a n n o t b e e x p e c te d t o p r o v id e

a r e l i a b l e e v a l u a t i o n o f i n v iv o r e a c t i v i t y and a r e t h e r e f o r e

o f l i t t l e r e l e v a n c e to t h e a s s e s s m e n t o f i n v iv o a l k y l a t i n g

r e a c t i v i t y i n t h e mammal.

S e v e r a l a t t e m p t s have b een made, w i t h v a r y i n g d e g r e e s o f

s u c c e s s , to m easu re t h e i n v iv o a l k y l a t i n g r e a c t i v i t y o f o rg a n o ­

p h o sp h o ru s p e s t i c i d e s . W ennerberg (1973) examined t h e DNA and

RNA f r a c t i o n s d e r i v e d from t h e lu n g s and l i v e r s o f r a t s and

14mice, exposed to [Me- C ] d i c h lo r v o s by i n h a l a t i o n o r i . p .

i n j e c t i o n s . W h i l s t l a b e l l i n g o f t h e p u r i n e r i n g sy s te m s v i a

- 32 -

t h e 1-C p o o l was r e a d i l y d e t e c t e d , no 7 -m e th y lg u a n in e was found

i n e i t h e r DNA o r RNA f r a c t i o n s . However, e x a m in a t io n o f th e

u r i n e from t h e s e and o t h e r e x p e r im e n ts (W ennerberg and L o f r o t h ,

1974) r e v e a l e d t h e p r e s e n c e o f r a d i o l a b e l l e d Nf- m e t h y I n i c o t i n ­

am ide , 7 -m e th y lg u a n in e and 3 -m e th y la d e n in e . The d e t e c t i o n o f

t h e s e r a d i o l a b e l l e d m e th y la t e d b a s e s , w hich w i t h t h e p o s s i b l e

e x c e p t io n o f 3- m e th y la d e n in e a r e no rm al e x c r e t i o n p r o d u c t s ,

was t a k e n as e v id e n c e f o r i n v iv o a l k y l a t i n g r e a c t i v i t y . The

b i o l o g i c a l h a l f - l i v e s f o r r a d i o l a b e l l e d 7 -m e th y lg u a n in e and

3 - m e th y la d e n in e w ere a p p ro x im a te ly 50 h o u rs and 20 h o u rs

r e s p e c t i v e l y .*

S u b s e q u e n t ly , a s i m i l a r a p p ro a c h has b e e n a d o p te d by Dedek

e t a l (1976) t o s tu d y t h e i n v iv o a l k y l a t i n g r e a c t i v i t y o f

t r i c h l o r p h o n e and b u t o n a t e ( F ig . 7 b & c) a d m i n i s t e r e d i n t r a -

p e r i t o n e a l l y to m ice a t h ig h d o se l e v e l s . T hese w o rk e rs w ere

a b l e t o d e t e c t t h e p r e s e n c e o f r a d i o l a b e l l e d 7 -m e th y lg u a n in e

i n a combined n u c l e i c a c id (DNA and RNA) f r a c t i o n d e r i v e d from

a number o f o r g a n s . However, t h e r a d i o a c t i v i t y had a s h o r t l i f e

i n t h e n u c l e i c a c i d f r a c t i o n , b e in g u n d e t e c t a b l e i n a n im a ls

k i l l e d 24 h o u r s o r l a t e r a f t e r a d m i n i s t r a t i o n o f t h e compound.

The o v e r a l l h a l f - l i f e o f 7 - m e th y lg u a n in e , as m easu red b y u r i n a r y

e x c r e t i o n , was <24 h f o l l o w in g t r i c h l o r p h o n and 2 W0 h f o l l o w i n g

b u t o n a t e . Thus, a l th o u g h t h e r e i s e v id e n c e w h ich i n d i c a t e s a

d e g re e o f m e t h y l a t i n g a c t i v i t y , t h e r e i s no e v id e n c e t o i m p l i c a t ei

DNA as t h e t a r g e t , f u r th e r m o r e s u c h an e x p e r i m e n t a l a p p ro a c h

would n o t d i s c r i m i n a t e b e tw een s p o n ta n e o u s and e n z y m a tic

m e t h y l a t i o n .

- 33 -

By a n a lo g y w i t h th e p o w e r fu l a l k y l a t i n g a g e n ts . th e demon­

s t r a t i o n o f t h e , a l b e i t weak, a l k y l a t i n g p r o p e r t i e s o f d i c h l o r v o s

l e d t o a s u s p i c i o n t h a t t h i s compound m ig h t be a m utagen and

a c a r c in o g e n ( i . e . g e n o t o x i c ) . However, a l t h o u g h v e r y h ig h con­

c e n t r a t i o n s o f d i c h l o r v o s , r e l a t i v e t o t h o s e e n c o u n te re d i n

p r a c t i c a l u se s i t u a t i o n s , can c a u se m u ta t io n s i n b a c t e r i a l c e l l s

t h e r e i s no e v id e n c e t h a t t h i s compound p ro d u c e s g e n o to x ic

e f f e c t s i n mammals (T a b le 4 ) . Thus, d i c h l o r v o s h a s b een th o r o u g h ly

e v a lu a t e d f o r m u t a g e n ic i t y and c a r c i n o g e n i c i t y i n mammals a t

dose l e v e l s o f up to a t l e a s t 100 t im es th o s e a c h ie v e d i n

p r a c t i c a l u s e c o n d i t i o n s and th e r e s u l t s o f t h e s e t e s t s have

b e e n e n t i r e l y n e g a t i v e (Voogd e t a l , 1972; B u s e lm a ie r e t a l ,

1972; Dean, 1972h; E p s t e i n e t a l , 1972; Dean and T h o rp e , 1 9 7 2 a ,1 9 7 2 b ;

W ith e ru p e t a l , 1 9 7 1 ) .

T e s t s f o r m u ta g e n ic e f f e c t s i n mammals exposed t o d i c h l o r v o s

have i n c l u d e d dom inan t l e t h a l a s s a y s i n m ale and fem a le m ice

( E p s t e i n e t a l , 1972; Dean and T h o rp e , 1972a; Dean and B l a i r , 1976)

i n v e s t i g a t i o n s o f t h e chromosomes o f bone marrow c e l l s and

s p e rm a to g o n ia i n m ice and C h in e se h a m s te r s (Dean and T ho rpe ,

1972b) and h o s t - m e d i a t e d a s s a y s (Voogd e t a l , 1972; B u s e lm a ie r , 1972;

Dean e t a l , 1 9 7 2 ) . None o f t h e s e s t u d i e s h a s g iv e n any i n d i c a t i o n

t h a t d i c h l o r v o s by any r o u t e o f e x p o s u re e x e r t s m u ta g e n ic e f f e c t s .

I n . v i t r o t e s t s em ploy ing mammalian c e l l s h av e a l s o f a i l e d

t o show any c o m p o u n d - re la te d chromosome a b e r r a t i o n s even a t

c o n c e n t r a t i o n s w h ich gave r i s e t o c y t o t o x i c i t y (Dean, 1 9 7 2 b ) .

- 34 -

T a b le 4 - M u ta g e n i c i t y T e s t s w i th D ic h lo r v o s

a . B a c t e r i a l sy s tem s

B a c t e r i a l s p e c i e s R e s u l t R e fe re n c e

E s c h e r i c h i a c o l i WP2 N e g a t iv e ( 1)E s c h e r i c h i a c o l i WP2 P o s i t i v e ( 2 ,3 ,4 )E s c h e r i c h i a c o l i W3110 P o s i t i v e (6 )E s c h e r i c h i a c o l i W3478 P o s i t i v e ( 6 )E s c h e r i c h i a c o l i WP67 P o s i t i v e (5)E s c h e r i c h i a c o l i CM561 N e g a t iv e (5)E s c h e r i c h i a c o l i WP12 N e g a t iv e (5)E s c h e r i c h i a c o l i CM57 N e g a t iv e (5)E s c h e r i c h i a c o l i CM611 N e g a t iv e (5)E s c h e r i c h i a c o l i K12 P o s i t i v e (7 ,9 )

S a lm o n e l la typh im urium h i s C l17 N e g a t iv e (5)S a lm o n e l la typh im urium h i s G46 N e g a t iv e (5)S a lm o n e l la typh im urium TA 1530 P o s i t i v e (5)S a lm o n e l la typh im urium TA 1535 P o s i t i v e ( 3 ,5 )S a lm o n e l la typh im urium TA 1536 N e g a t iv e (3)S a lm o n e l la typh im urium TA 1537 N e g a t iv e (3)S a lm o n e l la typh im urium TA 1538 N e g a t iv e (3)S a lm o n e l la typh im urium C 117 P o s i t i v e (8 )

Dean, 1972a (5) Hanna and D y e r , 1975B r id g e s e t a l , 1973 (6 ) R o se n k ra n z , 1973S h i r a s u e t a l , 1976 (7) Mohn, 1973Ashwood-Smith e t a l , 1972 (8 ) Dyer and Hanna, 1973

(9) Voogd e t a l , 1972

b . Mammalian sy s tem s

S p e c ie s T e s t sy s tem R e s u l t R e f e re n c e

Human lym phocy tes Chromosome a n a l y s i s N e g a t iv e ( 1)C h inese h a m s te r s A zaguan ine r e s i s t a n c e N e g a t iv e ( 2 )Mice (m ale) Dominant l e t h a l a s s a y N e g a t iv e (3)Mice (male and fem a le ) Dominant l e t h a l a s s a y N e g a t iv e (4)Mice (m ale) Dominant l e t h a l a s s a y N e g a t iv e (5)Mice H o s t -m e d ia te d a s s a y N e g a t iv e (6 )Mice H o s t -m e d ia te d a s s a y N e g a t iv e (7)Mice and h a m s te r s C y to g e n e t i c s t u d i e s N e g a t iv e (8 )

(1) Dean, 1972b (5) Dean and iThorpe, 1972a(2) Green e t a l , 1974 ( 6 ) Dean e t a l , 1972(3) E p s t e i n e t a l , 1972 (7) B u s e lm a ie r e t a l , 1972(4) Dean and B l a i r , 1976 (8 ) Dean and T h o rp e , 1972b

( 1)( 2 )(3)(4)

- 35 -

The c o n s i s t e n t l y n e g a t i v e r e s u l t s w i t h r e s p e c t t o m u t a t i o n a l

e v e n ts i n mammalian germ c e l l s and th e a b se n c e o f g e n e t i c e f f e c t s\

i n s u s p e n s io n s o f mammalian s o m a t ic c e l l s , i s complemented by

n e g a t i v e r e s u l t s i n mammalian c a r c i n o g e n i c i t y s t u d i e s * R a ts

w ere f e d d i e t s c o n t a i n i n g d i c h l o r v o s (up t o n o m in a l c o n c e n t r a t i o n s

o f 500 ppm) f o r 2 y e a r s and no c o m p o u n d - re la te d t i s s u e damage

o r c a r c i n o g e n i c r e s p o n s e s w ere o b s e rv e d (W itherup e t a l , 1971)„

A more r e c e n t 2 y e a r s tu d y ( B l a i r e t a l , 1976) w here r a t s w ere

exposed f o r 23 h o u rs d a i l y t o a tm o sp h e re s c o n t a i n i n g 0*05 , 0 .5

and 5 .0 y g d i c h l o r v o s p e r l i t r e , f a i l e d t o e s t a b l i s h any c a r c i n o ­

g e n ic e f f e c t s a t t r i b u t a b l e t o e x p o s u re t o d i c h l o r v o s .

I t h a s b e e n s u g g e s t e d (Lawley e t a l , 1974) t h a t . t h e f a i l u r e

o f d i c h l o r v o s t o in d u c e g e n o to x ic e f f e c t s i n mammals i s due

t o t h e l i m i t i n g e f f e c t o f t h e known r a p i d m e t a b o l i c d e g r a d a t i o n

o f t h i s compound on th e e x t e n t o f m e t h y l a t i o n o f DNA i n v i v o .

Such m e ta b o l i s m (H utson and H o a d le y , 1 9 7 2 a ,b ) l e a d s t o t h e

l o s s o f m e t h y l a t i n g p o t e n t i a l as e v id e n c e d by th e n e g a t i v e r e s u l t s

o b t a i n e d i n h o s t - m e d i a t e d a s s a y s (Voogd e t >a l , 1972; B u s e lm a ie r ,

1972; Dean e t a l , 1972) and t h e n o n - r e a c t i v i t y o f t h e m e t a b o l i c p r o ­

d u c t s , d e s - m e th y ld i c h lo r v o s and d im e th y lp h o s p h a te i n t h e 4 - ( 4 -

n i t r o b e n z y l ) p y r i d i n e c o lo u r r e a c t i o n (B e d fo rd and R o b in so n ,

1 972) .

- 36 -

1 .4 Aims o f t h e p r e s e n t s tu d y

The e x p e r im e n ta l work d e s c r i b e d i n t h i s t h e s i s was

s p e c i f i c a l l y d e s ig n e d to i n v e s t i g a t e , a t t h e m o le c u la r l e v e l ,

t h e g e n o to x ic p o t e n t i a l o f d i c h l o r v o s i n v i v o . I n c o m p a ra t iv e

s t u d i e s w i t h MMS i n t e r a c t i o n s w i t h DNA w ere a s s e s s e d i n b o th

c h e m ic a l and b i o l o g i c a l t e r m s , i . e . ex am in in g th e n a t u r e o f t h e

r e a c t i o n p r o d u c t s w i t h DNA and th e e f f e c t on DNA i n t e g r i t y

r e s p e c t i v e l y .

I n a d d i t i o n , a c o m p a ra t iv e e x a m in a t io n o f th e u r i n a r y

e x c r e t i o n o f m e th y la t e d b a s e s f o l l o w i n g e x p o su re t o a l k y l a t i n g

a g e n ts and 1-C p o o l don o rs was p e r fo rm e d . T hese l a t t e r e x p e r i ^

m ents w ere d e s ig n e d t o a s s e s s t h e j u t i l i t y ; o f m e th y la t e d u r i n a r y

p u r i n e m easu rem en ts i n t h e d e t e r m i n a t i o n o f i n v iv o a l k y l a t i n g

r e a c t i v i t y .

- 37 -

CHAPTER 2

M a t e r i a l s and s y n th e s e s

2 .1 M a t e r i a l s

Io n -e x c h a n g e r e s i n , Dowex AG 50 W x 8 ( +H form ; 100-200 mesh)

was p u rc h a s e d from Bio-Rad L a b o r a t o r i e s , B rom ley, K e n t . B lue

d e x t r a n , Sephadex G-10, 2 - a m i n o - 6 - c h l o r o p u r i n e , g l u t a t h i o n e

( r e d u c e d ) , S -m e th y 1c y s t e i n e , S - m e t h y l g l u t a t h i o n e , p r o t e a s e

( ty p e VI from S tre p to m y c e s g r i s e u s ) , r i b o n u c l e a s e A ( ty p e I I I

f rom b o v in e p a n c r e a s ) and DNA ( ty p e I , c a l f thymus) w ere p u r c h a s e d

from t h e Sigma C hem ical Co. L t d . t K in g s to n -u p o n -T h a m e s , S u r r e y .

The r e f e r e n c e b a s e s a d e n in e s u l p h a t e , 2 - m e th y la d e n in e h e m i s u lp h a t e ,

6 -m e th y la m in o p u r in e , g u a n in e , 7 - m e th y lg u a n in e , th y m in e , u r a c i l ,

c y t o s i n e , 5 - m e t h y l c y t o s i n e , x a n t h i n e and h y p o x a n th in e w ere a l s o

o b t a i n e d from Sigma C hem ical Co. L t d . The m e th y la t e d b a s e s ,

1-me t h y l a d e n i n e , 3 - m e th y la d e n in e , 7 - m e th y la d e n in e , and 9 - m e th y l -

a d e n in e w ere p u rc h a s e d from Cyclo C hem ica ls I n c . , Los A n g e le s ,

C a l i f o r n i a , USA. M ethy l m e th a n e s u lp h o n a te , d i m e th y l n i t r o s a m i n e

and d i e t h y l p y r o c a r b o n a te w ere o b t a i n e d from R a lph Eriianuel L t d . ,

Wembley, M id d le s e x . T r i - i s o p r o p y l n a p h t h a l e n e s u lp h o n a t e

was p u rc h a s e d from Kodak L t d . , K irk b y , L i v e r p o o l . m -C re s o l (GPR

g r a d e , r e d i s t i l l e d b e f o r e u s e ) , p h e n o l ( A n a l a r ) , 8- h y d r o x y q u i n o l i n e

( A n a la r ) , s u c r o s e ( A r i s t a r ) , sodium d o d e c y l s u l p h a t e ( ’ s p e c i a l l y

p u r e * g r a d e ) , h y d r o c h l o r i c a c id ( A n a l a r ) , p e r c h l o r i c a c i d (72%

w / v , A r i s t a r ) , ammonium fo rm a te ( A n a l a r ) , sodium 4 - a m i n o s a l i c y l a t e ,

_ t -b u ta n o l and’ m e th y l e t h y l k e to n e ( b u ta n o n e , s p e c i a l l y p u r e f o r

c h rom a tog raphy ) w ere p u rc h a s e d from B r i t i s h Drug Houses L t d . ,

JPoole, D o r s e t . A l l o t h e r c h e m ic a ls and r e a g e n t s w ere o f t h e

- 38 -

p u r e s t g ra d e a v a i l a b l e and w ere p u r c h a s e d from B r i t i s h Drug

Houses L t d . , o r Hopkin and W i l l i a m s , Romford, Essex* Chrom ato­

g rap h y p a p e r was o b t a i n e d from Whatman L a b S a le s L t d . , M a id s to n e ,

K e n t . T h i n - l a y e r c e l l u l o s e and s i l i c a g e l p l a t e s (Merck) w ere

o b t a i n e d th ro u g h Anderman and Co. L t d . , London.

2[M ethyl-C H ^ ] D ic h lo r v o s : T h is compound, s y n t h e s i s e d from d e u t e r o -

m e th a n o l and 2 , 2- d i c h l o r o v i n y l p h o s p h o d i c h l o r i d a t e was a g i f t

f rom D r . D. H. H u tso n .

D im ethy l n a p th y l p h o s p h a t e : S y n t h e s i s e d from 1 - n a p th o l and d im e th y l

p h o s p h o ry l c h l o r i d e was a l s o a g i f t from D r . D. H, H u tso n .

2 .2 R a d io c h e m ic a ls

[ M e th y l - ^ C ] m e th a n e s u lp h o n a te (56mCi.mmol ^) ; Di [M e th y l-^ 4C]

~ X X ̂ Xs u l p h a t e (46mCi.mol ) ; L - [M e th y l - C ]m e th io n in e (56mCi.mmol ) ;

sodium [^4C] f o rm a te (56mCi.mmol ^ ) ; [ 6- ^ C ] o r o t i c a c id m onohydra te

(57mCi.mmol ^ ) ; [8- ^4C ]a d e n in e (54.2mCi .mmol ^ ) ; [8- ^ C ] g u a n i n e

—1 3 . . —1s u l p h a t e (56mCi.mmol ) and [M ethy l- H ] th y m id in e (20Ci.mmol )

w ere p u rc h a s e d from th e R a d io c h e m ic a l C e n t r e , Amersham. B e fo re

u s e th e r a d io c h e m ic a l p u r i t y o f a l l m a t e r i a l s w i t h t h e e x c e p t io n

o f [14C]MMS and [U C]DMS was checked by p a p e r o r t h i n - l a y e r

ch ro m a to g rap h y (T a b le 5 ) .

[Me-^4C ] D ic h lo r v o s : T h is compound (113C i.m o l was s y n t h e s i s e d

a t S h e l l B io s c ie n c e s L a b o r a t o r y , S i t t i n g b o u m e R e s e a rc h C e n t r e ,

from [^4C ]m ethano l and 2 , 2 - d i c h l o r o v i n y l p h o s p h o d i c h l o r i d a t e .

14

+ 2 HC1

The r a d io c h e m ic a l was s t o r e d a t - 2 0 ° as a s o l u t i o n (2 mg.ml

i n d i ( 2- e t h y l h e x y l ) - a d i p a t e .

- 39 -

T a b le 5 - P u r i t y o f C om m erc ia l ly A v a i l a b l e R a d io c h e m ic a ls

R a d io c h e m ic a l Suppo rt S o lv e n t P u r i t y

[ 8- ̂ C ] Adenine P a p e r 1 ,2 99%

T h i n - l a y e r s i l i c a 3 99%

[ 8- ^ C ] Guanine P a p e r 1 ,2 99%

14L - [ m e th y l - C ]m e th io n in e TLC c e l l u l o s e 4 , 5 , 6k

99%

Sodium [ ^ C ] f o r m a t e TLC s i l i c a 7 99% v

C e l l u l o s e 8 99%

14[6- C ] o r o t i c a c id P a p e r 1 ,5 ,9 99%

3[m e th y l - H ] th y m id in e TLC s i l i c a 3 99%

A f t e r rem oval o f a p p ro x . 1% o f i m p u r i t y , p r o b a b ly s u lp h o x id e

S o l v e n t s :

System 1. i s o p r o p a n o l / w a t e r / h y d r o c h l o r i c a c i d ( 1 3 0 :3 7 :3 3 )System 2. t - b u t a n o l / m e t h y l e t h y l k e to n e /w a te r /a m m o n ia ( 4 : 3 : 2 : 1 ) System 3. n - b u ta n o l s a t u r a t e d w i t h w a te r System 4. n - b u t a n o l / w a t e r / a c e t i c a c i d ( 1 2 :5 :3 )System 5 . e th a n o l /a m m o n ia /w a te r ( 8 0 :4 :1 6 )System 6 . n - b u t a n o l / p y r i d i n e / w a t e r ( 1 : 1 : 1 )System 7. e th an o l /am m o n ia (3 :1 )System 8 . e th a n o l /2 N sodium h y d r o x id e / w a te r (1 4 4 :3 2 :1 4 )

- 40 -

Mass s p e c t r o m e t r y

F o r t h e s u b s e q u e n t e x p e r i m e n t a t i o n i t was n e c e s s a r y t o c o n f i rm14

th e i s o t o p i c abundance w i th r e s p e c t to [ Cj in t h e m e th y l g roups14

o f th e [Me- C ] d i c h l o r v o s . Mass s p e c t r o m e t r y p r o v id e d th e m ost

c o n v e n ie n t m ethod f o r m e a s u r in g th e i s o t o p i c d i s t r i b u t i o n . However,

. . . . . r 14owing to the very high specific radioactivity of the LMe- C]>

d i c h l o r v o s a f u l l s p e c t r a l a n a l y s i s was n o t p o s s i b l e . The i s o ­

t o p i c abundance i n t h e m e th y l g roups o f th e p a r e n t compound

was t h e r e f o r e a s s e s s e d by m u l t i p l e io n m o n i to r in g w h ich r e q u i r e d

a s m a l l e r q u a n t i t y o f compound and t h e r e f o r e p r e s e n t e d l e s s o f a

c o n ta m in a t io n h a z a r d . E x a m in a t io n s o f t h e GC-MS s p e c t ru m o b t a i n e d

w i t h an a n a l y t i c a l l y p u r e sam ple o f d i c h l o r v o s ( F ig . 8) showed,

i n common w i th many o r g a n o p h o s p h a te s , a v e r y weak p a r e n t io n

(S a fe and H u l z in g e r , 1 9 7 3 ) . The m ost a b u n d an t io n o c c u r r e d a t

M/e 109 c o r r e s p o n d in g t o [(CH^O)2P=0 ] + and was a t l e a s t t e n

t im es as i n t e n s e as th e p a r e n t i o n . A n a ly s i s o f d i c h l o r v o s con­

t a i n i n g d e u te r iu m i n t h e m e th y l g roups ( F ig . 9) showed a s h i f t

i n t h e p a r e n t io n o f 6 AMU. The i o n p r e s e n t a t M/e 115 was

2 +e x a c t l y t h a t e x p e c te d f o r th e [(C H ^ O ^ P ^ ] i o n . S u b se q u e n t

r 14 t . .a n a l y s i s o f t h e sam ple o f [Me- C] d i c h l o r v o s f o r i o n s o c c u r r i n g

a t M/e 109, M/e 111 and M/e 113 c o r r e s p o n d in g t o [ ( ^ C H ^ O ^ P ^ ] + ,

[ ( 12CH30) ( 14CH30 )P = 0 ]+ and [ ( 14CH30 ) 2P = 0]+ r e s p e c t i v e l y ( F i g . 10)

f a i l e d t o d e t e c t any n o n - r a d i o a c t i v e m o le c u le s . However, a p p ro x ­

i m a te l y 15% o f th e t o t a l m o le c u le s had o n ly one r a d i o l a b e l l e d

14m eth y l g ro u p . Thus th e i s o t o p i c abundance o f [ Cj i n t h e m e th y l

g roups a p p ro x im a te d to 92.5% o f t h e o r e t i c a l .

100

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100

(b) Deutero labelled100

(c) [14C-Me] dichlorvos

100

109 112 113 114 115110 111M/e

Fig 10 GC—Mass spectrum of dichlorvos obtained by multiple ion monitoring

- 44 -

-S~ [M ethyl-~^C] g l u t a t h i o n e : T h is compound was s y n t h e s i s e d by

r e a c t i n g g l u t a t h i o n e (16yM) i n 1ml 0»05M p h o s p h a te b u f f e r

. r l 4 ipH 7o6 w i t h d i - [ C Jm ethyl s u l p h a t e (10.88yM, 500yCi) i n 1ml

p h o s p h a te b u f f e r , f o r 2h a t 3 7 ° . A f t e r rem oval o f r e s i d u a l

d i m e th y l s u lp h a t e i n vacuo th e r e s i d u e was d i s s o l v e d i n 0 . 5ml

w a t e r and was p u r i f i e d by ch ro m a to g rap h y on Whatman 3MM p a p e r

i n b u t a n - l - o l / a c e t i c / a c i d w a te r ( 1 1 : 4 : 5 , v / v ; Rf 0 . 4 5 ) , The\

r a d i o a c t i v e band was e l u t e d w i t h w a t e r and th e e x t r a c t was

d r i e d i n a s t r e a m o f d ry n i t r o g e n . The p r o d u c t , o b t a i n e d i n

50% y i e l d (2 5 0 y C i) , c h ro m a to g ra p h ed as a s i n g l e s p o t c o i n c i d e n t

w i t h u n l a b e l l e d S - m e t h y l g l u t a t h i o n e on c e l l u l o s e TLC i n e t h a n o l /

w a te r /am m o n ia ( 9 0 :5 :5 v / v ) , and on s i l i c i c a c id TLC i n b u t a n -

1 - o l / a c e t i c / w a t e r ( 4 : 2 : 1 , v / v ) . F o l lo w in g h y d r o l y s i s i n 4M

HC1 f o r 2h a t 100° , a sam ple o f t h e 5 - [ ^ C ] m eth y l g l u t a t h i o n e

gave a s i n g l e s p o t c o i n c i d e n t w i t h m arked S - m e t h y l c y s t e i n e when

ch ro m a to g rap h ed on c e l l u l o s e t h i n - l a y e r s d e v e lo p e d w i t h b u t a n -

1 - b l / a c e t i c a c i d / w a t e r ( 4 : 2 : 1 , v / v ) 0

2 .3 S y n t h e s i s o f r e f e r e n c e compounds

3-M ethy1a d e n in e : T h is compound was p r e p a r e d by r e f l u x i n g a d e n in e

and t r i m e t h y lp h o s p h a te i n d im e th y lfo rm a m id e (Yamauchi e t a l ,

1 9 7 5 ) . The p r o d u c t o b t a i n e d i n 43% y i e l d was r e c r y s t a l l i s e d

from e th a n o l (Found: C 4 8 ,3 0 ; H 4 .7 3 ; N 4 6 ,9 5 ; C a lc : F o r C^H^N^;

C 4 8 ,3 2 ; H 4 .7 3 ; N 4 6 , 9 5 ) . Mass s p e c t r o m e t r y r e v e a l e d a p a r e n t

i o n a t M/e 149 and t h e s p e c t ru m ( F ig . 11) was i d e n t i c a l w i t h

t h a t o b t a i n e d w i t h t h e a u t h e n t i c compound. P a p e r c h ro m a to g ra p h y

(Whatman 3MM) i n _ t -b u t a n o l /m e th y l e t h y l k e to n e /am m o n ia . /w a te r

( 4 : 3 : 2 : ! , - v /v ) gave a s i n g l e s p o t , Rf 0 . 6 2 , Column c h ro m a to g ra p h y

o

CM

00

00

o

Aijsuaiu; 0AijB|ay

Fig

11 Ma

ss

spec

trum

of

3—m

ethy

l—6—

amin

o pu

rine

(8 m

ost

inte

nse

peak

s are

in

dica

ted)

- 46 -

on Sephadex G-10 e l u t e d w i t h 0.05M ammonium f o rm a te pH 6 .8

gave a s i n g l e peak e l u t i n g a t f r a c t i o n s 2 4 -2 9 . T h is component

b ehaved i d e n t i c a l l y w i t h th e a u t h e n t i c compound d u r in g co­

ch ro m a to g rap h y i n t h i s system,,

00 - M e th y lg u a n in e : T h is compound was s y n t h e s i s e d by r e f l u x i n g

2 - a m i n o - 6 - c h l o r o p u r i n e w i t h sod ium m eth o x id e ( B a l s i n g e r and

Montgomery, 1 9 6 4 ) . The p r o d u c t o b t a i n e d i n 80% y i e l d was

r e c r y s t a l l i s e d from w a t e r (Found: C 4 3 .6 ; H 4 .2 5 ; N 4 2 037;

C a lc : F o r C^H^N^O: C 4 3 .6 3 ; H 4 .2 5 ; N 4 2 . 3 7 ) . Mass s p e c t r o ­

m e try ( F ig . 12) showed a p a r e n t i o n a t M/e 165 and was0

i d e n t i c a l w i t h th e o f 0 - m e th y lg u a n in e o b t a i n e d by th e a l k y -

l a t i o n o f g u a n in e w i t h d ia z o m e th a n e (F riedm an e t a l , 1965) .

P a p e r ch ro m a to g rap h y (Whatman 3MM) i n t - b u t a n o l / m e t h y l e t h y l

k e to n e /a m m o n ia /w a te r ( 4 : 3 : 2 : 1 , v / v ) gave a s i n g l e s p o t (Rf 0 , 6 9 ) .

Column ch ro m a to g rap h y on Sephadex G-10 gave a s i n g l e peak

e l u t i n g a t f r a c t i o n s 9 5 -1 0 5 . T h is component behaved i d e n t i c a l l y

w i t h t h e a u t h e n t i c compound d u r in g c o -c h ro m a to g ra p h y i n t h i s

s y s te m ,

2 .4 Anim als

A lb in o r a t s o f t h e CFE s t r a i n , o r i g i n a l l y s u p p l i e d by

C arw orth Farm and th e n m a in ta in e d as a s p e c i f i c p a th o g e n f r e e

c o lo n y i n t h i s l a b o r a t o r y w ere u se d i n t h e s e s t u d i e s b Where

a p p r o p r i a t e p a r t i a l h e p a te c to m y was p e r fo rm e d on 10-12 week

o ld r a t s by th e m ethod o f H ig g in s and A nderson (1 9 3 1 ) , 60%

o f t h e l i v e r b e in g removed. L a b e l l i n g o f l i v e r DNA o c c u r r e d

d u r in g t h e p e r i o d o f r e s t o r a t i v e h y p e r p l a s i a f o l l o w i n g t h e

p a r t i a l h e p a te c to m y (as d e s c r i b e d i n s u b s e q u e n t s e c t i o n s )

( B r e s n i c k , 1971)„

o‘ CM

_Lo© Aiisuajuj aAijB|0y

Fig

12 Ma

ss

spec

trum

of

2—A

min

o—6—

met

hoxy

pu

rine

(8 m

ost

inte

nse

peak

s are

in

dica

ted)

- 48 -

CHAPTER 3

D i r e c t A l k y l a t i o n S tu d ie s

3 .1 I n t r o d u c t i o n

By a n a lo g y w i t h p o w e r fu l a l k y l a t i n g a g e n t s , t h e r e c o g n i t i o n

and s u b s e q u e n t d e m o n s t r a t io n o f t h e a l k y l a t i n g p r o p e r t i e s o f

d i c h l o r v o s s u g g e s t e d t h a t t h i s mixed t r i e s t e r o f p h o s p h o r ic

a c id m ig h t b e a m utagen and a c a r c in o g e n . A l th o u g h t h e r e s u l t s

o f mammalian c a r c i n o g e n i c i t y and m u t a g e n i c i t y s t u d i e s have

b e e n c o n s i s t e n t l y n e g a t i v e i t w as , n e v e r t h e l e s s , im p o r ta n t

t o d e te r m in e w h e th e r d i c h l o r v o s m e th y la t e d DNA i n v i v o .

The e x p e r im e n ta l work d e s c r i b e d i n t h i s C h a p te r was s p e c i f i ­

c a l l y d e s ig n e d t o i n v e s t i g a t e t h i s p o s s i b i l i t y . I n t h e p r a c t i c a l

u se s i t u a t i o n , t h e i n h a l a t i o n r o u t e i s t h e m a jo r r o u t e o f

e x p o su re t o d i c h l o r v o s . C o n s e q u e n t ly t h e i n h a l a t i o n r o u t e was

ch o sen f o r t h e e x p e r im e n ta l programme. The a i r c o n c e n t r a t i o n em­

p lo y e d i n t h e s e s t u d i e s was e q u i v a l e n t t o a t y p i c a l h ig h p r a c t i c a l

u s e c o n c e n t r a t i o n ( E lg a r and S t e e r , 1972) and i n o r d e r to m axi­

m ise t h e s e n s i t i v i t y o f d e t e c t i o n o f DNA a l k y l a t i o n , i t was

d e c id e d t h a t t h e DNA from t h e t o t a l s o f t t i s s u e s o f exposed

a n im a ls would be exam ined f o r th e p r e s e n c e o f 7 -m e th y lg u a n in e .

P i l o t s t u d i e s w ere co n d u c te d w i t h p o s i t i v e c o n t r o l a g e n ts (MMS

and DMS) i n o r d e r to e s t a b l i s h t h e v a l i d i t y and s e n s i t i v i t y

o f t h e e x p e r im e n ta l p ro c e d u r e s employed i n t h i s s t u d y .

- 49 -

3 8 ^ M ethods

A l k y l a t i o n o f DNA i n v i t r o

C a l f thymus DNA ( c a . 50mg; ty p e I ) was d i s s o l v e d i n 8ml

o f 8mM sodium p h o s p h a te b u f f e r , pH 7 06 c o n t a i n i n g 2mM EDTA a t

4 ° . A s o l u t i o n o f t h e a l k y l a t i n g a g e n t , ([^C]MMS (250yC i;14

4 ,8 y m o le s ) o r [ C ] d i c h lo r v o s (250yC i; 2„2ym oles) i n 2 ml o f

b u f fe r* w a s added and th e m ix tu r e i n c u b a t e d f o r 4h a t 37° •

A f t e r i n c u b a t i o n 0 .5m l o f 2M NaCl was added , and t h e DNA was

p r e c i p i t a t e d by t h e a d d i t i o n o f e th a n o l (1 2 m l)„ The m e th y la t e d

DNA was c o l l e c t e d on a g l a s s - r o d and w ashed s u c c e s s i v e l y w i t h

e t h a n o l 0.25M sodium a c e t a t e s o l u t i o n ( 3 :1 , v / v ) , f i v e t i m e s ,

e t h a n o l , e t h a n o l / d i e t h y l e t h e r ( 1 : 1 , v / v ) , and , f i n a l l y w i t h

d i e t h y l e t h e r . A f t e r d r y in g o v e r P 2°5 anc ̂ p a r a f f i n wax ( t o

remove t r a c e s o f e t h e r ) t h e m e th y la t e d DNA was s t o r e d , d e s i c c a t e d

as a f e l t * a t - 20° . '

H y d r o ly s i s o f DNA

M arker 7 -m e th y l g u a n in e (a p p ro x . lOOyg p e r mg DNA) was added

to a l l DNA sam p les p r i o r to h y d r o l y s i s . Two m ethods o f h y d r o l y s i s

w ere em ployed, ( i ) I n some o f t h e i n v i t r o e x p e r im e n t s , sam p les

o f DNA w ere h y d r o ly s e d i n 0.1M HC1 (250y l p e r mg DNA) a t 70°

f o r l h ; ( i i ) I n t h e i n v iv o e x p e r im e n ts and i n some o f t h e

i n v i t r o e x p e r im e n ts sam ples o f DNA w ere h y d r o ly s e d i n 72%

(w/v) p e r c h l o r i c a c i d (5 0 y l p e r mg DNA) f o r lh a t 100°C. P r i o r

t o ch rom atog raphy t h e s e l a t t e r h y d r o l y s a t e s w ere d i l u t e d t o

a p p ro x im a te ly lM ,w ith r e s p e c t t o t h e p e r c h l o r i c a c id ^ b y th e

a d d i t i o n o f d i s t i l l e d w a t e r .

- 50 -

F r a c t i o n a t i o n o f DNA h y d r o l y s a t e s

Two m ethods o f column ch ro m a to g rap h y w ere employed i n t h e s e

s t u d i e s . P e r c h l o r i c a c id h y d r o l y s a t e s and some h y d r o c h l o r i c

a c i d h y d r o l y s a t e s w ere ch ro m a to g rap h ed on Dowex 50 . Dowex AG50

■f"W x 8 , (100-200 mesh) H form was washed e x h a u s t i v e l y w i t h 4M

HC1 and th e n t o n e u t r a l i t y w i t h g l a s s d i s t i l l e d w a t e r p r i o r

t o p a c k in g i n t o g l a s s columns (1 .5cm x 12cm o r 35cm x 3.5cm)

f i t t e d w i th a s i n t e r e d g l a s s d i s c . The DNA h y d r o l y s a t e s were

a p p l i e d t o t h e columns i n 1M p e r c h l o r i c a c id and w ashed on

t o t h e r e s i n w i t h d i s t i l l e d w a t e r (10ml f o r t h e s m a l l column

and 50ml f o r t h e l a r g e c o lu m n ) . The columns w ere t h e n e l u t e d

w i th a l i n e a r g r a d i e n t o f 1 to 4M HC1 a t a f low r a t e o f 25 ml

h I n d i v i d u a l f r a c t i o n s o f 99 d ro p s (6 .4 m l) w ere c o l l e c t e d

u s in g an LKB U l t r o r a c f r a c t i o n c o l l e c t o r (LKB, Croydon , S u r re y )

eq u ip p e d w i t h a d rop c o u n t e r . The a b s o rb a n c e o f e a ch f r a c t i o n

was m easu red a t 254nm u s in g a Unicam SP 800 r e c o r d i n g s p e c t r o ­

p h o to m e te r . A l i q u o t s (1ml) o f each f r a c t i o n w ere removed f o r

t h e d e t e r m i n a t i o n o f r a d i o a c t i v i t y . The re m a in d e r o f each o f

t h e f r a c t i o n s c o n s t i t u t i n g r a d i o a c t i v i t y peaks w ere p o o le d ,

d r i e d i n a s t r e a m o f d ry n i t r o g e n , and r e d i s s o l v e d i n 0.1M HC1

(5ml) .

C hrom atography o f t h e 0.1M HC1 h y d r o l y s a t e s a nd , i n some

e x p e r im e n t s , r e c h ro m a to g ra p h y o f t h e 7 -m e th y lg u a n in e peaks from

Dowex ch ro m a to g rap h y ( d e s c r ib e d above) w ere p e rfo rm e d on

Sephadex G-10. The Sephadex was s t i r r e d i n d i s t i l l e d w a t e r con­

t a i n i n g 0 . 1% sodium a z id e f o r 48 h o u r s a t room t e m p e r a t u r e .

I t was t h e n packed i n t o an a l l - g l a s s column (95cm x 1.5cm)

- 51 -

f i t t e d w i t h a s i n t e r e d g l a s s d i s c , and e q u i l i b r a t e d w i th 0.05M

ammonium f o rm a te , pH 6 .8 ( g e n e r a l l y 4 l i t r e s ) . A f t e r t h e a d d i t i o n

o f b l u e d e x t r a n ( t o mark th e v o id volume) and any o t h e r m ark e r

b a s e s t h a t w ere r e q u i r e d , t h e sam ples i n 0.1M HC1 w ere a p p l i e d

d i r e c t l y t o t h e column as a s o l u t i o n i n 0.1M HC1 and w ere th e n

e l u t e d w i t h 0.05M ammonium fo rm a te pH 6 . 8 . I n d i v i d u a l f r a c t i o n s

o f 99 d ro p s (5 .9 m l) w ere c o l l e c t e d , and th e a b s o rb a n c e o f each

f r a c t i o n was d e te rm in e d as d e s c r i b e d above .

M easurem ent o f r a d i o a c t i v i t y

R a d i o a c t i v i t y was d e te rm in e d by l i q u i d s c i n t i l l a t i o n s p e c t r o

m e try w h ich was p e rfo rm e d i n a P a c k a rd T r i - C a r b (Model 526

o r 3003) s p e c t r o m e t e r . Samples (1ml) o f n u c l e i c a c id h y d r o l y s a t e s

and th e column e l u a n t f r a c t i o n s w ere b le n d e d w i t h NE 260

s c i n t i l l a t o r m ix tu r e (1 0 m l) . Such sam p les r e g u l a r l y c o u n te d

w i t h an e f f i c i e n c y o f 60-65% as d e te rm in e d by th e c h a n n e ls r a t i o

m ethod .

Animal e x p e r i m e n t a t i o n

The a p p a r a tu s u s e d , i n t h e i n h a l a t i o n e x p e r im e n ts w i t h [Me-

14 . .C J d ic h lo r v o s , c o n s i s t e d o f f i v e s e p a r a t e a tm o sp h e re g e n e r a t o r s

s u p p ly in g f i v e i n d i v i d u a l r a t h o l d e r s . Each h o l d e r was s e a l e d

i n t o a p o ly th e n e bag to m in im is e t h e r i s k o f r a d io c h e m ic a l con­

t a m i n a t i o n . The g e n e r a to r s c o n s i s t e d o f p a p e r - w ic k s a t u r a t o r s

c o n ta in e d i n g l a s s t u b i n g , 14cm in l e n g t h and 1.5cm o u t s i d e

d i a m e te r . A s o l u t i o n (1 .5 m l) o f [ M e - ^ C ] d ic h l o r v o s i n d i - ( 2 -

e t h y l h e x y l ) - a d i p a t e (0 .116m g.m l ^) was a p p l i e d t o each w ic k .

The a tm osphe re g e n e r a te d from t h e s e w ic k s , c o n t a i n i n g 0 . 0 6 4 y g . l ^

- 52 -

d i c h l o r v o s (2 2 ° , f low r a t e , 0 .4 6 lom in as m easu red by g „ l .„ c B,

was b r o u g h t im m e d ia te ly i n t o c o n t a c t w i t h t h e b r e a t h i n g zones

o f t h e r a t s a t a c o n s t a n t f low r a t e ( 0 .4 6 1 ,min ^) . The a tm o sp h e re

was drawn o u t o f th e head s e c t i o n o f t h e r a t h o l d e r , a t a p o i n t

j u s t above t h e head o f t h e r a t a t a f low r a t e (0 .5 6 l .m i n )

t h a t was m a r g i n a l l y g r e a t e r th a n th e r a t e o f s u p p ly . As a con­

s e q u e n c e , t h e h o l d e r s w ere u n d e r a s l i g h t n e g a t i v e p r e s s u r e

w hich ca u se d them t o l e a k in w a rd s , r e d u c in g even f u r t h e r th e

r i s k o f r a d io c h e m ic a l c o n ta m in a t io n w h i l s t e n s u r i n g e f f i c i e n t

rem ova l o f r e s p i r e d g a s e s . T h is e x i t a tm o sp h e re was drawn s u c ­

c e s s i v e l y th ro u g h a w a t e r - s e a l e d b u b b l e r , a s i l i c a g e l d r y in g

tu b e , a f low r e s t r i c t o r and f i n a l l y an a c t i v a t e d c a rb o n f i l t e r

t o t r a p t h e r a d i o a c t i v e w a s te ( F ig . 1 3 ) .

Two r e p l i c a t e e x p e r im e n ts w ere p e r fo rm e d . I n each e x p e r i ­

m en t , two groups o f f i v e r a t s w ere exposed t o th e a tm o sp h e re

c o n t a i n i n g 0 .064 iJg» l ^ [ M e - ^ C ] d ic h l o r v o s f o r a 12h p e r i o d .

Based on a m in u te volume (Vm) o f 0 .1 3 l .m i n ^ (R o d e r ic k , H.

p e r s o n a l com m unica tion) t h e t o t a l i n h a l e d d ose o f d i c h l o r v o s

amounted t o 6pg p e r r a t . The a n im a ls w ere k i l l e d by d e c a p i t a t i o n

im m e d ia te ly a t t h e end o f t h e e x p o s u re p e r i o d . The o rg a n s ( b r a i n ,

h e a r t and lu n g , l i v e r , s p l e e n , k id n e y and t e s t e s ) w ere r a p i d l y

d i s s e c t e d from th e c a r c a s s e s , p o o le d a c c o r d in g t o t y p e , e . g .

l i v e r , f r o z e n i n l i q u i d n i t r o g e n and s t o r e d a t - 2 0 ° „ The u r i n e ,

w hich c o l l e c t e d i n t h e p o l y th e n e b ags d u r in g th e e x p o s u re p e r i o d

o f th e f i r s t e x p e r im e n t , was a d j u s t e d t o pH 2 by a d d i t i o n o f

d i l u t e s u l p h u r i c a c id and was s t o r e d a t - 20°.

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- 54 -

14The a p p a r a tu s u sed f o r t h e L CJMMS e x p e r im e n t w as , w i th

th e e x c e p t io n o f t h e a tm o sp h e re g e n e r a t o r , i d e n t i c a l t o t h a t

d e s c r i b e d above ( F i g . 1 3 ) . The g e n e r a t o r c o n s i s t e d o f a s i n g l e

p a p e r - w ic k ty p e s a t u r a t o r c o n ta in e d i n a g l a s s tu b e 25.5cm x

3 .0cm ( F ig . 1 4 ) . A s o l u t i o n (5 .0 m l) o f [Me-^CjMMS i n d i - ( 2 -

e t h y l h e x y l ) - a d i p a t e (0 .106m g.m l ^) was a p p l i e d t o t h e w ic k .

The a tm o sp h e re g e n e r a te d from t h i s w ick c o n ta in e d 0 .0 6 4 y g . l ^

MMS (2 2 ° , f lo w r a t e 0 .355 l .m in ^) c a l c u l a t e d from d e t e r m i n a t i o n

o f r a d i o a c t i v i t y . T h is a tm o sp h e re was t h e n u sed t o s u p p ly f i v e

i n d i v i d u a l r a t h o l d e r s , i n r e p l i c a t e . e x p e r i m e n t s , a s ab o v e .

I n v iv o a l k y l a t i o n s t u d i e s w i t h [ m e t h y l - ^ C jm e th a n e s u lp h o n a t e

Two i n v iv o i n t r a p e r i t o n e a l a l k y l a t i o n s t u d i e s w ere p e r ­

fo rm ed . I n t h e f i r s t s tu d y 10 m ale r a t s w ere i n j e c t e d i n t r a -

14p e r i t o n e a l l y w i t h 24yg [ CjMMS p e r kg body w e ig h t (2 .5 y C i

p e r r a t ) i n i s o t o n i c s a l i n e and i n t h e seco n d e x p e r im e n t 10

rl4m ale r a t s w ere i n j e c t e d m t r a p e r i t o n e a l l y w i t h 2„4mg L CJMMS

p e r k g body w e ig h t (250yCi p e r r a t ) . At t h e end o f t h e f o u r

h o u r e x p o s u re p e r i o d t h e a n im a ls w ere k i l l e d by d e c a p i t a t i o n

and th e b r a i n , h e a r t and l u n g s , l i v e r , s p l e e n , k i d n e y , t e s t e s

and a l i m e n t a r y c a n a l , w ere r a p i d l y d i s s e c t e d from th e c a r c a s s e s .

The t i s s u e s w ere p o o le d a c c o r d in g to ty p e and w ere f r o z e n i n

l i q u i d n i t r o g e n . These t i s s u e p o o l s w ere s t o r e d a t -20°C u n t i l

r e q u i r e d .

r 14 *iI n v iv o a l k y l a t i o n s tu d y w i t h d iL m e th y l - C j s u lp h a t e

A s i n g l e s tu d y u s in g 5 m ale r a t s was p e r fo rm e d . Each a n im a l

r e c e i v e d 530yg p e r k g body w e ig h t (125yCi p e r r a t ) by i n t r a ­

p e r i t o n e a l i n j e c t i o n and was k i l l e d f o u r h o u rs l a t e r . The s o f t

t i s s u e s w ere removed and t r e a t e d as d e s c r i b e d a bove .

S19 ball O ___ SI 9 socket

Air flow

B 34/35

Filter paper wicks ^supporting

[14C]MMS

225

All dimensions in mm

Fig 14 Paper wick-type saturator used to generate the atmosphere of [14 C] MMS

- 56 -

r 14 ,L a b e l l i n g o f n e o n a t a l r a t DNA w i th sodium L C j fo rm a te

L i t t e r s o f CFE r a t s , g e n e r a l l y 14-15 p e r l i t t e r , were

re d u c e d i n s i z e to 10 i n o r d e r t o e n s u re maximum s u r v i v a l amongst

t h e t r e a t e d n e o n a t e s . Commencing 24 h o u rs a f t e r b i r t h t h e pups

w ere i n j e c t e d , s u b c u ta n e o u s ly f o r t h e f i r s t 5 days and th e n

r14m t r a p e r i t o n e a l l y , w i t h 2 .5 y C i o f sodium L C Jfo rm a te i n s a l i n e

( s t e r i l i s e d by f i l t r a t i o n ) d a i l y f o r 3 w e e k s . The t o t a l c u m u la t iv e

dose o f r a d i o a c t i v i t y was 105yCi p e r r a t . The l i t t e r s w ere k i l l e d

by d e c a p i t a t i o n 1 h o u r a f t e r t h e l a s t i n j e c t i o n . The b r a i n s ,

h e a r t and l u n g s , l i v e r s , s p l e e n s , k id n e y s a nd , w here a p p l i c a b l e

t e s t e s w ere r a p i d l y d i s s e c t e d from t h e c a r c a s s e s , p o o le d a c c o r d ­

in g t o t y p e , f r o z e n i n l i q u i d n i t r o g e n and s t o r e d a t - 20° .

L a b e l l i n g l i v e r DNA f o l lo w in g p a r t i a l h e p a te c to m y14 14

A t o t a l o f 50yCi o f sodium [ C] fo rm a te o r [m e th y l- C]

m e th io n in e was a d m i n i s t e r e d t o each p a r t i a l l y h e p a te c to m is e d

r a t by means o f a s e r i e s o f i n t r a p e r i t o n e a l i n j e c t i o n s (e a c h

o f 2 05yCi) a t 4 h o u r ly i n t e r v a l s commencing 16 h o u rs a f t e r t h e

o p e r a t i o n . I n a l l c a s e s th e a n im a ls w ere k i l l e d by d e c a p i t a t i o n

4 h o u rs a f t e r th e f i n a l i n j e c t i o n .

I s o l a t i o n o f DNA

A f t e r th a w in g , t h e t i s s u e p o o l s w ere b l o t t e d t o remove

e x c e s s m o i s t u r e and w e ig h e d . These, and ( u n le s s s t a t e d o t h e r w i s e )

a l l s u b s e q u e n t p r o c e d u r e s , w ere c a r r i e d o u t a t 4 ° . Each p o o l

was t h e n p a s s e d th ro u g h a p r e - c o o l e d s t a i n l e s s s t e e l t i s s u e - p r e s s

i n t o a p r e - c o o l e d , p re -w e ig h e d g l a s s h o m o g en is in g v e s s e l .

H om ogenising medium, sodium p - a m i n o s a l i c y l a t e / b u t a n - 2 - o l / t r i -

i s o p r o p y l n a p h t h a l e n e s u l p h o n a t e / d i s t i l l e d w a t e r ( 6 : 6 : 1 : 8 7 w /w ) ,

- 57 -

was th e n added (5ml. gm p r e s s e d t i s s u e w e i g h t ) . A f t e r f i l t e r i n g

th ro u g h a n y lo n m esh, t h e hom ogenate was a d j u s t e d t o 10% w /v ,

by a d d i t i o n o f ho m o g en is in g medium.

An e q u a l volume o f p h e n o l r e a g e n t ( p h e n o l /w a te r /m r - c r e s o l /

8- h y d r o x y q u i n o l i n e ; 5 0 0 : 5 5 : 7 0 : 0 . 5w/w) was added and t h e m ix tu r e

s t i r r e d v i g o r o u s l y a t room t e m p e r a tu r e f o r 20min u s in g a m a g n e t ic

s t i r r e r . The r e s u l t a n t e m u ls io n was b ro k e n by c e n t r i f u g a t i o n

a t 10 ,000 x g f o r 45min a t 4° i n a S o r v a l l RC2-B r e f r i g e r a t e d

c e n t r i f u g e e q u ip p e d w i t h a GS-3 r o t o r . The p h e n o l p h a se was

s e p a r a t e d from t h e aqueous p h a s e .

The aqueous p h a s e , r e t a i n e d a f t e r t h e p h e n o l e x t r a c t i o n ,

was a d j u s t e d t o 1%‘ w i t h r e s p e c t t o sodium c h l o r i d e , w ashed w i t h

one h a l f o f i t s volume o f p h e n o l r e a g e n t and r e - c e n t r i f u g e d

as d e s c r i b e d above . A f t e r c a r e f u l l y rem ov ing th e aqueous p h a s e ,

i t was mixed w i th an e q u a l volume o f 2- e th o x y e th a n o l w h ich

r e s u l t e d i n t h e fo rm a t io n o f a vo lum inous f i b r o u s p r e c i p i t a t e

o f im pure DNA. T his was r e c o v e r e d by w in d in g o n to a g l a s s r o d .

A f t e r w a sh in g i n 75% aqueous e t h a n o l t h e im pure DNA was d i s s o l v e d

i n 2% aqueous sodium a c e t a t e c o n t a i n i n g 1.5% sodium c h l o r i d e

( c a 2 5 m l) .

R ib o n u c le a s e , p r e v i o u s l y h e a t e d t o 80° f o r 10 min and

s lo w ly c o o le d t o d e s t r o y d e o x y r ib o n u c le a s e a c t i v i t y , was t h e n

added ( lm g.m l ^DNA s o l u t i o n ) . The m ix t u r e was i n c u b a t e d a t 37°

f o r 30 m in . C o n ta m in a t in g p r o t e i n was removed by i n c u b a t i n g

w i t h p r o n a s e (lmg.ml ̂ DNA s o l u t i o q ) f o r l h a t 3 7 ° . The DNA

was r e - p r e c i p i t a t e d w i t h 2 - e t h o x y e t h a n o l , w ashed i n 75% aqueous

e t h a n o l and th e n r e d i s s o l v e d i n 2% aqueous sodium a c e t a t e con­

t a i n i n g 1.5% sodium c h l o r i d e . R e s id u a l p o l y s a c c h a r i d e m a t e r i a l

- 58 -

was removed by c e n t r i f u g a t i o n a t 106 ,000 x g f o r l h a t 4 ° i n

an MSE s u p e r - s p e e d 75 r e f r i g e r a t e d c e n t r i f u g e .

The p u r i f i e d DNA was p r e c i p i t a t e d from s o l u t i o n w i t h 2 -

e th o x y e t h a n o l , washed s u c c e s s i v e l y w i t h 75%, 85%, 90%, 95%

and 100% e t h a n o l , a n d f i n a l l y w i t h d i e t h y l e t h e r b e f o r e d r y in g

i n vacuo o v e r p h o sp h o ru s p e n t o x i d e . Samples o f a p p ro x im a te ly

100yg o f each DNA f r a c t i o n w ere removed f o r th e d e t e r m i n a t i o n

o f DNA c o n te n t and r a d i o a c t i v i t y .

E s t im a t i o n o f DNA

DNA was e s t i m a t e d u s in g a m o d i f i c a t i o n o f t h e d ip h e n y la m in e

p r o c e d u r e ( B u r to n , 1956) d e s c r i b e d b y G i l e s and Myers (1 9 6 5 ) .

C a l f thymus DNA, (h igh m o le c u la r w e ig h t ) was u sed i n t h e

c o n s t r u c t i o n o f t h e s t a n d a r d c u r v e .

R ecovery o f 7 - m e th y lg u a n in e from th e s c i n t i l l a t i o n m ix tu r e

To t h e p o o l o f s c i n t i l l a t i o n m ix tu r e c o n t a i n i n g th e 7 - m e th y l -

g u a n in e f r a c t i o n d e r i v e d from th e f i r s t e x p e r im e n t , and i n

p a r t t h e seco n d e x p e r im e n t , was added 1M s i l v e r n i t r a t e (20ml)

and th e m ix tu r e s t o r e d a t room t e m p e r a tu r e i n t h e d a rk f o r

2 d a y s . At t h e end o f t h i s p e r i o d , an e q u a l volume o f r e d i s t i l l e d

a c e to n e was added and th e m ix tu r e s t o r e d a t 4 ° f o r 3 d a y s .

The r e s u l t i n g " o f f - w h i t e " p r e c i p i t a t e was c o l l e c t e d by c e n t r i ­

f u g a t i o n i n an MSE m in o r c e n t r i f u g e (lO min; 1200rpm ), w ashed

f o u r t im es w i t h r e d i s t i l l e d a c e to n e and th e n h y d r o ly s e d i n 0.1M

HC1 (20m l; 30m in; 1 0 0 ° ) , A f t e r rem ova l o f t h e i n s o l u b l e s i l v e r

s a l t by f i l t r a t i o n , t h e c l e a r f i l t r a t e was t a k e n t o d r y n e s s

i n a s t r e a m o f d r y n i t r o g e n . A f t e r r e d i s s o l v i n g i n 0.1M HC1

(5ml) t h e " r e c o v e r e d 7 - m e th y lg u a n in e " was p u r i f i e d by

- 59 -

ch ro m a to g rap h y on Sephadex G-10.

P a p e r ch rom atog raphy

The u . v . a b s o r b in g and th e r a d i o a c t i v e peaks w ere s u b j e c t e d

t o p a p e r c h ro m a to g ra p h y on Whatman N o . l o r 3MM p a p e r d e v e lo p e d

i n t h e f o l l o w i n g s o l v e n t s : m e t h a n o l / h y d r o c h l o r i c a c i d / w a t e r

( 7 : 3 : 1 , v / v ) ; i s o p r o p a n o l / h y d r o c h l o r i c a c i d / w a t e r ( 1 7 0 :4 1 :3 9 ,

v / v ) ; t - b u t a n o l / m e t h y l e t h y l k e to n e /a m m o n ia /w a te r ( 4 : 3 : 2 : 1 ,

v / v ) .

3 .3 R e s u l t s

I n v i t r o s t u d i e s

Two m ethods o f a c id h y d r o l y s i s w ere i n v e s t i g a t e d , w i t h

a v iew t o em ploy ing column ch ro m a to g rap h y on Sephadex G-10

o r Dowex 5 0 , t o r e s o l v e t h e a l k y l a t e d b a s e s . F i g s . 15 and 16

show th e r e s o l u t i o n o f m ix t u r e s o f r e f e r e n c e b a s e s i n t h e s e

s y s te m s . Samples o f c a l f thymus DNA, a l k y l a t e d i n v i t r o w i t h

[m ethy l-^C ]M M S, w ere h y d r o ly s e d i n 0.1M HC1 o r 12N HCIO^

and t h e h y d r o l y s a t e s w ere ch ro m a to g ra p h ed on Dowex 50 co lum ns .

The c h ro m a to g ra p h ic p r o f i l e s a r e shown i n F i g s . 17 and 18

r e s p e c t i v e l y . I n b o th c a s e s t h e m a jo r p o r t i o n o f t h e r a d i o ­

a c t i v i t y was a s s o c i a t e d w i t h th e m a rk e r 7 - m e th y lg u a n in e ,

a c c o u n t in g f o r 66% and 70.97% r e s p e c t i v e l y o f t h e r e c o v e r e d

r a d i o a c t i v i t y . No a t t e m p t was made i n t h e s e e a r l y e x p e r im e n ts

t o s e p a r a t e 3 - m e th y la d e n in e and 7 -m e th y lg u a n in e w h ich a r e n o t

r e s o l v e d by ch rom a tog raphy on Dowex 50 co lum ns. B ased on t h e

amount o f r a d i o a c t i v i t y r e c o v e r e d i n t h e 7 - m e th y lg u a n in e f r a c t i o n s ,

( u n c o r r e c t e d f o r 3 -m e th y la d e n in e ) and a ssum ing t h a t g u a n in e

254

T3 O)JC

■D

1.5

ao

0.5

50 100Fraction No.

Fig15 Chromatography of Purine bases on Sephadex G—10 Eluted with 0.05 M ammonium formate pH 6.8

0Uiuen6|Aq*0|/\|-£

©©

inif)

Fig

16 Ch

rom

atog

raph

y of

DNA

base

s on

Dowe

x 50

[H+]

elute

d w

itha

1—4

M HC

I gr

adie

nt

20,000 7 —Methylguanine

15,000

v*

I 10,000o.o

5,000

1,000

10 30 4020 50 60 70 80

Fraction No.

Fig 17 Dowex 50[+ H] chromatography of the 0.1m HCI hydrolysate of DNA alkylated in vitro with [Methyl—14C] MMS

Cpm

ml-

25000

20000

15000

10000

5000

50 100 150

Fraction No.

Fig 18 Ion-exchange chromatogram of DNA alkylated in vitro with [Methyl—14C] MMS hydrolysed 72% pechloric acid

- 64 -

c o n t r i b u t e s 10% o f t h e w e ig h t o f DNA, th e y i e l d s o f 7 - m e th y l -

g u a n in e r e p r e s e n t e d 0,12% and 0.13% o f t h e a v a i l a b l e DNA g u a n in e

r e s p e c t i v e l y .

A f u r t h e r sam ple o f i n v i t r o a l k y l a t e d DNA was h y d r o ly s e d

i n 0.1M HC1 and th e h y d r o l y s a t e was ch ro m a to g rap h ed on Sephadex

G-10 i n o r d e r t o i n v e s t i g a t e t h e r e s o l u t i o n o f 3 - m e th y la d e n in e

and 7 - m e th y lg u a n in e . The c h ro m a to g ra p h ic p r o f i l e i s shown i n

F i g . 19. Of t h e r a d i o a c t i v i t y r e c o v e r e d , 75.69% was a s s o c i a t e d

w i th t h e 7 -m e th y lg u a n in e m a rk e r , 9.96% w i th t h e 3 - m e th y la d e n in e

m a rk e r and a p p ro x im a te ly 0.4% w i t h t h e 0 - m e th y lg u a n in e m a r k e r .

Making th e same a s su m p t io n s as above , 0.157% o f t h e a v a i l a b l e

g u a n in e i n t h i s sam ple o f DNA had b e e n a l k y l a t e d . A t te m p ts t o

f r a c t i o n a t e p e r c h l o r i c a c id h y d r o l y s a t e s ( n e u t r a l i s e d

w i t h p o ta s s iu m h y d ro x id e ) on Sephadex G-10 f a i l e d t o p r o v id e

a d e q u a te r e s o l u t i o n . The r e s o l v i n g power d e c r e a s e d s t i l l f u r t h e r

when th e amount o f h y d r o l y s a t e (b o th h y d r o c h l o r i c a c id and

p e r c h l o r i c a c id ) a p p l i e d to t h e Sephadex G-10 column was i n c r e a s e d .

B ecause o f t h e r e l a t i v e l y l a r g e amounts o f DNA t o be a n a ly s e d

i n t h e i n v iv o e x p e r im e n t s , t h e te c h n iq u e , c h o sen

was Dowex 50 ch ro m a to g rap h y o f 72% p e r c h l o r i c a c id h y d r o l y s a t e s .

C hrom atography o f t h e p e r c h l o r i c a c id h y d r o l y s a t e o f a

sam ple o f c a l f thymus DNA, a l k y l a t e d i n v i t r o w i t h [ m e t h y l - ^ C ]

d i c h l o r v o s gave t h e p r o f i l e shown i n F i g . 20 . I n m arked c o n t r a s t

t o t h e e l u t i o n p r o f i l e s o b t a i n e d w i t h [methyl-^C]M M S a l k y l a t e d

DNA, t h e p r o f i l e from th e d i c h l o r v o s - a l k y l a t e d DNA d e m o n s t r a te d

t h e p r e s e n c e o f l a r g e q u a n t i t i e s o f r a d i o a c t i v i t y e l u t i n g w i t h

t h e v o id volume o f th e column. U s ing p a p e r ch ro m a to g rap h y

Cpm

ml-

UJ

7—Methylguanine

40000

30000

s>05

20000 100

10000

10 15020 100Fraction No.

Fig 19 Separation of 0.1 M HCI hydrolysate of DNA alkylated in vitro with [Methyl—14 C]MMS on Sephadex G-10 eluted with 0.05M ammonium formate pH 6.8

o

o •LO .

©

inr—

t _|UI Ulda

Fig

20 Do

wex

50[+

H]

elut

ion

prof

ile

of the

ac

id hy

drol

ysat

e of

calf

thym

us

DN

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s

- 67 -

(H utson e t a l , 1971) (Whatman 3MM d e v e lo p e d i n b u t a n o l / a c e t i c /

w a t e r , 1 1 : 5 :4 , v / v ) , t h i s r a d i o a c t i v i t y was shown t o be l a r g e l y

a s s o c i a t e d w i t h d im e th y lp h o s p h a te and d e s m e th y l d i c h l o r v o s .

R a d io a s s a y o f th e 7- m e th y lg u a n in e f r a c t i o n i n d i c a t e d t h a t 0.004%

o f t h e t o t a l DNA g u a n in e had b e e n m e th y la t e d by d i c h l o r v o s .

I n v iv o s t u d i e s

The p ro p o s e d i n h a l a t i o n s t u d i e s w i t h [ m e t h y l - d i c h l o r v o s

r e p r e s e n t e d a d e p a r t u r e from th e c l a s s i c a l s t u d i e s . The a tm os­

p h e r i c c o n c e n t r a t i o n employed was low* e q u a t i n g w i th t h a t o f

a t y p i c a l l y h ig h p r a c t i c a l u s e c o n c e n t r a t i o n . A s e r i e s o f e x p e r i ­

m ents w i th low d o ses o f [m ethyl-^CjM M S and d i [ m e t h y l - ^ C ] s u l p h a t e

were t h e r e f o r e p e rfo rm e d as p o s i t i v e c o n t r o l s .

T a b le 6 shows th e y i e l d s and s p e c i f i c r a d i o a c t i v i t i e s o f

t h e DNA f r a c t i o n s i s o l a t e d from th e s o f t - t i s s u e s o f r a t s 4 h o u rs14

a f t e r a s i n g l e i . p . i n j e c t i o n o f [m e th y l- CjMMS (24yg p e r

kg body w e i g h t ) . R a d i o a c t i v i t y was a s s o c i a t e d w i t h t h e DNA

o f a l l o f th e s o f t t i s s u e p o o l s . The c h ro m a to g ra p h ic p r o f i l e

(Dowex 50) o f th e a c i d h y d r o l y s a t e o f t h e t o t a l s o f t t i s s u e

DNA i s shown i n F i g . 21. I n a d d i t i o n t o 7 -m e th y lg u a n in e , r a d i o ­

a c t i v i t y was a s s o c i a t e d w i t h t h e n a t u r a l l y o c c u r r i n g b a s e s

th y m in e , a d e n in e and g u a n in e . B ased on t h e t o t a l r a d i o a c t i v i t y

a s s o c i a t e d w i t h t h e 7 - m e th y lg u a n in e p e a k , and assum ing t h a t

a l l o f t h e r a d i o a c t i v i t y r e s i d e d i n t h e m e th y l g roup and a l s o

t h a t t h e r e was no c o n ta m in a t io n w i t h 3 - m e th y la d e n in e , t h e o v e r -

-9a l l e x t e n t o f a l k y l a t i o n was 387 x 10 mol 7 - m e th y lg u a n m e

-1 -1 -1 mol •DNA-P.pmol MMS.kg body w e i g h t .

- 68 -

T a b le 6 - Y ie ld s and s p e c i f i c r a d i o a c t i v i t i e s o f DNA

o b t a i n e d from t h e s o f t t i s s u e s o f r a t s exposed t o -14 -1

24yg [ C]MMS kg body w e ig h t by i n t r a p e r i t o n e a l i n j e c t i o n

Organ Y ie ld DNA mg0g n f l f r e s h w e ig h t

S p e c i f i c r a d i o a c t i v i t y Dpnumg“ l

B r a in 1 .9 5 2 .6 8

H e a r t and lu n g - -

L i v e r 2 .1 4 1 .77

S p le e n 5 .7 4 5 .7

Kidney 2 .5 7 3 .9 2

T e s t i s 1 .95j

3 .2 4

150

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X wlL< to<DO >-S ^^ v> X g| 2o °Qt— wCM g.2* 8 IL '13

L-|W uido

- 70- -

The y i e l d s and s p e c i f i c r a d i o a c t i v i t i e s o f t h e DNA from

. . r 14t h e s o f t t i s s u e p o o l DNA,from r a t s i n j e c t e d w i th [m e th y l- CJ

MMS ( 2 .4 mg p e r kg body w e ig h t ) , a r e shown i n T a b le 7 . Chrom ato­

g rap h y o f t h e t o t a l p o o le d DNA h y d r o l y s a t e on Dowex 50 gave

t h e e l u t i o n p r o f i l e shown i n F i g . 22 . Heavy l a b e l l i n g o f t h e

7 -m e th y lg u a n in e f r a c t i o n was o b t a i n e d , b u t t h i s was accom panied

by an e l e v a t e d b ack g ro u n d r a d i o a c t i v i t y . C a l c u l a t i o n o f t h e

e x t e n t o f th e r e a c t i o n b a s e d on t o t a l r a d i o a c t i v i t y i n th e

7 -m e th y lg u a n in e p eak i n d i c a t e d an o v e r a l l e x t e n t o f r e a c t i o n- 9 - i - i - x

o f 511 x 10 mol 7 -m e th y l g u a n in e mol DNA-P.ymol MMS .k g

body w e i g h t .

D u r in g t h i s p a r t i c u l a r e x p e r im e n t a t t e m p t s w ere made t o

i s o l a t e a sam ple o f DNA from t h e a l i m e n t a r y t r a c t s o f t h e exp o sed

a n im a ls . These a t t e m p t s w ere n o t e n t i r e l y s u c c e s s f u l . Thus

t h e a d d i t i o n o f 2- e t h o x y - e t h a n o l t o t h e aqueous p h a s e o f t h e

p h e n o l e x t r a c t i o n p ro d u c e d a f l o c c u l e n t p r e c i p i t a t e r a t h e r

th a n th e no rm al f i b r o u s s t r a n d s . H y d r o ly s i s o f th e p r e c i p i t a t e

and s u b s e q u e n t c h ro m a to g ra p h y (Dowex 50) gave th e p r o f i l e

shown i n F i g . 23 . Compared w i t h t h e DNA from th e t o t a l s o f t

t i s s u e p o o l much h i g h e r amounts o f r a d i o a c t i v i t y w ere a s s o c i a t e d

w i t h t h e n a t u r a l l y o c c u r r i n g b a s e s o f t h e a l i m e n t a r y t r a c t

DNA. T h is r e s u l t was c o n s i s t e n t w i t h th e r a p i d t u r n o v e r o f

t h e c e l l s o f t h i s l a t t e r t i s s u e . The e x t e n t o f a l k y l a t i o n was

-9 . - 1e s t i m a t e d t o b e 933 x 10 mol 7 - m e th y lg u a n m e .m o l DNA-P.

ym * MMS.kg ^ body w e i g h t .

An a n a lo g o u s e x p e r im e n t was c a r r i e d o u t w i t h a n o th e r

14 —1a l k y l a t i n g a g e n t , d i - [ C ]m ethyl s u l p h a t e (53 0 y g ,k g , 125yCi

- 71 -

T a b le 7 - Y ie ld s and s p e c i f i c r a d i o a c t i v i t i e s o f DNA

o b t a i n e d from th e s o f t t i s s u e s o f r a t s exposed to

2 .4 mg [i^C] MMS, kg~4 body w e ig h t by i n t r a p e r i t o n e a l i n j e c t i o n

OrganY ie ld DNA

mgogm” ! f r e s h w e ig h tS p e c i f i c r a d i o a c t i v i t y

| Dpmomg” ^

B r a in 2 .1 1 111

H e a r t and lu n g 4 .4 1 4 .4 4

L i v e r 2 .0 4 1 95 .5

S p le e n 5 .0 1 2 8 .4

Kidney 1 .9 2 268

T e s t i s 2 .3 17 .37

— I Z . —

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ts

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74 -

p e r r a t ) , t h e t i s s u e d i s t r i b u t i o n i s shown i n T a b le 8 , Examin­

a t i o n o f t h e DNA h y d r o l y s a t e by ch rom a tog raphy on Dowex 50

gave th e p r o f i l e shown i n F i g . 24 . R a d i o a c t i v i t y was a s s o c i a t e d

w i t h th e n a t u r a l l y o c c u r r i n g p u r in e s as w e l l as t h e 7 -m e th y l ­

g u a n in e f r a c t i o n . Based on th e r a d i o a c t i v i t y a s s o c i a t e d w i t h

t h e 7 -m e th y lg u a n in e f r a c t i o n th e o v e r a l l e x t e n t o f m e t h y la t i o n

- 9 . - 1 - 1 - 1was 356 x 10 mol 7 -m e th y l g u a n in e mol DNA-P.ymol DMS, kg

body w e i g h t .

H aving e s t a b l i s h e d t h a t low l e v e l s o f r a d i o l a b e l l e d a l k y l ­

a t i n g a g e n ts a d m i n i s t e r e d by th e i . p . r o u te l e a d t o m e a s u r a b le

a l k y l a t i o n o f DNA, t h e i n h a l a t i o n e x p e r im e n ts w ere p e r fo rm e d .

The y i e l d s and s p e c i f i c r a d i o a c t i v i t i e s o f t h e DNA f r a c t i o n s ,

i s o l a t e d from th e i n d i v i d u a l s o f t t i s s u e p o o ls o f 10 r a t s exposed

t o an a i r c o n c e n t r a t i o n o f 0 . 6 4 y g . l ^ [ m e t h y l - ^ C ] MMS f o r 12

h o u rs a r e shown i n T ab le 9 . I o n exchange ch ro m a to g rap h y o f

t h e p o o le d DNA h y d r o l y s a t e s gave t h e e l u t i o n p r o f i l e shown in

F i g . 25 . I n c o r p o r a t i o n o f c o n s i d e r a b l e amounts o f r a d i o a c t i v e l y

l a b e l l e d c a rb o n i n t o th y m in e , g u a n in e and a d e n in e was o b t a i n e d .

T here was a l s o an i n d i c a t i o n o f r a d i o a c t i v i t y a s s o c i a t e d w i t h

t h e 7 - m e th y lg u a n in e m a rk e r p e a k s . The f r a c t i o n s c o m p r is in g

t h e 7 - m e th y lg u a n in e peak from t h e Dowex column w ere p o o le d

and d r i e d i n a s t r e a m o f d ry n i t r o g e n . A f t e r r e d i s s o l v i n g t h e

r e s i d u e i n 0.1M HC1 i t was re c h ro m a to g ra p h e d on Sephadex G -10.

The e l u t i o n p r o f i l e o b t a i n e d i n t h i s e x p e r im e n t i s shown i n

F i g . 26 . R a d i o a c t i v i t y was a s s o c i a t e d w i t h t h e 7 -m e th y lg u a n in e

p e a k as w e l l as w i t h th e a d e n in e and g u a n in e p e a k s . I n a d d i t i o n

a s m a l l amount o f r a d i o a c t i v i t y was a s s o c i a t e d w i t h t h e

- 75 -

T a b le 8 - Y i e ld and s p e c i f i c r a d i o a c t i v i t i e s o f DNA

o b t a i n e d from th e s o f t t i s s u e s o f r a t s exposed to

530ng [ 1^C]DMS kg" 1 body w e ig h t by i n t r a p e r i t o n e a l i n j e c t i o n

Organ Y ie ld DNA mg.gm f r e s h w e ig h t

S p e c i f i c r a d i o a c t i v i t y Dpm.mg“ l

B r a in • 2 .1 9 95

H e a r t and lung 4 .57 3 .1

L i v e r 1 .97 1 72 .5

S p leen 5 .2 4 21 .3

K idney 3 .1 3 140

T e s t i s 2 .07 9 .0 6

Cpm

ml-

O)

500

400

300

200

100

50 200100 150

Fraction No.

Fig 24 Dowex 50 [+ H] elution profile of the acid hydrolysate of rat soft tissue DNA alkylated in vivo with [14C}dimethyl sulphate

- 77 -

T a b le 9 - Y i e ld s and s p e c i f i c r a d i o a c t i v i t i e s o f DNA

o b t a i n e d from t h e s o f t t i s s u e s o f r a t s exposed f o r

12 h t o an a tm o sp h e re c o n t a i n i n g 0 .0 6 4 u g [-^C]MMS I"-*-

Organ Y ie ld DNA mg.gm“ l f r e s h w e ig h t

S p e c i f i c r a d i o a c t i v i t y Dpm.mg“ l

B r a in 2 .4 1 3 .1

H e a r t and lung 4 .3 5 3 .9 5

L i v e r 2 .23 1 .0 5

S p le e n : 4 .9 5 3 .1 5

K idney 3 .2 6 3 .0 4

T e s t i s 2 .2 4 0 .9 3

— / o —

O

If)

LOm in

Fig

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10 rat

s ex

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0.06

4 jug

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50 [+

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Dpm

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60

30 40 50 60 7010 20 80 90

Fraction No.

Fig 26 Rechromatography on sephadex G10 of the “ 7—Methylguanine" peak from Dowex 50 [H+ ] chromatography of the DNA from the soft tissue pool of

10 rats exposed to 0.064 jug I-1 [14C]MMS j

- 8 0 -

3 - m e th y la d e n in e p e a k . The e x t e n t o f a l k y l a t i o n p ro d u ce d i n t h i s

-9e x p e r im e n t was c a l c u l a t e d to be 2 8 .5 x 10 mol 7 - m e th y lg u a n m e .

-1 -1 -1 mol DNA-P.fjtmol MMS kg body w e i g h t .

The y i e l d s and s p e c i f i c r a d i o a c t i v i t i e s o f t h e DNA f r a c t i o n s ,

i s o l a t e d from th e i n d i v i d u a l s o f t t i s s u e p o o l s , f rom b o th th e

14f i r s t and second [m e th y l- C ] d i c h l o r v o s e x p e r im e n ts a r e shown

i n T a b le 10. These r e s u l t s show a good a g re e m e n t , w i t h r e s p e c t

t o b o th th e y i e l d s o f DNA and th e s p e c i f i c r a d i o a c t i v i t i e s o f

th e DNA, b e tw een t h e two e x p e r im e n t s . A l l o f th e s o f t t i s s u e

DNA p o o ls were found t o c o n t a i n r a d i o a c t i v i t y . I n b o th e x p e r i ­

m en ts t h e DNA from s p l e e n had th e h i g h e s t s p e c i f i c r a d i o a c t i v i t y

w h i l s t t h a t from l i v e r had t h e lo w e s t s p e c i f i c r a d i o a c t i v i t y .

The t o t a l o rg a n p o o l DNA h y d r o l y s a t e from each e x p e r im e n t

was exam ined by io n -e x c h a n g e c h ro m a to g ra p h y . The e l u t i o n p r o f i l e s

a r e shown i n F ig . 27 ( e x p e r im e n t 1) and F i g . 28 ( e x p e r im e n t 2 ) .

B o th columns r e v e a l e d r a d i o l a b e l l e d m a t e r i a l e l u t i n g w i t h t h e

v o id volum e. However, i n no c a s e was t h e r e e v id e n c e o f a s p e c i f i c

a s s o c i a t i o n o f r a d i o a c t i v i t y w i t h any u . v . - a b s o r b i n g com ponent,

e . g . 7 -m e th y lg u a n in e . The f r a c t i o n s c o m p r is in g t h e 7 -m e th y l ­

g u a n in e p e a k from th e Dowex columns were p o o le d a c c o r d in g to

th e e x p e r im e n t and d r i e d i n a s t r e a m o f d ry n i t r o g e n . A f t e r

r e - d i s s o l v i n g i n 0.1M HC1, t h e 7 -m e th y lg u a n in e was r e c h r o m a to ­

g rap h ed on Sephadex G-10. The e l u t i o n p r o f i l e s a r e shown i n

F ig . 29 (e x p e r im e n t 1) and F i g . 30 (e x p e r im e n t 2 ) . R e c o v e r ie s

o f th e 7 - m e th y lg u a n in e a p p l i e d to t h e s e columns w e re 93% and

94% r e s p e c t i v e l y . As w i t h t h e Dowex co lum ns , no r a d i o a c t i v i t y

- 81 -

T a b le 10 - Y ie ld s and s p e c i f i c r a d i o a c t i v i t i e s o f DNA o b t a i n e d

from t h e s o f t t i s s u e s o f r a t s exposed f o r 12h t o an

a tm o sp h e re c o n t a i n i n g 0 .0 6 4 u g [ m e th y l - - ^ C ] d ic h lo r v o s 1~^(E x p e rim e n ts 1 and 2 )

Organ

E x p er im en t 1 E x p e r im en t 2 jY ie ld DNA

m g.gnT1 f r e s h w e ig h t

S p e c i f i cr a d i o a c t i v i t y

Dpm.mg” -*-

Y ie ld DNA mg. gnT1

f r e s h w e ig h t

S p e c i f i c j r a d i o a c t i v i t y j

Dpm.mg“ l |

B ra in 3 .3 3 2 .3 2 3 .0 5 2 .2

H e a r t and lung 4 .9 9 1 .5 9 4 .5 1 .61

L iv e r 1 .99 0 .2 1 2 .2 0 .2 2 5

S p leen 4 .0 5 9 .8 8 3 .9 5 7 .5

Kidney 1.04 8 .8 4 1 .2 7 .8

T e s t i s 2 .0 1 0 .8 3 2 .1 0 .7 9

Gua

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Fraction No.50 100

Fig 29 Rechromatography on Sephadex G—10 of the "7—Methylguanine" fraction derived

from Dowex chromatography of DNA from [Methyl—14Cjdichlorvos treated rats(Experiment 1)

u U i_ Oloj re o> -c.■E > -E Jpt- »- “TO TT re 'SO S> o | j

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Fraction No.

Fig 30 Rechromatography on Sephadex G-IO of the "7— Methylguanine' fraction derived from Dowex chromatography of DNA from

[Methyl— 14 Cjdichlorvostreated rats (Experiment 2)

- 8 6 t

was s p e c i f i c a l l y a s s o c i a t e d w i t h t h e 7 -m e th y lg u a n in e f r a c t i o n s .

The 7 -m e th y lg u a n in e r e c o v e r e d from t h e Sephadex columns

(e x p e r im e n ts 1 and 2 . ; i n p a r t r e c o v e r e d from s c i n t i l l a t o r )

was r e p u r i f i e d by Sephadex G-10 c h ro m a to g ra p h y . The o v e r a l l

r e c o v e r i e s o f 7 - m e th y lg u a n in e , e x p r e s s e d as a p e r c e n ta g e o f

t h a t added t o th e DNA f r a c t i o n s p r i o r t o h y d r o l y s i s , w ere 72,98%

and 74,55% r e s p e c t i v e l y , c o r r e s p o n d in g t o 496mg and 650mg DNA

r e s p e c t i v e l y . F r a c t i o n s c o m p r is in g th e 7 -m e th y lg u a n in e peaks

from b o th e x p e r im e n ts w ere p o o le d , t r a n s f e r r e d t o a s c i n t i l l a t i o n

v i a l , d r i e d i n a s t r e a m o f d r y n i t r o g e n and a s sa y e d f o r t h e

p r e s e n c e o f r a d i o a c t i v i t y by r e p e a t e d 7 h o u r c o u n t s . The b l a n k

f o r t h i s p r o c e d u r e com prised an e q u i v a l e n t q u a n t i t y o f u n l a b e l l e d

7 -m e th y lg u a n in e from th e same b a t c h sam ple t h a t was added as

m ark e r to t h e o r i g i n a l DNA f r a c t i o n s , The r e s u l t s o f t h i s r a d i o ­

a s s a y a r e shown i n T a b le 11. No s i g n i f i c a n t d i f f e r e n c e was

d e t e c t e d be tw een t e s t and b l a n k sa m p le . S t a t i s t i c a l a n a l y s i s

o f t h e d a t a , u s in g a o n e - s id e d t e s t , i n d i c a t e d t h a t a d i f f e r e n c e

o f 0 ,5 3 2 c ,p ,m , would have b e e n s i g n i f i c a n t a t t h e 5%: l e v e l .

Based on t h i s f i g u r e , t h e l i m i t s o f d e t e c t i o n o f m e t h y l a t i o n

o f n u c l e i c a c id s i n t h e c u r r e n t s tu d y w ere e s t i m a t e d t o be one

11 -12 m e th y l group p e r 5 .7 x 10 n u c l e o t i d e u n i t s (< 1 ,7 9 x 10

mol 7 - m e th y lg u a n in e , mol ^ DNA-P.ymol ^ d i c h l o r v o s kg ^ body

w e i g h t ) ,

One o f th e i n t e r e s t i n g f a c e t s o f t h e above e x p e r im e n ts

was t h e i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o t h e p u r i n e r i n g .

I t seemed t h a t such i n c o r p o r a t i o n o n ly o c c u r r e d i n t h e e x p e r i ­

m ents t h a t a l s o y i e l d e d r a d i o l a b e l l e d 7 - m e th y lg u a n in e , T hese

- 87 -

T a b le 11 - Mean c o u n ts f o r t h e p o o le d 7 - m e th y lg u a n in e f r a c t i o n

i s o l a t e d from 1 . 146g DNA from s o f t t i s s u e s o f 20 r a t s exposed

t o an a tm o sp h e re c o n t a i n i n g [ m e t h y l - 1 4 c ] d i c h lo r v o s f o r 12h

Number o f Mean c o u n t C o n f id e n c e l i m i t7h r e p l i c a t e s (cpm) D « u o

(P< 0 .0 5 )

Sample 20 2 1 .1 4 ( 1 8 .2 - 2 4 .3 ) 0 .2 2 2 + 0 .4 5

B lank 20 2 1 .2 3 ( 1 8 .1 - 2 4 .6 ) 0 .2 2 2 + 0 .4 5

R e s u l t s i n p a r e n t h e s e s g iv e t h e r a n g e o f v a lu e s o b t a i n e d d u r in g r e p e a t e d c o u n t i n g .

The mean b a c kg round c o u n t o f t h e v i a l s p r i o r t o a d d i t i o n o f t e s t and c o n t r o l 7 -m e th y lg u a n in e was 20 .95 cpm ( 1 8 .3 - 2 3 .9 )

The d i f f e r e n c e be tw een th e t e s t sam ple and t h e c o n t r o l was 0 .0 9 5 cpm w i t h a s t a n d a r d e r r o r o f 0 .3 1 4 3 on 32 d e g re e s o f freedom

- 8 8 -

f i n d i n g s r a i s e d c e r t a i n d o u b ts as t o t h e n a t u r a l o c c u r r e n c e

o f t h i s b a s e i n no rm al DNA and , i n o r d e r t o p ro b e t h i s a r e a ,

-14 ip a r t i a l l y h e p a te c to m is e d r a t s w ere t r e a t e d w i th sod ium I CJ:

f o rm a te o r [ m e t h y l - ^ C ] m e th io n in e . Dowex 50 e l u t i o n p r o f i l e s

o f t h e DNA from t h e l i v e r s o f t h e s e r a t s a r e shown i n F i g s .14

31, 32 and 33 r e s p e c t i v e l y . T re a tm e n t w i t h b o th sod ium [ C j .

fo rm a te and [ m e t h y l - ^ C ] m e th i o n i n e gave r i s e to r i n g l a b e l l i n g

o f th y m in e , g u a n in e and a d e n in e . W ith t h e m e th io n in e s i g n i f i c a n t

amounts o f r a d i o a c t i v i t y w ere a l s o a s s o c i a t e d w i t h t h e 5 - m e t h y i -

c y t o s i n e m a r k e r . The f r a c t i o n s c o m p r is in g th e 7 -m e th y lg u a n in e

(m arker) peaks from th e Dowex column w ere c o n c e n t r a t e d and

w ere th e n re c h ro m a to g ra p h e d on Sephadex G-10 ( F i g s . 34 and

35 r e s p e c t i v e l y ) . No r a d i o a c t i v i t y was found t o be s p e c i f i c a l l y

a s s o c i a t e d w i t h t h e 3 - m e th y la d e n in e o r 7 -m e th y lg u a n in e m a r k e r s .

Based on t h e s p e c i f i c r a d i o a c t i v i t y o f t h e i s o l a t e d g u a n in e ( 86mCi.

mol 1) from t h e l i v e r DNA o f r a t s exposed t o sodium [ 1^ C ] fo rm a te ,

and assum ing t h a t 1 d .p .m . would h av e b e e n s i g n i f i c a n t , t h e 7- m e th y l

g u a n in e .a c c o u n te d f o r l e s s t h a n 0.00024% by w e ig h t o f t h e DNA. Based

on th e s p e c i f i c r a d i o a c t i v i t y o f g u a n in e (5 .03m C i.m ol_ 1 ) from

th e l i v e r DNA o f r a t s exposed t o [ m e t h y l - ^ C ] m e t h i o n i n e ,

7 - m e th y lg u a n in e a c c o u n te d f o r l e s s t h a n 0.0006% o f t h e DNA

by w e i g h t .

DNA, i s o l a t e d from a group o f r a t s t h a t w ere t r e a t e d14 .

n e o n a t a l l y w i th sodium [ C ] f o rm a te , gave t h e e l u t i o n p r o f i l e

shown i n F i g . 36. R a d i o l a b e l l i n g o f th y m in e , g u a n in e and a d e n in e

was e v i d e n t , b u t no r a d i o a c t i v i t y was a s s o c i a t e d w i t h t h e 7 - m e th y l ­

g u a n in e f r a c t i o n . F o l lo w in g c o n c e n t r a t i o n , r e c h ro m a to g ra p h y

1500

a>

oo

t—|tu uido

Fig

31

Dowe

x 50

[H+

] ch

rom

atog

raph

y of

the

liver

DN

A hy

drol

ysat

e of

an

adul

t fe

mal

e rat

do

sed

with

[1

4C]

form

ate

follo

win

g pa

rtial

hep

atec

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y

o

CO

JC

to

o

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Fig

32 Do

wex

50[H

+ ]

chro

mat

ogra

phy

of the

liv

er

DNA

hydr

olys

ate

of an

adul

t m

ale

rat

dose

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th

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] fo

rmat

e fo

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ing

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epat

ecto

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9U|UBnB|Aq;e|/\|—1_

8U!soiAo|Ame|/\j-g

euiuiAqj.

oo oo

t —|UJ u id o

Fig

33 DN

A is

olat

ed

from

the

soft

tissu

es

of rat

s la

belle

d wi

th

[Met

hyl—

14C

] m

ethi

onin

e fo

llow

ing

parti

al h

epat

ecto

my

10 20 30 40 50 60 70 80 90 100

Fraction No.

Fig 34 Sephadex G—10 Rechromatography of the "7 — Methylguanine fraction" from the Dowex 50 [H+ ] chromatography of the soft-tissue pool derived from 10 rats

treated neonatally with [14C] formate

Dpm

per

frac

tion

■o

100

50

40 100Fraction No.

Fig 35 Rechromatography on Sephadex G—10 of the "7—Methylguanine" fraction derived from Dowex chromatography of DNA from [Methyl—14 C] methionine-

treated partially hepatectomised rats

oIT)CM

OoCJo'

oo

L_ |iu UidQ

Fig

36 Ch

rom

atog

raph

y on

Dowe

x 50

[H+

] of

the

acid

hydr

olys

ate

of

soft-

tissu

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A de

rived

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] fo

rmat

e

- 95 -

o f t h e 7 -m e th y lg u a n in e c o n t a i n i n g f r a c t i o n s , on Sephadex G-10

gave th e p r o f i l e shown i n F i g . 37. A g a in , no r a d i o a c t i v i t y

was a s s o c i a t e d w i t h e i t h e r th e 3 - m e th y la d e n in e o r th e 7 - m e th y l -

g u a n in e f r a c t i o n s . The s p e c i f i c r a d i o a c t i v i t y o f t h e g u a n in e

i s o l a t e d from t h e s e t i s s u e s was 4.94mCi,moT ^ g u a n in e . I n t h i s

s tu d y 7-m e th y lg u a n in e a c c o u n te d f o r l e s s th a n 0.00004% o f t h e

DNA by w e i g h t .

Dpm

per

frac

tion

— y v

c n

■O

200

100

40 60 8020 100

Fraction No.

Fig 37 Rechromatography on Sephadex G—10 of the "7—Methylguanine" fraction derived from Dowex chromatography of DNA from sodium [14C]

formate treated partially hepatectomised rats

- 97 -

CHAPTER 4

M e th y la te d P u r in e s i n U r in e

4 .1 I n t r o d u c t i o n

The d e t e c t i o n o f sp o n ta n e o u s i n v iv o a l k y l a t i n g r e a c t i v i t y

o f a m e t h y l a t i n g a g e n t n e c e s s i t a t e s t h e u n e q u iv o c a l d e m o n s t r a t io n

o f non-enzym e m e d ia te d t r a n s f e r o f an i n t a c t m e th y l group from

t h e m e t h y l a t i n g a g e n t t o a t i s s u e n u c l e o p h i l e . The a n a l y t i c a l

p r o c e d u r e m ust t h e r e f o r e d i s c r i m i n a t e b e tw een sp o n ta n e o u s m e th y l

t r a n s f e r r e a c t i o n s and n a t u r a l i n c o r p o r a t i o n o f t h e p a r t i a l l y

o r c o m p le te ly d e h y d ro g e n a te d e l e c t r o p h i l i c m e th y l g roups i n t o

t a r g e t n u c l e o p h i l e s v i a one c a rb o n (1-C) p o o l s . Such d i f f e r e n t i a ­

t i o n i s m ost r e a d i l y a c h ie v e d by c h o o s in g t a r g e t s t h a t y i e l d

p r o d u c t s , e . g . 7 -m e th y lg u a n in e m o i e t i e s i n norm al mammalian

DNA, f o r w hich no n a t u r a l b i o s y n t h e t i c pathw ay e x i s t s .

T h is d e f i n i t i v e t e c h n i q u e h as b e e n employed t o d e m o n s t r a t e

t h a t t h o s e m e t h y la t i n g a g e n ts t h a t a r e e s t a b l i s h e d mammalian

m utagens o r c a rc in o g e n s d i r e c t l y m e t h y la t e a v a r i e t y o f n u c l e o -

p h i l i c c e n t r e s i n t h e DNA o f mammalian t i s s u e s i n v iv o (Swann

and M agee, 1971; C raddock , 1973; M a i t r a and F r e i , 1975; F r e i

and L aw ley , 1975; 1976 ) . W ith a v iew tow ards s i m p l i f y i n g t h e

a n a l y t i c a l p r o c e d u r e s , some s t u d i e s have r e c e n t l y b e e n c o n d u c te d

t o a s s e s s t h e u t i l i t y o f m o n i to r in g u r i n a r y e x c r e t i o n p r o d u c t s

f o r t h e d e t e c t i o n o f i n v iv o a l k y l a t i n g r e a c t i v i t y (Chu and

L aw ley , 1973, 1975a, 1975b ) . I n t h i s c o n t e x t , Lb’f r o t h and

W ennerberg (1974) and L o f r o t h e t a l (1 9 7 4 ) , have p ro p o se d t h a t

t h e e x c r e t i o n o f r a d i o l a b e l l e d 7 - m e th y lg u a n in e and 3 - m e th y la d e n in e

- 98 -

c o n s t i t u t e s e v id e n c e f o r t h e sp o n ta n e o u s a l k y l a t i o n o f g u a n in e

and a d e n in e m o i e t i e s by m e th y l l a b e l l e d d i c h l o r v o s and d i m e th y l -

s u l p h a t e i n v i v o . More r e c e n t l y Dedek e t a l (1976) have drawn

s i m i l a r c o n c lu s i o n s from a n a lo g o u s e x p e r im e n ts w i t h t r i c h l o r p h o n e

and b u t o n a t e . F o r such a c o n c lu s io n t o be v a l i d , s i g n i f i c a n t

amounts o f r a d i o a c t i v i t y m ust n o t b e i n c o r p o r a t e d i n t o t h e s e

m e th y la t e d p u r in e s v i a n a t u r a l m e t a b o l i c p a th w a y s .

In t h e e x p e r im e n ts d e s c r i b e d i n t h i s c h a p te r t h e t r a n s f e r

o f r a d i o l a b e l l e d m e th y l g roups from b i o l o g i c a l l y a c t i v e m e th y l ­

a t i n g a g e n ts and d i c h l o r v o s i s exam ined . I n a d d i t i o n t h e e x i s t e n c e

o f a n a t u r a l b i o s y n t h e t i c mechanism i s d e m o n s t ra te d w hereby

th e m e th y l c a rb o n atoms o f d i c h l o r v o s can become i n c o r p o r a t e d ,

w i t h p a r t i a l r e t e n t i o n o f h y d ro g e n , i n t o t h e h e t e r o c y c l i c r i n g s

and i n t o t h e m e th y l group o f u r i n a r y 7 -m e th y lg u a n in e a f t e r e n t e r ­

in g 1-C p o o l i n v i v o . P r e l i m i n a r y d a t a i n d i c a t i n g a s i m i l a r

pathw ay f o r 3 - m e th y la d e n in e i s a l s o p r e s e n t e d ,

4 .2 Methods

Animal e x p e r im e n ts

r l 4A l i t t e r o f n e o n a te s was dosed w i t h sod ium L C j fo rm a te

as d e s c r i b e d p r e v i o u s l y ( S e c t i o n 3 .2 ) „ U r in e was c o l l e c t e d f o r

t h r e e weeks commencing im m e d ia te ly a f t e r w e a n in g .

P a r t i a l l y h e p a te c to m is e d r a t s w ere dosed w i t h a t o t a l o f

160yCi o f sodium [ ^ C ] f o rm a te o r [methy 1 - ^ C ] m e t h i o n i n e e q u i v a l e n t

t o 194yg and 426yg r e s p e c t i v e l y . The i . p i n j e c t i o n s , each o f

10 y C i,w ere a d m i n i s t e r e d a t 9 a .m . , 12 n oon , 3 p .m . and 5 p .m .

on f o u r c o n s e c u t iv e d a y s , commencing 16 h o u rs a f t e r t h e o p e r a t i o n .

- 99 -

A d u l t r a t s w ere dosed i n t r a p e r i t o n e a l l y w i t h [ m e t h y l - ^ C ]— 1 1 /

MMS (250yC i, 1 ,57mCi .mmol ) , [m e th y l- C ] d ic h lo r v o s (250yC i,

3.25mCi.mmol ^ ) . [8- ^ C ] A denine and [8- ^ C ] g u a n i n e (56mCi,mmol

w ere a d m i n i s t e r e d to a d u l t r a t s a t a d o se l e v e l o f 50yCi p e r

r a t . 5 - [ m e t h y l - ^ C ] g l u t a t h i o n e (46mCi.mmol was i n j e c t e d i . p .

i n t o a d u l t m ale r a t s by a s e r i e s o f f o u r i n j e c t i o n s (15yC i)

a t h o u r l y i n t e r v a l s .

C o l l e c t i o n o f u r i n e and e x t r a c t i o n o f u r i n a r y p u r i n e s

In a l l o f t h e u r i n a r y p u r i n e e x p e r im e n ts c o n d u c te d w i t h

a d u l t r a t s , t h e a n im a ls w ere ho u sed s i n g l y i n a l l - g l a s s m e ta b o l i s m

cag es (M etabowl, J e n co n s L t d . ) as d e s c r i b e d by W rig h t e t a l

( 1 9 6 5 ) . These cages a r e d e s ig n e d f o r t h e s e p a r a t e c o l l e c t i o n

o f u r i n e and f a e c e s . In t h e e x p e r im e n ts w i t h a n im a ls t r e a t e d14 . .

f o r 6 weeks n e o n a t a l l y w i t h sod ium I CJ f o rm a te , t h e a n im a ls

w ere weaned a t t h r e e weeks o f age and u r i n e was c o l l e c t e d d u r in g

th e f o l l o w i n g t h r e e weeks o f t h e e x p o s u re p e r i o d . T hese a n im a ls

w ere m a in ta in e d i n p a i r s d u r in g t h e u r i n e c o l l e c t i o n . I n a l l

c a s e s t h e r a t s had f r e e a c c e s s to food and w a t e r .

U r in e was c o l l e c t e d o v e r t o l u e n e and i n th e p r e s e n c e o f

1M; s u l p h u r i c a c id (2 m l) . The c o l l e c t i n g f l a s k s w e re changed

a t 9 a .m . e v e ry day* and th e u r i n e sam ples s t o r e d a t - 2 0 ° u n t i l

r e q u i r e d .

A f t e r th a w in g , u r i n e sam ples from e a ch e x p e r im e n t w ere

f i l t e r e d t o remove any i n s o l u b l e m a t e r i a l t h a t had p r e c i p i t a t e d

d u r in g s t p r a g e . Sm all q u a n t i t i e s ( l-10m g) o f u n l a b e l l e d

x a n t h i n e , h y p o x a n th in e , a d e n in e , g u a n in e and 7 -m e th y lg u a n in e

w ere added as m ark e rs t o each u r i n e p o o l , fo l lo w e d by 20ml o f

- 100 -

1M s i l v e r n i t r a t e s o l u t i o n (C raddock and Magee, 1 9 6 7 ) , These

m ix tu r e s w ere s t o r e d f o r two days i n t h e d a rk a t room t e m p e r a t u r e ,

t o p r e c i p i t a t e t h e s i l v e r p u r i n a t e s and th e n , f o r two days in

t h e d a rk a t 4 ° t o e n s u r e c o m p le te p r e c i p i t a t i o n . The s i l v e r

p u r i n a t e s w ere h a r v e s t e d by c e n t r i f u g a t i o n and w ashed (x 3 )

w i th d i s t i l l e d w a t e r . The p u r i n e s w ere r e c o v e r e d by h y d r o l y s i s

i n 0.5M HC1 f o r 20min a t 100° . Th is h y d r o l y s i s s t e p was r e p e a t e d

t w i c e .

F r a c t i o n a t i o n o f u r i n a r y p u r in e s

The i n i t i a l c h ro m a to g ra p h ic s e p a r a t i o n o f t h e u r i n a r y p u r in e s

w a s , u n l e s s s t a t e d o t h e r w i s e , e f f e c t e d on a Dowex AG50 Wx8 (200

mesh, H+ form) column (15cm x 1.5cm o r 35cm x 1 .8 c m ) , d e v e lo p e d

w i t h a l i n e a r g r a d i e n t o f HC1 (1 t o 4M). F r a c t i o n s o f 99 d ro p s

(6 .4 m l) were c o l l e c t e d u s in g an LKB f r a c t i o n c o l l e c t o r . A l i q u o t s

( lm l) o f each f r a c t i o n w ere removed f o r t h e d e t e r m i n a t i o n o f

r a d i o a c t i v i t y . The a b s o rb a n c e o f each f r a c t i o n was d e te r m in e d

a t 254nm u s in g a Unicam SP 800 r e c o r d i n g s p e c t r o m e t e r .

I n some e x p e r im e n ts t h e r e m a in d e r o f each o f t h e e l u a n t

f r a c t i o n s from t h e Dowex 50 column c o r r e s p o n d in g t o t h e m a rk e r

7 - m e th y lg u a n in e w ere p o o le d , d r i e d i n vacuo and r e d i s s o l v e d

i n a minimum q u a n t i t y o f 0.1M HC1. T h is s o l u t i o n was t h e n a p p l i e d ,

t o g e t h e r w i th m ark e r 3 -m e th y la d e n in e and 1 -m e th y l a d e n in e , to

a Sephadex G-10 column (85cm x 1.5cm) and e l u t e d w i t h 0.05M

ammonium f o rm a te , pH 6 . 8 . I n d i v i d u a l f r a c t i o n s o f 99 d ro p s (5 .9 m l)

w ere c o l l e c t e d and t h e a b s o rb a n c e o f e a ch f r a c t i o n d e te r m in e d

as d e s c r i b e d a bove .

- 101 -

D e te r m in a t io n o f r a d i o a c t i v i t y

Samples (1ml) o f each f r a c t i o n w ere b le n d e d w i t h 10ml o f

s c i n t i l l a t o r s o l u t i o n (NE 260) and r a d i o a c t i v i t y was d e te rm in e d

i n a P a c k a rd (Model 526) l i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r .

C o u n t in g e f f i c i e n c i e s w ere d e te rm in e d by th e C hanne ls r a t i o

m ethod , and w ere i n t h e r a n g e o f 60-65%. The p r e s e n c e o f h y d r o ­

c h l o r i c a c id from t h e Dowex column had no s i g n i f i c a n t e f f e c t

on th e c o u n t in g e f f i c i e n c y .

P a p e r ch rom a tog raphy

P a p e r c h ro m a to g ra p h y was c a r r i e d o u t on Whatman 3MM p a p e r

( d e s c e n d in g deve lopm en t) i n t h e f o l l o w i n g s y s te m s . System I :

t - b u t a n o l / m e t h y l e t h y I k e t o n e / f o r m i c a c i d / w a t e r ( 4 : 3 : 1 . 5 : 1 . 5 ,

v / v ) . System I I : t -b u ta n o l /m e th y le th y I k e to n e /a m m o n iu m h y d r o x id e

( s p . g r . 0 . 8 8 ) / w a t e r ( 4 : 3 : 2 : 1 , v / v ) . System I I I : n - b u t a n o l / a c e t i c

a c i d / w a t e r ( 2 : 1 : 1 , v / v ) . System IV: p r o p a n - 2 - o l / w a t e r / l l . 6M

h y d r o c h l o r i c a c id ( 5 6 : 1 8 . 4 : 1 6 . 6 , v / v ) •

T h i n - l a y e r ch rom a tog raphy

C e l l u l o s e t h i n - l a y e r ch ro m a to g rap h y was p e rfo rm e d u s in g

t h e f o l l o w in g s y s te m s : System I : t - b u t a n o l / m e t h y l e t h y l k e t o n e /

fo rm ic a c i d / w a t e r ( 4 0 :3 0 : 1 5 :1 5 , v / v ) . System I I : m e th a n o l /

h y d r o c h l o r i c a c i d / w a t e r ( 7 : 2 : 1 , v / v ) . Sys tem I I I : t - b u t a n o l /

m e th y le th y lk e to n e /a m m o n iu m h y d ro x id e ( s p . g r . 0 . 88 ) / w a t e r

(4 : 3 : 2 : 1 , v / v ) .

- 102 ^

4.3 Results

At t h e dose l e v e l s employed i n t h i s e x p e r im e n t a l l o f

t h e MMS t r e a t e d r a t s s u r v i v e d , b u t f o u r o u t o f s i x d i c h l o r v o s

t r e a t e d r a t s showed symptoms o f a c u t e o rg a n o p h o s p h a te p o i s o n i n g

and d i e d w i t h i n 10 m in u te s o f d o s in g .

The Dowex 50 e l u t i o n p r o f i l e o f t h e u r i n a r y p u r i n e f r a c t i o n s

14 14from r a t s t r e a t e d w i t h [Me- CJMMS and [Me- C J d ic h lo r v o s a r e

shown i n F i g s . 38 and 39 r e s p e c t i v e l y . I n b o th c a s e s r a d i o a c t i v i t y

was a s s o c i a t e d w i t h m ark e r x a n t h i n e , h y p o x a n th in e , 7 -m e th y lg u a n in e

and i n t h e p o s i t i o n o f 1-m e th y la d e n in e ( t h i s m ark e r was n o t

a d d e d ) . The r a d i o a c t i v i t y r e c o v e r e d i n th e 7 -m e th y lg u a n in e f r a c t i o n

r e p r e s e n t e d 0.055% o f t h e a d m i n i s t e r e d r a d i o a c t i v i t y ( 8 .8 x

10 ® m ol. m e th y l ca rb o n ) f o l l o w i n g t r e a tm e n t w i t h [Me-"^C]MMS_8

and 0.025% o f t h e a d m i n i s t e r e d r a d i o a c t i v i t y ( 3 .7 8 x 10 m o l.14

m e th y l ca rb o n ) f o l l o w in g t r e a tm e n t w i t h [Me- C J d i c h lo r v o s .

R e -c h ro m ato g rap h y on Sephadex G-10 o f t h e p o o le d 117 -m e th y lg u a n in e

f r a c t i o n s ” from th e two s u r v i v i n g r a t s t r e a t e d w i t h [M e-^ C ]

d i c h l o r v o s i s shown i n F i g . 40 . I n a d d i t i o n t o t h e r a d i o a c t i v i t y

e l u t e d w i t h t h e v o id volume (m easu red u s in g D e x t ra n b l u e ) ,

a peak o f r a d i o a c t i v i t y was e l u t e d w i t h t h e m ark e r 1- m e t h y la d e n in e .

A t h i r d peak o f r a d i o a c t i v i t y e l u t e d m a r g i n a l l y ahead o f m a rk e r

3 - m e th y la d e n in e . T h is m a t e r i a l ( l a b e l l e d 3 -m e th y la d e n in e i n

F i g . 40) was f u r t h e r examined by t h i n - l a y e r ch ro m a to g ra p h y

on c e l l u l o s e p l a t e s . When d e v e lo p e d w i t h System s I •■arid I I I

(Rf 0 .5 6 and 0 .6 1 r e s p e c t i v e l y ) i t was i n d i s t i n g u i s h a b l e from

t h e 3 - m e th y la d e n in e m a rk e r . However, i n System I I i t r a n a h ead

o f t h e m ark e r (Rf 0 . 9 4 ) . I n t h i s p a r t i c u l a r f r a c t i o n a t i o n , t h e

U)

8UjuenB|Ai))a|/\|—i

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9000

8000

7000

6000

5 0 0 0 -

4000

3000 -

2000

1000

Fraction No.

Fig 40 Sephadex G—10 chromatography of the urinary purine fraction derived from two rats dosed with [Methyl—14C] dichlorvos

- 106 -

r e s o l u t i o n o f r a d i o a c t i v i t y i n t h e a r e a o f t h e 7 ~ m e th y lg u an in e

r e g i o n was p o o r . Thus m ark e r 7 -m e th y lg u a n in e e l u t e d e a r l i e r

th a n w ould have b een p r e d i c t e d on th e b a s i s o f t h e r e s u l t s

o f p r e v io u s f r a c t i o n a t i o n s .

R echrom atography o f t h e u r i n a r y " 7 -m e th y l g u a n in e 11 f r a c t i o n s

( i n c l u d i n g th e 1- m e th y la d e n in e f r a c t i o n ) from r a t s t r e a t e d w i t h

[Me-^C]MMS gave th e p r o f i l e shown i n F i g . 41 . I n t h i s i n s t a n c e

r 14- 1t h e r e s o l u t i o n was s l i g h t l y b e t t e r th a n w i t h th e [Me- C J d ic h lo r v o s

s t u d y . R a d i o a c t i v i t y was a s s o c i a t e d w i t h m arke r 1 - m e th y la d e n in e ,

3 -m e th y la d e n in e and 7 - m e th y lg u a n in e .

F u r t h e r i n v e s t i g a t i o n o f t h e 7 - m e th y lg u a n in e p e a k , from

Dowex 50 c h ro m a to g rap h y o f t h e u r i n a r y p u r i n e s from r a t s t r e a t e d

w i t h [Me-^C]MMS, by p a p e r ch ro m a to g rap h y i s shown i n F i g . 4 2 .

I n a d d i t i o n t o 7 -m e th y lg u a n in e , r a d i o a c t i v i t y was a s s o c i a t e d

w i t h t h e 3 -m e th y la d e n in e m a r k e r . I n o r d e r t o i n v e s t i g a t e t h e

. . . r l 4o r i g i n o f t h e 3 - m e th y la d e n m e two r a t s , p r e - l a b e l l e d w i t h [ C]

f o rm a te as p r e v i o u s l y d e s c r i b e d ( S e c t i o n 3 . 2 ) , w ere m a i n t a i n e d

on a no rm al d i e t f o r n i n e months to a l lo w th e l e v e l o f r a d i o r . ;

a c t i v i t y n o t a s s o c i a t e d w i th DNA t o d e c a y . P a p e r ch ro m a to g ra p h y

o f t h e u r i n a r y p u r i n e f r a c t i o n from t h e s e a n im a ls , a f t e r t r e a t ­

ment w i t h n o n - r a d i o a c t i v e MMS, by p a p e r c h ro m a to g rap h y gave

th e p r o f i l e shown i n F i g . 43 . R a d i o a c t i v i t y was c l e a r l y a s s o c i a t e d

w i t h t h e 7 -m e th y lg u a n in e m a rk e r , b u t l i t t l e i f a ny , was a s s o c i a t e d

w i t h t h e 3 - m e th y la d e n in e m a rk e r . The peak o f r a d i o a c t i v i t y

ru n n in g i n f r o n t o f th e 3 - m e th y la d e n in e m ark e r (R^ 0 .7 : i n s y s te m

I I I ) rem a in s t o b e i d e n t i f i e d .

Dpm

per

frac

tion

7—Methylguanine

5000

~a 73

3000

1000

10 20 30 40 50 60 70

Fraction No.

Fig 41 Sephadex G—10 chromatography of the urinary purine fraction derived from a rat dosed with [Methyl—14C]MMS

-L U O

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A n a ly s i s o f t h e DNA, from th e a n im a ls u sed i n t h e e x p e r i ’-

m ents d e s c r i b e d above , by th e m ethods d e s c r i b e d i n S e c t i o n 3*2

gave t h e p r o f i l e s shown i n F i g . 44 ( f o l l o w in g [ M e - ^ C ] d i c h l o r v o s ;

F i g . 45 ( f o l l o w in g [Me-^C]MMS) and F i g . 46 ( f o l l o w in g u n l a b e l l e d

MMS t o a p r e - l a b e l l e d r a t ) . I n t h e c a s e o f [ M e - ^ C ] d ic h l o r v o s

no r a d i o a c t i v i t y was a s s o c i a t e d w i t h t h e 7 - m e th y lg u a n in e o r

3 - m e th y la d e n in e m a r k e r s , and t h i s was c on firm ed by p a p e r c h ro m a to ­

g ra p h y . However, l a b e l l i n g o f t h e b a s e s th y m in e , g u a n in e and

a d e n in e , each o f w hich p ro v ed t o be homogenous on p a p e r c h ro m a to ­

g ra p h y , i n d i c a t e d t h e i n c o r p o r a t i o n o f t h e m e th y l c a rb o n from

d i c h l o r v o s i n t o t h e 1-C p o o l .

. L a b e l l i n g o f n a t u r a l l y o c c u r r i n g b a s e s v i a t h e 1-C p o o l

was a l s o n o te d i n t h e c a se o f [Me-^C]MMS F i g . 45 . A s m a l l

q u a n t i t y o f r a d i o a c t i v i t y was found t o b e a s s o c i a t e d w i t h t h e

7 -m e th y lg u a n in e p e a k . T h is was c a l c u l a t e d to be e q u i v a l e n t t o

483 x 10 mol.CH^.mol DNA—P.ym ol HMS.kg ̂ body .w e ig h t .

Assuming a b i o l o g i c a l h a l f - l i f e f o r 7 -m e th y lg u a n in e o f t h r e e

days would i n d i c a t e an i n i t i a l e x t e n t o f r e a c t i o n o f a p p ro x ._ q _i - i - i

230 x 10 mol.CH^.mol DNA-P.ymol MMS.kg body w e i g h t , no

3 - m e th y la d e n in e c o u ld b e d e t e c t e d . I n t h e f i n a l p r o f i l e t h a t

from th e p r e - l a b e l l e d r a t t r e a t e d w i t h u n - l a b e l l e d MMS ( F i g . 46)

r a d i o a c t i v i t y vas a g a in a s s o c i a t e d w i t h t h e 7 - m e th y lg u a n in e r e g i o n

b u t no 3 - m e th y la d e n in e was d e t e c t e d .

r 14 n .I n t h e f i r s t i n h a l a t i o n e x p e r im e n t w i t h [Me- C ] d i c h l o r v o s

a u r i n e sa m p le , u n c o n ta m in a te d w i th f a e c e s , was o b t a i n e d .

F r a c t i o n a t i o n o f t h i s sam ple p ro d u ce d th e p r o f i l e shown i n F i g .

4 7 . More t h a n 98% o f t h e r a d i o a c t i v i t y a p p l i e d to t h e column

was e l u t e d i n t h e f i r s t 15 f r a c t i o n s . No e v id e n c e was o b t a i n e d

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Cpm

ml-

- j . j . j

05tc

180

150

100

20 40 60 100 20018080 120 140 160

Fraction No.

Fig 46 Dowex 50 [+ H] elution profile of the acid hydrolysate of the soft tissue DNA from pre-labelled rats treated with 50 mg k g -1 MMS

(killed after 7 days)

15 Carrier 7—Methylguanine

2 10

inCM

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10 20 30 40 50 60 70 80 90 100Fraction No.60

50

LL 30CL

20

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10 50 100Fraction No.

Fig 47 Dowex 50 chromatogram of the urinary purine fraction from rats exposed to 0.064 jug l“ 1 [Methyl—14C]dichlorvos for 12 h

- 115 -

f o r t h e p r e s e n c e o f r a d i o a c t i v i t y a s s o c i a t e d s p e c i f i c a l l y w i t h

7 - m e th y lg u a n in e . The e a r l y e l u t i n g m a t e r i a l was i d e n t i f i e d as

p r e d o m in a n t ly d im e th y l p h o s p h a te , b u t s m a l l q u a n t i t i e s o f d e sm e th y l

d i c h l o r v o s w ere a l s o p r e s e n t ( s e e S e c t i o n 3 . 3 ) .

The r e s u l t s g e n e r a te d i n th e above s t u d i e s i n d i c a t e d t h a t

r i n g l a b e l l i n g o f t h e DNA b a s e s o c c u r r e d f o l lo w in g t h e a p p l i c a t i o n

o f m e t h y l a t i n g a g e n t s . The p a t t e r n o f l a b e l l i n g c l o s e l y f o l lo w e d

t h a t d e s c r i b e d i n S e c t i o n 3*3 . 7 -M e th y lg u a n in e i s a known com­

p o n e n t o f no rm al u r i n e , w h ereas t h e o c c u r r e n c e o f 3 - m e th y la d e n in e

i n u r i n e h a s n o t b e e n r e p o r t e d . S t u d ie s on th e n a t u r a l o c c u r r e n c e

o f t h e s e b a s e s i n u r i n e w ere t h e r e f o r e i n i t i a t e d i n o r d e r to

g a in a b e t t e r u n d e r s t a n d in g o f t h e s i g n i f i c a n c e o f t h e i r e x c r e t i o n

i n a n im a ls t r e a t e d w i t h a l k y l a t i n g a g e n t s .

The Dowex 50 e l u t i o n p r o f i l e s o f t h e u r i n a r y p u r i n e s o b t a i n e d

r 14 • 14from r a t s dosed w i t h [8- C ]a d e n in e and [8- C ]g u a n in e a r e shown

i n F i g s . 48 and 49 r e s p e c t i v e l y . I n b o th c a se s r a d i o a c t i v i t y

was a s s o c i a t e d w i t h t h e x a n t h i n e , h y p o x a n th in e , g u a n in e and

147 -m e th y lg u a n in e m a r k e r s . In t h e c a se o f L8- C ] a d e n in e b u t n o t

r 14 nw i t h L8- C ]g u a n in e , r a d i o a c t i v i t y was a s s o c i a t e d w i t h t h e

u r i n a r y a d e n in e f r a c t i o n . R echrom atography on Sephadex G-10

o f th e r a d i o l a b e l l e d 7 -m e th y lg u a n in e f r a c t i o n i s o l a t e d by Dowex

r 14chrom atog raphy o f th e u r i n a r y p u r in e s from L8- C ja d e n in e t r e a t e d

r a t s , r e v e a l e d t h e p r e s e n c e o f a s i g n i f i c a n t q u a n t i t y o f r a d i o ­

a c t i v i t y a s s o c i a t e d w i th th e 1 - m e th y la d e n in e , 3 -m e th y la d e n in e

and 7 -m e th y lg u a n in e m ark e rs ( F ig . 5 0 ) . E x a m in a t io n o f f r a c t i o n s

15-45 from t h i s e l u t i o n p r o f i l e by p a p e r c h ro m a to g rap h y ( s y s te m

I I I ) gave th e p r o f i l e shown i n F i g . 5 1 . A gain r a d i o a c t i v i t y

Cpm

— J L J -U —

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8000

7000

6000

5000

4000

3000

2000

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10 20 30 40 50 60 70 80 90 100 110

Fraction No.

Fig 48 Urinary purine profile of two male rats following a single i.p. injection of 50 /zCi [8—14C] adenine

d>Cc

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Fraction No.

Fig49 Urinary purine profile of two male rats following a single i.p. injection of 50 juCi [8—14C] guanine

Dpm

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4000

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3000

2000

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10 20 30 40 50 60 70 80 90 100Fraction No.

Fig 50 Rechromatography on SephadexG-10 of the 7—Methyl guanine fraction from a Dowex 50( H + ) chromatogram of rat urine following

i.p. injection of [8-1'4Cj adenine

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was a s s o c i a t e d w i t h 1 - m e th y la d e n in e , 3 - m e th y la d e n in e and 7 -m e th y l -

g u a n in e m a r k e r s . E x a m in a t io n o f th e s o f t t i s s u e DNA h y d r o l y s a t e

f rom a n im a ls u se d i n t h e above s tu d y ( F ig . 52) f a i l e d to r e v e a l

any r a d i o a c t i v i t y s p e c i f i c a l l y a s s o c i a t e d w i t h th e 7 -m e th y lg u a n in e

o r 3 - m e th y la d e n in e f r a c t i o n s . However, a d e n in e and g u a n in e w ere

l a b e l l e d .

Thus i t seemed t h a t b o th 3 - m e th y la d e n in e and 7 - m e th y lg u a n in e

c o u ld o c c u r n a t u r a l l y i n r o d e n t u r i n e . I f t h i s w ere th e c a s e , t h e n

th e y m ust have b e e n s y n t h e s i s e d by m e t h y l a t i o n o f th e p r e - f o rm e d

p u r i n e . A s e r i e s o f e x p e r im e n ts w i t h f o rm a te and m e th io n in e

( n a t u r a l c o n t r i b u t o r s t o t h e 1-C p o o l ) w ere t h e r e f o r e p e r fo rm e d .

Two model s y s te m s , d e s ig n e d to examine th e u r i n a r y p u r i n e l e v e l

d u r in g r a p i d c e l l d i v i s i o n , w ere u s e d . I n t h e f i r s t , u r i n a r y

14p u r in e s from n e o n a t a l r a t s i n j e c t e d w i t h sodium [ C ] f o r n a t e

( a s d e s c r i b e d i n S e c t i o n 3 .2 ) was exam ined by io n -e x c h a n g e

ch rom a tog raphy on Dowex 50 . T h is p r o f i l e i s shown i n F i g . 53 .

R a d i o a c t i v i t y was a s s o c i a t e d w i t h th e 7 -m e th y lg u a n in e and g u a n in e ,

and , i n a d d i t i o n , r a d i o a c t i v i t y e l u t e d i n t h e a r e a o f 1- m e t h y l ­

a d e n in e . R echrom atography o f t h e p o o l o f f r a c t i o n s 145-180 on

Sephadex G-10 r e a f f i r m e d th e d i s t r i b u t i o n o f r a d i o a c t i v i t y i n

t h e s e b a s e s ( F ig . 5 4 ) . I n t h e seco n d s y s te m , p a r t i a l l y h e p a -

14te c t o m i s e d r a t s , t r e a t e d w i t h e i t h e r [m e th y l - C jm e th io n in e o r

14sodium [ C jfo rm a te gave r i s e to r a d i o l a b e l l e d 7 -m e th y lg u a n in e

i n t h e i r u r i n e ( F i g s . 55 and 56 r e s p e c t i v e l y ) . The p r e s e n c e o f

3 - m e th y la d e n in e was c o n f i rm e d by p a p e r c h ro m a to g ra p h y .

R a d i o a c t i v i t y from th e 1-C p o o l c a n t h e r e f o r e b e i n c o r p o r a t e d

i n t o t h e u r i n a r y 7 - m e th y lg u a n in e f r a c t i o n and p o s s i b l y 3 - m e th y l ­

a d e n in e f r a c t i o n . O rganophosphorus e s t e r s and a l k y l a t i n g a g e n t s

b o t h g iv e r i s e to S - m e t h y l g l u t a t h i o n e d u r in g t h e i r m e t a b o l i c

— JL Z .1

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500

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Fig 53 Dowex 50 [* H] chromatography of the urinary purine fraction from 10 rats treated neonatally with [14C]formate (—3 week urine sample)

Dpm

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400

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20 30 40 50 60 7010 90

Fraction No.

Fig 54 Sephadex G10 chromatogram of the 7—Methylguanine fraction from the Dowex 50 [H+ ] separation of urinary purine fraction from rats treated

neonataliy with [14C]formate

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Fraction IMo.

Fig 56 Dowex 50[H+ ] chromatogram of the urinary purine fraction derived from 1 male and 1 female rat dosed with [14C]formate following partial hepatectomy

- 126 -

d e g ra d a t io n * The m e th y l group o f m a t e r i a l when i n j e c t e d i n t o

r a t s i s l a r g e l y e x c r e t e d as CO^ (H o l l in g w o r th , 1970)* I n t r a -

p e r i t o n e a l i n j e c t i o n o f S - m e t h y l g l u t a t h i o n e (60yCi) i n t o m ale

14r a t s gave r i s e t o t h e e x c r e t i o n o f 65% o f t h e do se as CO^

w i t h i n t h e f i r s t t h r e e d a y s . A n a ly s i s o f t h e u r i n a r y p u r in e

f r a c t i o n gave t h e e l u t i o n p r o f i l e shown i n F i g . 57 . R ad io ­

a c t i v i t y was a s s o c i a t e d w i t h th e 7 -m e th y lg u a n in e f r a c t i o n w hich

when examined by p a p e r c h ro m a to g ra p h y , w ere a l s o found t o con­

t a i n r a d i o a c t i v i t y ru n n in g w i t h t h e 1- m e th y la d e n in e m a rk e r ,

b u t n o t w i t h t h e 3 - m e th y la d e n in e m a rk e r . E x a m in a t io n o f t h e

s o f t t i s s u e DNA from t h i s e x p e r im e n t ( F ig . 58) r e v e a l e d l a b e l ­

l i n g o f th y m in e , g u a n in e and a d e n in e m o i e t i e s v i a t h e 1-C p o o l .

The 113 - m e th y la d e n in e " p ro d u ce d i n t h e above s t u d i e s w ere

h e a v i l y d i l u t e d w i t h m ark e r 3 - m e th y la d e n in e and w ere t h e r e f o r e

n o t s u i t a b l e f o r mass s p e c t r o m e t r y , and t h e t im e a v a i l a b l e

d id n o t p e r m i t t h e c o l l e c t i o n o f s u f f i c i e n t m a t e r i a l t o o b t a i n

a p o s i t i v e i d e n t i f i c a t i o n . However, t h e mass s p e c t r a l c o r r e l a t i o n s

shown i n F i g . 59 w ere o b t a i n e d w i th a r a n g e o f m e th y la t e d

d e r i v a t i v e s o f a d e n in e . The s p e c t r a o f t h e s e r e f e r e n c e s t a n d a r d s

showed s u f f i c i e n t i n d i v i d u a l d i f f e r e n c e s t o a l lo w t h e i r p o s i t i v e

mass s p e c t r o m e t r i c i d e n t i f i c a t i o n .

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Fig 59 Mass spectra of adenine and methylated adenines (8 most intense peaks are indicated)

- 130 -

CHAPTER 5

DNA S t r a n d Damage

5 .1 I n t r o d u c t i o n

Damage to DNA, p ro d u c e d b y c h e m ica l o r p h y s i c a l a g e n t s ,

i s r e p a i r a b l e i n m ost ty p e s o f c e l l . D u r in g th e e x c i s i o n o f

th e damaged r e g i o n a " p a t c h " o f up t o 100 b a s e s i s removed

(R o b e r ts e t a l , 1971; C le a v e r , 197 5 ) , . and r e p l a c e d u s i n g th e

undamaged s t r a n d as th e t e m p la t e . S i n g l e - s t r a n d b r e a k s i n th e

DNA w i l l h av e a t r a n s i e n t e x i s t e n c e d u r in g th e e x c i s i o n r e p a i r

p r o c e s s . The p r e s e n c e o f t h e s e b re a k s may be d e t e c t e d by

u s in g a l k a l i n e s u c r o s e g r a d i e n t s (McGrath and W i l l i a m s , 1966)

t o d e te r m in e t h e m o le c u la r w e ig h t o f s i n g l e - s t r a n d DNA.

L e t t e t a l (1967) employed t h i s t e c h n iq u e t o m o n i to r X -ra y

damage t o DNA i n mammalian c e l l s i n c u l t u r e . More r e c e n t l y '

t h e t e c h n iq u e has b een a p p l i e d t o m easure DNA s t r a n d damagej

i n v iv o (Cox e t a l , 1973a; Dam jarov e t a l , 197 3 ) .

I n t h i s C h a p te r t h e r e s u l t s o f a s e r i e s o f e x p e r im e n ts

a r e d e s c r i b e d i n w h ich t h i s t e c h n iq u e has b een a p p l i e d t o mea­

s u r e t h e c o m p a ra t iv e r e a c t i v i t y o f an o rg an o p h o sp h o ru s t r i e s t e r

and MMS. The t r i e s t e r chosen f o r t h e s e s t u d i e s was d im e th y l -

n a p h th y lp h o s p h a te w h ic h l i k e d i c h l o r v o s h a s p ro v e n a l k y l a t i n g

r e a c t i v i t y i n 4 - ( 4 - n i t r o b e n z y l ) p y r i d i n e t e s t sy s te m (B e d fo rd

and R o b inson , 1 9 7 2 ) . However, u n l i k e d i c h l o r v o s , t h i s model

compound does n o t have th e added c o m p l ic a t io n o f t h e p o s s i b l e

r e a c t i v i t y o f a v i n y l g r o u p in g .

- 131 -

5 .2 Methods

L a b e l l i n g o f l i v e r DNA

3L i v e r DNA was l a b e l l e d by i n j e c t i n g [m e th y l- H ] th y m id in e

d u r in g th e p e r i o d o f maximum DNA s y n t h e s i s f o l l o w in g p a r t i a l

h e p a te c to m y . A t o t a l o f 500yCi o f r a d i o l a b e l l e d th y m id in e

was a d m i n i s t e r e d t o each r a t i n a s e r i e s o f f o u r h o u r l y i . p .

i n j e c t i o n s ( ea c h o f 5 0 y C i ) , commencing 16 t o 17 h o u r s a f t e r

t h e o p e r a t i o n .

These r a t s w ere u t i l i s e d f o r s t r a n d damage e x p e r im e n ts

a f t e r a minimum r e c o v e r y p e r i o d o f 14 d a y s , by w hich t im e th e

l i v e r h a d r e t u r n e d t o i t s q u i e s c e n t s t a t e (B u c h e r , 1 9 6 3 ) . At

t h i s t im e th e l e v e l s o f a c i d s o l u b l e r a d i o a c t i v i t y i n t h e l i v e r

had f a l l e n to i n s i g n i f i c a n t l e v e l s i . e . l e s s t h a n 0 . 1% o f

t o t a l r e c o v e r a b l e r a d i o a c t i v i t y (Wooder and Holm es, 1975;

u n p u b l i s h e d o b s e r v a t i o n s ) .

L a b e l l i n g o f l i v e r p r o t e i n and RNA

-14 nL i v e r p r o t e i n s w ere l a b e l l e d w i t h [ C j l e u c i n e two weeks

_ 14 i ,a f t e r p a r t i a l h e p a te c to m y . The L C J l e u c in e was a d m i n i s t e r e d

by i . p . i n j e c t i o n a t a dose l e v e l o f lOOyCi p e r r a t e v e ry h o u r

f o r f o u r h o u r s . Anim als w ere k i l l e d by d e c a p i t a t i o n one h o u r

a f t e r t h e l a s t i n j e c t i o n .14 .

RNA was l a b e l l e d w i th [ C ] o r o t i c a c i d a d m i n i s t e r e d by

i . p . i n j e c t i o n commencing 19 h o u rs a f t e r p a r t i a l h e p a te c to m y .14

Each r a t r e c e iv e d lOOyCi o f I C ] o r o t i c a c id e v e ry h o u r f o r

f o u r h o u r s . The a n im a ls w ere k i l l e d b y d e c a p i t a t i o n 30min a f t e r

t h e l a s t i n j e c t i o n .

- 132 -

T re a tm e n t o f a n im a ls

MMS was d i l u t e d w i t h 0.9% NaCl s o l u t i o n (1ml) and a d m in i -

. . . -1 -1s t e r e d im m e d ia te ly by i . p . i n j e c t i o n a t 30mg.kg ; 60mg.kg ;- 1 - i

120mg.kg and 200mg.kg body w e i g h t . The a n im a ls w ere k i l l e d

by d e c a p i t a t i o n 1 , 4 o r 16 h o u rs a f t e r a d m i n i s t r a t i o n o f t h e

MMS. The v e h i c l e c o n t r o l f o r t h e s e e x p e r im e n ts was 0.09% NaCl

( l m l ) . I n a d d i t i o n , t h e r e l e v a n c e o f t h e m ethy l group i n t h i s

r e a c t i o n was i n v e s t i g a t e d u s in g m ethane s u lp h o n i c a c i d ad m in i­

s t e r e d as a s a l i n e s o l u t i o n ( lm l) by th e i . p . r o u t e a t 500 and

lOOOmg.kg ^ body w e ig h t .

R a ts w ere i n j e c t e d w i t h a s o l u t i o n o f d im e th y ln a p h th y lp h o s -

p h a te i n c o m o i l (0 .3 m l) a t dose l e v e l s o f lOOmg p e r k g ,

500mg p e r kg and lOOOmg p e r kg body w e i g h t . The r a t s i n t h e

lOOmg p e r kg and 500mg p e r kg g roups w ere k i l l e d by d e c a p i t ­

a t i o n a t 1, 4 and 16 h o u r s , and t h o s e i n t h e lOOOmg p e r kg

group a t 1 and 4 h o u r s a f t e r a d m i n i s t r a t i o n o f t h e compound.

The c o n t r o l e x p e r im e n ts f o r t h e s e s t u d i e s w ere p e r fo rm e d u s in g

0 . 3 - 1 . 0ml o f c o m o i l .

P r e p a r a t i o n o f l i v e r c e l l s u s p e n s io n

A f t e r d e c a p i t a t i o n , th e l i v e r was r a p i d l y removed from

t h e c a r c a s s . A 2g sam ple o f t h e l i v e r was p la c e d i n t o a s m a l l

b e a k e r w i th 2ml o f i c e - c o l d h o m o g en is in g medium (0.024M Na£

EDTA; 0.075M NaCl (pH 7 . 5 ) . The t i s s u e was g e n t l y s q u a sh e d

w i t h t h e b l u n t - e n d o f a s p a t u l a and th e r e s u l t i n g s u s p e n s i o n

c e n t r i f u g e d , f o r lm in a t 200 r e v /m in i n a MSE m in o r bench

c e n t r i f u g e , t o s e d im e n t th e c o n n e c t iv e t i s s u e . D i l u t i o n o f th e

s u p e r n a t a n t 1 :1 0 w i t h i c e - c o l d h o m o g en is in g medium y i e l d e d

- 133 -

t h e c e l l s u s p e n s io n f o r g r a d i e n t a n a l y s i s ( e q u i v a l e n t to

2 .0 - 1 0 y g DNA p e r 1 .0 m l) .

S e d im e n ta t io n a n a l y s i s

L i n e a r a l k a l i n e s u c r o s e g r a d i e n t s (5ml; 5-20%; c o n t a i n i n g

0.9M NaCl and 0.3N NaOH) w ere p r e p a r e d o v e r a lm l s h e l f o f

2.3M s u c r o s e i n 16ml p o ly p ro p y le n e c e n t r i f u g e tu b e s (MSE).

The s u c r o s e s o l u t i o n s u se d t o p r e p a r e t h e g r a d i e n t w ere t r e a t e d

w i t h d i e t h y l p y r o c a r b o n a t e t o d e s t r o y n u c l e a s e a c t i v i t y . G r a d ie n t s

w ere p r e p a r e d u s in g a m o d i f ie d T e c h n ic o n A u to a n a ly s e r pump

( F ig . 6 0 ) . The sh ap e o f t h i s g r a d i e n t , as m easu red i n term s

o f r e f r a c t i v e in d e x i s shown i n F i g . 61.

Onto each g r a d i e n t was c a r e f u l l y l a y e r e d 0 .3m l o f l y s i n g

s o l u t i o n (0.3M NaOH, 0.03M EDTA, 0.1M T r is -H C l b u f f e r and

0.5% sodium d o d e c y l s u l p h a t e , pH 1 0 ) . To t h i s was added 0 .1m l

o f c e l l s u s p e n s io n ( e q u i v a l e n t t o 0 . 2 - 1 .0 y g DNA) a nd , to e n s u re

c o m p le te l y s i s , a f u r t h e r 0 . 1ml o f l y s i n g s o l u t i o n was added

( F ig . 62) (Cox e t a l , 1973a). A f t e r f i l l i n g t h e r e m a in d e r o f

t h e tu b e w i t h l i q u i d p a r a f f i n , t h e tu b e s w ere c e n t r i f u g e d a t

1 1 0 ,000 x g f o r 45min a t 20° i n an MSE SS75 r e f r i g e r a t e d

c e n t r i f u g e e q u ip p e d w i t h a 6 x 15ml a lum in ium s w in g -o u t r o t o r .

I n a l l c a s e s t h e c e n t r i f u g e was d e c e l e r a t e d t o a s to p w i t h o u t

t h e u se o f th e b r a k e .

Each g r a d i e n t was f r a c t i o n a t e d i n t o 48 f r a c t i o n s e ach

o f 10 d ro p s by upward d i s p la c e m e n t w i t h 2.5M s u c r o s e . The

f r a c t i o n a t i o n a p p a r a tu s c o n s i s t e d o f a MSE d i s p l a c e m e n t s t a n d

a ssem b ly i n c o n ju n c t io n w i t h an LKB 1200 V a r io - P e r p e x p e r i ­

s t a l t i c pump ( F ig . 6 3 ) . The f r a c t i o n s w ere c o l l e c t e d d i r e c t l y

i n t o s c i n t i l l a t i o n v i a l s .

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MSE 15 ml polyproplene centrifuge tube

Liquid paraffin

0.1 ml lysing solution

0.1 ml of sample 0.3 ml of lysing solution

5% w/v to 20% w/v linear alkaline sucrose gradient (5 ml)

Scale 5 mm : 1 cm 2.3M sucrose cushion (1 ml)

Fig 62 Centrifuge tube and contents before centrifugation

Bub

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- 138 -

D e te r m in a t io n o f r a d i o a c t i v i t y

Each f r a c t i o n was b le n d e d w i t h 10ml o f s c i n t i l l a t o r

m ix tu r e (NE 260)„ The q u a n t i t y o f r a d i o a c t i v i t y p r e s e n t i n

each f r a c t i o n was th e n d e te rm in e d by l i q u i d s c i n t i l l a t i o n

c o u n t in g i n a P a c k a rd T r i c a r b 2450 s p e c t r o m e t e r . Such sam ples

3r e g u l a r l y c o u n te d w i t h an e f f i c i e n c y f o r [ H] o f 38 to 40%

as d e te rm in e d by th e C hannels r a t i o m ethod .

I n v e s t i g a t i o n o f t h e r a p i d l y s e d im e n t in g m a t e r i a l

In an e x p e r im e n t u s in g r a t l i v e r c e l l s l a b e l l e d w i t h

3[ H ] th y m id in e , t h e r a p i d l y s e d im e n t in g m a t e r i a l i s o l a t e d from

20 a l k a l i n e s u c r o s e g r a d i e n t s was p o o le d and d i a l y s e d f o r

16 h o u rs a g a i n s t 2% aqueous sodium a c e t a t e c o n t a i n i n g 1.5%

sod ium c h l o r i d e . The t o t a l volume was th e n re d u c e d by f o r c e d

d i a l y s i s a g a i n s t p o l y e t h y l e n e g l y c o l (> 220 ,000 m .w t ) . A f t e r

a d j u s t i n g th e t o t a l vo lum e, t o a h a l f o f t h e o r i g i n a l volume

o f c e l l s u s p e n s io n a p p l i e d t o t h e g r a d i e n t , w i t h 0.01M

p h o s p h a te b u f f e r pH 7 . 5 , t h e m a t e r i a l was d i v id e d i n t o f o u r

e q u a l f r a c t i o n s . A f t e r t h e a d d i t i o n o f an e q u a l volume o f

b u f f e r , f r a c t i o n one was i n c u b a t e d a t 37° f o r 1 h o u r . To

f r a c t i o n s two, t h r e e and f o u r was added an e q u a l volume o f

e i t h e r p r o n a s e (lm g.m l \ p r e v i o u s l y a u t o d i g e s t e d S e c t i o n 3 .2 )

r i b o n u c l e a s e (lm g.m l \ p r e v i o u s l y h e a t e d t o d e s t r o y deoxy­

r i b o n u c l e a s e , S e c t i o n 3 .2 ) o r d e o x y r ib o n u c le a s e (lm g.m l ^ i n

b u f f e r c o n t a i n i n g 0.015M MgCl^) and th e m ix tu re s i n c u b a t e d

a t 37° f o r 1 h o u r . At t h e end o f t h e i n c u b a t i o n p e r i o d , sam p les

o f t h e f o u r f r a c t i o n s w ere exam ined , i n d u p l i c a t e , by a l k a l i n e

g r a d i e n t c e n t r i f u g a t i o n . The r e m a in d e r o f e a c h f r a c t i o n was

- 139 -

th e n exam ined f o r r e t e n t i o n on m i l l i p o r e c e l l u l o s e a c e t a t e

f i l t e r s ( i . e . h ig h m o le c u la r w e ig h t DNA) e l u t e d w i t h 0«05M

p h o s p h a te b u f f e r pH 7 .6 (Kohn e t a l , 1 9 7 6 ) .

R e p a i r o f s i n g l e - s t r a n d damage

To fo l lo w t h e t im e c o u r s e o f th e i n v iv o rem ova l o f

s i n g l e - s t r a n d damage ( r e p a i r ) a group o f e i g h t r a t s w ere

i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 60mg MMS p e r kg body w e i g h t .

T h is d ose was chosen b e c a u s e , i n a d d i t i o n t o p r o d u c in g a

m e a s u ra b le d e g re e o f damage, i t was c o n s i s t e n t w i t h t h e

s u r v i v a l o f t h e a n im a ls . P a i r s o f a n im a ls w ere exam ined f o r

l i v e r DNA damage a t 4 , 16, 25 and 48 h o u r s a f t e r d o s in g .

5 .3 R e s u l t s

A l k a l i n e s u c r o s e g r a d i e n t p r o f i l e s o f ly s e d l i v e r c e l l s

d e r i v e d from r a t s p r e - l a b e l l e d w i t h [m e th y l- H ] th y m id in e

i n d i c a t e d t h a t t h e b u l k o f th e r a d i o a c t i v i t y a s s o c i a t e d w i t h

th e DNA was c o n c e n t r a t e d i n a compact peak a t t h e i n t e r f a c e

o f t h e 5-20% g r a d i e n t and th e 2.3M s h e l f ( F ig . 6 4 ) . E l u t i o n

_ 14p r o f i l e s o f l i v e r c e l l l y s a t e s from r a t s t r e a t e d w i t h [ CJ

r l4 n .o r o t i c a c id o r L C J le u c in e a r e shown m F i g s » 65 and 66

r e s p e c t i v e l y . The r a d i o a c t i v i t y a s s o c i a t e d w i t h b o t h t h e RNA

and t h e p r o t e i n was l o c a t e d i n t h e u p p e r r e g io n s o f t h e g r a d i e n t

and d id n o t o v e r l a p w i t h th e c o n t r o l DNA p e a k . T h e r e f o r e n e i t h e r

p r o t e i n n o r RNA s h o u ld s i g n i f i c a n t l y a f f e c t t h e q u a n t i t a t i o n

o f t h e DNA p e a k . T re a tm e n t o f t h e r a p i d l y s e d im e n t in g m a t e r i a l

w i t h p r o n a s e and r i b o n u c l e a s e (b o th p r e t r e a t e d as i n S e c t i o n 3 . 2 )

c a u se d no s i g n i f i c a n t change i n s e d im e n ta t i o n c h a r a c t e r i s t i c s .

— 1 4 U —

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Fig 66 Alkaline sucrose gradient profile of rat liver cells labelled with [14C] —Leucine

- 143. t*

I n c o n t r a s t d e o x y r ib o n u c le a s e cau sed a s h i f t t o t h e to p o f

th e g r a d i e n t (T a b le 1 2 ) .

T re a tm e n t o f r a t s w i th MMS p ro d u ce d a b r o a d e n in g o f

t h e DNA peak accom panied by a change i n i t s p o s i t i o n i n t h e

g r a d i e n t , i n d i c a t i v e o f th e f o rm a t io n o f a h e te r o g e n o u s popu­

l a t i o n o f p o l y n u c l e o t i d e m a t e r i a l . T h is e f f e c t was dose

r e l a t e d i n th e r an g e 30-200mg MMS p e r kg body w e ig h t ( F i g . 6 7 ) .

At t h e low er dose t h e e f f e c t was m a n i f e s t e d i n a ' b i p h a s i c d i s t ­

r i b u t i o n o f r a d i o a c t i v i t y , p a r t o f w hich s e d im e n te d w i t h th e

c h a r a c t e r i s t i c s o f c o n t r o l DNA. At t h e h i g h e r d o s e , 200mg MMS

p e r kg body w e ig h t a co m p le te r e d u c t i o n i n m o le c u la r w e ig h t

was s e e n . Th is d e g re e o f damage p ro v ed t o be t h e maximum t h a t

c o u ld be d e t e c t e d w i th t h e s e g r a d i e n t s . Thus i n c r e a s e s i n

do se above 200mg MMS p e r kg body w e ig h t w h i l s t a c u t e l y t o x i c

t o th e r a t s , f a i l e d t o p ro d u c e any f u r t h e r s h i f t i n t h e p o s i t i o n

o f t h e r a d i o a c t i v e p e a k . M e th a n e s u lp h o n ic a c i d , t h e d e m e t h y la t i o n

p r o d u c t o f MMS, f a i l e d to p ro d u c e m e a s u r a b le s t r a n d damage

a t do se l e v e l s up t o lOOOmg MMS p e r kg body w e ig h t and f o r

e x p o s u re p e r i o d s o f up t o 16 h o u r s . The f u l l r a n g e o f r e s u l t s

a r e g iv e n i n T a b le 13.

The r e v e r s i b i l i t y o f t h i s damage, t o l i v e r c e l l DNA,induced

by MMS, was shown u s in g g roups o f r a t s exposed t o 60mg MMS

p e r kg body w e ig h t . At f o u r h o u r s a f t e r d o s in g t h e p r e s e n c e

o f damage was c l e a r l y v i s i b l e , b u t t h i s d i s a p p e a r e d w i t h t im e

u n t i l a t 48h no d i f f e r e n c e be tw een t r e a t e d and c o n t r o l a n im a ls

was d i s c e r a a b l e . F i g s . 68-71 show p o i n t s i n t h e t im e c o u r s e

o f t h i s " r e p a i r p r o c e s s " . As ju d g e d by e x a m in a t io n o f s i m i l a r l y

- 144 -

T a b le 12 - N a tu re o f t h e R a p id ly S e d im e n t in g M a t e r i a l

T re a tm e n tD e v i a t i o n from c o n t r o l

on a l k a l i n e g r a d i e n tR e ta in e d on

M i l l i p o r e f i l t e r

P ro n a s e N e g a t iv e P o s i t i v e

RNAse N e g a t iv e P o s i t i v e

DNAse P o s i t i v e N e g a t iv e

Frac

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0.4

0.2

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(ii) 30 mg kg 1 MMS .

0.1

10 20 30 40Fraction No.

Fig 67 Alkaline sucrose gradient profile of liver cell DNA from rats treated with MMS

Frac

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0.1

0.05

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(v) 200 mg kg 1 MMS

0.1

20 3010 40Fraction No.

Fig 67 cont.

- 147 -

T a b le 13 - E f f e c t o f M ethy l M e th a n e s u lp h o n a te and D im e th y l-

n a p th y lp h o s p h a te on t h e I n t e g r i t y o f L i v e r C e l l DNA i n v iv o

Compound Dose R oute E xposuret im e E f f e c t

1 ml kg” ^ i ®P ° 4 h N e g a t iv e

S a l i n e1 ml kg“ ̂

1 ml kg” l

O ra l

i . p .

4 h

16 h

N e g a t iv e

N e g a t iv e

1 ml kg“ l O ra l 16 h N e g a t iv e

30 mg kg” ^' i . p . ( s a l i n e ) 4 h P o s i t i v e

MMS 60 mg kg” f i . p . ( s a l i n e ) 4 h P o s i t i v e

120 mg kg-1 i . p . ( s a l i n e ) 4 h P o s i t i v e

200 mg kg” f i . p . ( s a l i n e ) 4 h P o s i t i v e

MMS 120 mg kg” l O ra l 4 h P o s i t i v e

M e th a n e s u lp h o n ic 500 mg kg” ^ X o P e ( s a l i n e ) 1 , 4 , 16 h N e g a t iv e

a c id 1000 mg k g " ! i . p . ( s a l i n e ) 1 , 4 , 16 h N e g a t iv e

D im e th y l- 100 mg kg” -*- i . p . Corn o i l 1 , 4 , 16 h N e g a t iv e

n a p h t h y l - 500 mg k g ” l i . p . Corn o i l 1 , 4 , 16 h N e g a t iv e

p h o s p h a te 1000 mg kg” l i . p . Corn o i l 1 , 4 h N e g a t iv e

Corn o i l 1 mg kg 1 i . p . 1 , 4 , 16 h N e g a t iv e

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- 152 _

t r e a t e d a n im a ls th e r e p a i r was s t a b l e f o r a t l e a s t t h r e e weeks

(Wooder, 1976, u n p u b l i s h e d o b s e r v a t i o n s ) .

D im e th y ln a p h th y lp h o s p h a te a t dose l e v e l s o f 100 and 500mg

p e r kg body w e ig h t ca u se d no m e a s u ra b le DNA damage a t 1, 4

o r 16 h o u rso At a dose l e v e l o f lOOOmg p e r kg body w e i g h t ,

w h ich ca u se d d e a th w i t h i n 12 h o u r s o f d o s in g , no damage was

d e t e c t a b l e a t 1 o r 4 h o u r s 0 FigSo 72 and 73 show t y p i c a l

a l k a l i n e g r a d i e n t p r o f i l e s o f l i v e r c e l l DNA f o l lo w in g a d m in i­

s t r a t i o n o f d i m e th y ln a p th y lp h o s p h a te , The f u l l r a n g e o f th e

r e s u l t s o b t a i n e d a r e shown i n T a b le 1 3 . No a t t e m p t was made

i n any o f t h e above s t u d i e s t o d e te r m in e t h e m o le c u la r w e ig h t

o f th e DNA s t r a n d s 0

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- 155 -

CHAPTER 6

D i s c u s s i o n

6 °1 D i r e c t i n t e r a c t i o n s w i th . DNA

Assuming t h a t a l l o f t h e r a d i o a c t i v i t y i n t h e 7-m e th y 1 -

g u a n in e f r a c t i o n , was a s s o c i a t e d w i t h t h e -methyl g ro u p , t h e

e x t e n t s o f m e t h y l a t i o n . a t W-7 o f DNA g u a n in e m o i e t i e s i n

g roups o f r a t s e x p o s e d . i n t r a p e r i t o n e a l l y t o 24yg and 2,4mg

r l 4 .. . - 9 - 9[ C]MMS p e r kg body w e ig h t w ere 387 x 10 and 511 x 10 mol

- 1 - 1 - 1 .m ethylom ol oDNA-P8ymol MMS,kg body w e i g h t , r e s p e c t i v e l y .

These r e s u l t s , o b t a i n e d w i t h p o o le d t i s s u e DNA, w e re n o t d i s ­

s i m i l a r t o t h o s e r e p o r t e d f o r th e DNA o f i n d i v i d u a l t i s s u e s .

F o r exam p le , K le ih u e s and Magee (1973) r e p o r t e d an o v e r a l l

-9 - 1 - 1e x t e n t o f r e a c t i o n o f .312 x 10 mol m e th y l ,m o l ,DNA-P,ymol

MMSokg ^ body w e ig h t i n th e DNA from r a t - b r a i n f o l l o w i n g a

s i n g l e i n t r a v e n o u s i n j e c t i o n o f 100 mg MMS p e r kg body w e i g h t ,

r l4 nI n an an a lo g o u s e x p e r im e n t to t h o s e d e s c r i b e d above f o r I C]

MMS, [1^ G ] d im e th y l s u lp h a t e gave an o v e r a l l e x t e n t o f r e a c t i o n

-9 - 1 - 1 - 1o f 356 x 10 mol m e th y l ,m o l 0DNA-P,ymol DMS,kg body

w e ig h t f o l l o w i n g a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n o f 530yg

p e r kg body w e i g h t . As was p r e d i c t a b l e s i g n i f i c a n t amounts

o f r a d i o a c t i v i t y w ere p r e s e n t i n th e DNA b a s e s as a c o n seq u e n c e

o f n a t u r a l i n c o r p o r a t i o n , .. The p r o p o r t i o n a l c o n t r i b u t i o n o f

t h i s i n c o r p o r a t i o n i n c r e a s e d w i t h i n c r e a s i n g d o se l e v e l s .

T h u s , u s in g i n c o r p o r a t i o n i n t o th e g u a n in e m o le c u le as an

ex am p le , a s p e c i f i c r a d i o a c t i v i t y 1 1 ,04yC i,m ol ^DNA g u a n in e

- 156 -

14 p. . ■ •was o b t a i n e d w i t h t h e low d o ses o f L CjMMS w h i l e t h e c o r ­

r e s p o n d in g r e s u l t o b t a i n e d , a t t h e h i g h e x p o su re was 7 6 97yCi

p e r mol g u a n in e „ W h i l s t t h i s i n c o r p o r a t i o n d id n o t mask th e

d e t e c t i o n o f a l k y l a t i o n i t was a t l e a s t p a r t l y r e s p o n s i b l e

f o r t h e d i f f e r e n t e x t e n t s o f r e a c t i o n m easu red i n t h e two

MMS e x p e r im e n ts 0 „

F o l lo w in g a 12h i n h a l a t i o n e x p o su re t o an a tm o sp h e re

c o n t a i n i n g 0 o064yg 1 ^ [ m e t h y l - ^ C jm e th a n e s u lp h o n a t e , t h e

-9d e g re e o f a l k y l a t i o n o f s o f t t i s s u e DNA was 2 8 „5 x 10 molo

-1 -1 -1m e th y l im o l 0DNA-P.ymol MMS0kg body w e ig h t „ The maximum

c u m u la t iv e i n h a l e d d o s e , a ssum ing a m in u te volume (Vm) o f

0 o13 l 0min ^ (R o d e r ic k s , H0 p e r s o n a l com m unica tion) and

q u a n t i t a t i v e a b s t r a c t i o n o f MMS from th e lu n g would have

amounted to 6yg p e r r a t ( i 0e 0 24yg MMS p e r kg body w e i g h t ) „

A c o m par ison o f t h e t o t a l e x t e n t o f r e a c t i o n a t DNA i n t h e

i n h a l a t i o n s tu d y w i t h t h o s e o b t a i n e d by i n t r a p e r i t o n e a l

i n j e c t i o n a t t h e same dose l e v e l , shows a s i g n i f i c a n t l y

g r e a t e r r e a c t i o n i n t h e l a t t e r case® Even a l l o w in g f o r t h a t

p r o p o r t i o n o f t h e 7 - m e th y lg u a n in e d e p u r i n a t e d d u r in g th e

e x p o s u re p e r i o d , u s in g t h e f o l l o w i n g e q u a t i o n ( L b f r o t h , Go

p e r s o n a l communication)®

K x t w here c f = c o r r e c t i o n f a c t o rc f ----- -— t = t im e i n days

t -K t K = 0 o 231 d“ l (b ase d ont i 7 -m e th y lg u a n in e o f 3 days M arg iso n e t a l ,

= 1 o06 1 9 7 3 ) .

- 157 -

-9The e x t e n t o f r e a c t i o n o n ly amounts t o 3 0 .2 x 10 mol m e th y l .

-1 -1 -1 mol DNA-P.M-mol MMS.kg body w e i g h t , i . e . 7.8% o f t h a t found

f o l lo w in g i n t r a p e r i t o n e a l e x p o s u re (9.7% o f r e p r e s e n t a t i v e

l i t e r a t i v e v a l u e s ) .

In c a l c u l a t i n g th e t o t a l i n h a l e d d o s e , i t was assumed t h a t

a l l o f t h e compound e n t e r i n g t h e r e s p i r a t o r y t r a c t would have

b een a b s o rb e d . By a n a lo g y w i th t h e r e s u l t s o b t a i n e d w i t h s a r i n

where a p p ro x im a te ly 90% o f t h e d ose was r e t a i n e d in r a b b i t ,

monkey and man (A in sw o rth and S h e p h a rd , 1 9 6 1 ) , i t seems u n l i k e l y

t h a t p oo r a b s o r p t i o n was t h e m a jo r f a c t o r a c c o u n t in g f o r t h e

d i s p a r a t e e x t e n t s o f a l k y l a t i o n o b t a i n e d . I t seems more l i k e l y

t h a t a d m i n i s t r a t i o n o f MMS by t h e i n t r a p e r i t o n e a l r o u t e a l lo w e d

f a c i l e p e n e t r a t i o n , and h e n c e a l k y l a t i o n , o f t h e m a jo r o rg a n s

i n t h e p e r i t o n e a l c a v i t y . C e r t a i n l y , a t l e a s t d u r in g t h e i n i t i a l

p h a s e o f e x p o s u re , t h e MMS r e a c h in g t h e n o n - h e p a t i c t i s s u e s i n t h e

p e r i t o n e a l c a v i t y would n o t have p a s s e d th ro u g h t h e l i v e r w hich

i s , q u a n t i t a t i v e l y t h e m ost im p o r ta n t s i t e o f MMS m e ta b o l i s m

( P i l l i n g e r e t a l , 1 9 6 8 ) . In c o n t r a s t , e x p o s u re v i a t h e i n h a l a t i o n

r o u t e m ig h t be e x p e c te d t o y i e l d a q u a n t i t a t i v e d i s p o s i t i o n o f

MMS ( i n t i s s u e s o t h e r t h a n lu n g ) s i m i l a r t o t h a t r e s u l t i n g from

c o n t in u o u s i n t r a v e n o u s i n f u s i o n o f t h e compound. However, i t i s

p o s s i b l e t h a t some d e t o x i f i c a t i o n o f MMS o c c u r s i n lu n g c e l l s

(G ro v e r , 1974; Hook and Bend, 1 9 7 6 ) . The d i s t r i b u t i o n o f MMS i n

t h e b lo o d s t r e a m would p r o v id e a d e q u a te o p p o r t u n i t y f o r t h e g e n e r ­

a t i o n o f 5 - m e t h y l g l u t a t h i o n e d u r in g i t s t r a n s p o r t a t i o n to o t h e r

o r g a n s . T h is c o n c e p t may g a in some s u p p o r t from t h e f a c t t h a t t h e

e x t e n t o f r i n g l a b e l l i n g f o l l o w i n g t h e i n h a l a t i o n e x p o s u re was

- 158 -

g r e a t e r t h a n t h a t a f t e r t h e same t o t a l dose a p p l i e d a s a s i n g l e

i n t r a p e r i t o n e a l i n j e c t i o n . Thus s p e c i f i c r a d i o a c t i v i t i e s o f DNA

g u a n in e o f 1 1 .04 and 2 0 .8 u C i .m o l ^ w ere o b t a i n e d f o l l o w i n g

i n t r a p e r i t o n e a l i n j e c t i o n and i n h a l a t i o n e x p o s u re r e s p e c t i v e l y .

The t h e s i s t h a t t h e q u a n t i t a t i v e d i s p o s i t i o n o f a r e a c t i v e

compound a d m i n i s t e r e d by t h e i n h a l a t i o n r o u t e would d i f f e r from

t h a t o b t a i n e d when t h e same d o s e , ( t o t a l i n h a l e d d o se ) i s a d m in i ­

s t e r e d by a s i n g l e i n t r a v e n o u s o r i n t r a p e r i t o n e a l i n j e c t i o n i s a l s o

s u p p o r te d by t h e f o l l o w i n g e v id e n c e . The e x t e n t o f m e t h y l a t i o n o f

W-7 of g u a n in e m o i e t i e s i n t h e t e s t i c u l a r DNA o f m ice exposed to

14 -1a h ig h i n t r a p e r i t o n e a l d o se l e v e l o f [ C]MMS (140mg.kg body

-9 . -1 -1w e ig h t) was 137 x 10 mol 7-m e th y lg u a n in e .m o l DNA-P.fi.mol

-1 .MMS.kg body w e ig h t . At low i n t r a p e r i t o n e a l d o s e s o f MMS

(12-80|ig.MMS.kg ^ body w e ig h t) t h e e x t e n t o f m e t h y l a t i o n r a n g e d

-9 -9 . - 1 -1from 62 x 10 to 84 x 10 mol 7 -m e th y lg u a n in e .m o l DNA-P.umol

MMS.kg body w e ig h t (van S i t t e r t e t a l , 1977 ) .

However, t h e e x t e n t o f m e t h y l a t i o n o f DNA i n t h e i n h a l a t i o n

e x p e r im e n t w i th MMS ( t o t a l t h e o r e t i c a l i n h a l e d d o se 24ug MMS

-1 -9 . -1kg body w e ig h t ) was 2 8 .5 x 10 mol 7 -m e th y lg u a n m e .m o l

DNA-P.umol ^MMS-.kg ̂ body w e ig h t . The r e s u l t s o f t h e s e e x p e r i ­

m en ts u s in g a n a lo g o u s t e c h n i q u e s f o r t h e a s s e s s m e n t o f m e t h y l a t i o n

o f DNA a l b e i t i n two d i f f e r e n t s p e c i e s and i n d i f f e r e n t o r g a n s ,

d e m o n s t r a te t h a t p r o t r a c t e d e x p o s u re o f a low c o n c e n t r a t i o n o f

MMS by th e i n h a l a t i o n r o u t e r e s u l t s i n a m ark e d ly low er e x t e n t

o f m e t h y la t i o n o f DNA th a n when t h e t o t a l t h e o r e t i c a l i n h a l e d

- 159 -

d o se i s a d m i n i s t e r e d as a s i n g l e i n t r a p e r i t o n e a l i n j e c t i o n .

T h i s r e d u c t i o n was a t l e a s t 75%. I t seems c l e a r t h a t th e

low er e x t e n t o f r e a c t i o n o b s e rv e d i n t h e i n h a l a t i o n e x p e r i ­

m ent was a d i r e c t co n seq u e n c e o f a s m a l l e r t o t a l d o se o f MMS

a r r i v i n g a t t h e g e n e t i c m a t e r i a l .

The a b sen c e o f r a d i o l a b e l l e d 7 -m e th y lg u a n in e m o i e t i e s i n

t h e DNA o f t h e i n t e r n a l o rg a n s o f r a t s exposed t o an a tm os­

p h e re c o n t a i n i n g 0 .0 6 4 p g [ m e t h y l - ^ C j d i c h l o r v o s p e r l i t r e f o r

12 h o u rs i n d i c a t e t h a t t h i s p h o s p h o r i c a c id t r i e s t e r does n o t

m e t h y la t e t h e DNA o f mammalian t i s s u e s when i t i s i n h a l e d

c o n t i n u o u s ly a t p r a c t i c a l u s e c o n c e n t r a t i o n . T h is c o n c lu s i o n

i s b a s e d on two p r e c e p t s . F i r s t l y , a l t h o u g h a v a r i e t y o f

n u c l e o p h i l e s i n t h e c o n s t i t u e n t b a s e s o f DNA can r e a c t w i t h

e l e c t r o p h i l i c m e th y l c a rb o n , r e a c t i o n a t t h e W-7 atom o f

g u a n in e m o i e t i e s i s a lw ays p re d o m in a n t i r r e s p e c t i v e o f w h e th e r

t h e m echanism o f t h e n u c l e o p h i l e s u b s t i t u t i o n i s ' S ^ l o r S ^2 .

S e c o n d ly , t h e e x p o su re p e r i o d employed i n t h i s s tu d y c o n s t i t u t e d

a s i g n i f i c a n t f r a c t i o n o f th e i n v iv o h a l f - l i f e o f 7 - m e th y l ­

g u a n in e m o i e t i e s i n DNA (3 d ay s ) (M arg ison e t a l , 1 9 7 3 ) .

The l i m i t s o f d e t e c t i o n o f m e t h y l a t i o n o f n u c l e i c a c id s i n

th e c u r r e n t s tu d y w ere one m e th y l group p e r 5 .7 x 1 0 ^

n u c l e o t i d e u n i t s i n DNA. The p r o p o r t i o n o f t h e a d m i n i s t e r e d do se

t h a t would have b een consumed i n su c h h y p o t h e t i c a l r e a c t i o nt

w i t h DNA w ould be 0.000001% . The r e s u l t s o f e x p e r im e n t s i n

m ic e , a l th o u g h l a c k i n g th e v e r y h ig h s e n s i t i v i t y o f t h e c u r r e n t

w ork , i n d i c a t e t h a t much h i g h e r d o se s o f d i c h l o r v o s a l s o f a i l

t o g iv e s i g n i f i c a n t m e t h y la t i o n o f n u c l e i c a c id s i n v i v o .

160 -

T hus, no r a d i o l a b e l l e d 7-m e th y lg u a n in e m o i e t i e s w ere d e t e c t e d

i n th e l i v e r o r lu n g s o f m ice exposed f o r 4 ,5 h o u rs t o an

a tm o sp h e re c o n t a i n i n g [ m e t h y l - ^ C ] d i c h l o r v o s (2 2 07mCi,mmol

a t a c o n c e n t r a t i o n e x c e e d in g d o m e s t ic u s e c o n c e n t r a t i o n s by a t

l e a s t a f a c t o r o f 100, I n a d d i t i o n no r a d i o a c t i v i t y was r e p o r t e d

to be p r e s e n t i n t h e p u r i n e f r a c t i o n s i s o l a t e d from t h e lu n g

and l i v e r DNA o f m ice a f t e r i , p , i n j e c t i o n o f [ m e t h y l - ^ C j d i c h l o r v o s

(3o7mCi»mmol >lQmg0kg ^ body w e ig h t (W ennerberg , 1973)

The a p p a r e n t l a c k o f r e a c t i v i t y to w a rd s mammalian DNA demon­

s t r a t e d i n t h e v a r i o u s i n v iv o s t u d i e s w i t h d i c h l o r v o s was a l s o su p ­

p o r te d by t h e r e s u l t s o f t h e c u r r e n t s t r a n d damage e x p e r im e n t s con­

d u c te d w i th d im e th y ln a p h th y lp h o s p h a te . I n t h e s e e x p e r im e n t s MMS

was shown t o p ro d u c e d o se r e l a t e d changes i n t h e r a t e o f s e d i ­

m e n ta t i o n o f DNA i n l i n e a r a l k a l i n e s u c r o s e g r a d i e n t s . No such

changes w ere o b s e rv e d w i t h d im e th y ln a p h th y lp h o s p h a te even a t v e r y

h ig h d o s e s . The o b s e rv e d b r e a k s may n o t a c t u a l l y o b t a i n i n v iv o

and may be a c o n seq u en ce o f t h e a c t i o n o f t h e a l k a l i n e g r a d i e n t

on th e i n i t i a l l e s i o n s g e n e r a te d by MMS, The f a c t t h a t v e ry

h i g h d o ses o f m e th a n e s u lp h o n ic a c i d , t h e d e m e t h y la t io n p r o d u c t '

o f MMS, gave n e g a t i v e r e s u l t s i n v iv o i n d i c a t e d t h a t t h e

m e t h y l a t i o n o f s i t e s , p resu m a b ly i n DNA, was i n f a c t a p r e ­

r e q u i s i t e f o r th e i n d u c t i o n o f s t r a n d damage. The damage

in d u ce d i n r a t l i v e r DNA by MMS does n o t a p p e a r t o c o n s t i t u t e

a pe rm anen t e f f e c t . I n th e c u r r e n t s t u d i e s th e s e d im e n ta t i o n

r a t e o f MMS-damaged DNA had r e t u r n e d t o t h e c o n t r o l l e v e l

w i t h i n 48 h o u r s o f th e i n i t i a l damage. However, a l t h o u g h t h e

damage may b e r e p a i r e d , th e f i d e l i t y - o f ' t h i s p r o c e s s c a n n o t

- 161 -

be a s s e s s e d i n t h i s ty p e o f e x p e r i m e n t a t i o n . I n r e s p e c t o f t h e

r a p i d r e p a i r th e c u r r e n t d a t a c o r r e l a t e s w i th o t h e r r e p o r t s

t h a t r e p a i r o f c a r c in o g e n - in d u c e d damage i n t h e DNA o f t a r g e t

o rg a n s i s l e s s r a p i d l y r e p a i r e d th a n damage p ro d u ced i n non ­

t a r g e t o rg a n s (Sarma e t a l , 1 9 7 5 ) . T h e l i v e r a p p e a rs n o t t o

b e a t a r g e t f o r th e c a r c i n o g e n i c a c t i o n o f MMS (Magee, 1969;

Clapp e t a l , 1 9 6 8 ) ,

S i m i l a r s t r a n d damage s t u d i e s co n d u c te d i n E, c o l i and

C h in e se h a m s te r c e l l s have shown t h a t d i c h l o r v o s i s c a p a b le o f

i n d u c in g DNA s t r a n d damage (B r id g e s e t a l , 1973; G reen e t a l ,

1 9 7 4 ) , I n t h e E„ c o l i sy s te m , t h e g r e a t e s t damage was p ro d u ce d

i n s t r a i n s p o s s e s s i n g a f u n c t i o n a l p o s t - r e p l i c a t i o n a l r e p a i r

sy s te m ( e r r o r p r o n e ) , a c o r r e l a t i o n w h ich a l s o h o l d s f o r m u ta ­

g e n i c i t y i n E, c o l i o These f i n d i n g s i n d i c a t e t h a t n e i t h e r t h e

i n i t i a l m e t h y la t i o n n o r t h e e n s u in g s t r a n d b r e a k a r e u l t i m a t e l y

r e s p o n s i b l e f o r t h e m u ta g e n ic e f f e c t . Dose r e l a t e d damage c o u ld

n o t be d e t e c t e d i n E, c o l i WP2, At v e r y h ig h c o n c e n t r a t i o n s ,

how ever , damage was m a n i f e s t e d as an a l l o r none phenomenon.

S i m i l a r i n c h a r a c t e r i s t i c s t o t h a t o b s e rv e d w i t h i o d o a c e ta m id e

a compound p o s s e s s i n g l i t t l e r e a c t i v i t y tow ards DNA, T h is ty p e

o f r e s p o n s e i s c o n s i s t e n t w i t h th e h y p o t h e s i s t h a t t h e t o x i c

a c t i o n o f th e compound on th e c e l l ca u se d r e l e a s e o f n u c l e a s e s .

I n t h e c a se o f d i c h l o r v o s d im e th y l p h o s p h o r y l a t i o n , p r o b a b ly

o f p r o t e i n may have b een r e s p o n s i b l e f o r t h e m e d ia t i o n o f t h e

c y t o t o x i c i t y .

The r e s u l t s o b t a i n e d i n C h in e s e h a m s te r c e l l s show d i c h l o r v o s

to b e a t l e a s t 10 t im e s l e s s e f f i c i e n t as MMS a t m e d i a t i n g t h i s

- 162 -

ty p e o f r e s p o n s e . I n t h i s p a r t i c u l a r c e l l l i n e th e m u t a g e n i c i t y

o f d i c h l o r v o s c o u ld n o t be d e m o n s t ra te d even a t n e a r c y t o t o x i c

doses (Green e t a l , 1 9 7 4 ) .

The f a i l u r e o f t h e s e o rg a n o p h o s p h a te e s t e r s t o m e t h y la t e

and damage DNA o f mammalian t i s s u e s i s a d i r e c t c o n seq u en ce

o f th e c h e m is t r y o f t h e s e compounds, F o r exam p le , as a m ixed

t r i e s t e r o f p h o s p h o r ic a c i d , d i c h l o r v o s ’p o s s e s s e s - two m e th y l J

g roups and an e l e c t r o n - w i t h d r a w i n g d i c h l o r o v i n y l g ro u p . The

e l e c t r o n w i th d ra w in g c a p a c i t y o f th e a t t a c h e d oxygen atoms and

o f th e d i c h l o r o v i n y l g roup r e s u l t i n a m arked r e s i d u a l p o s i t i v e

c h a rg e a t p h o s p h o ru s . The e l e c t r o p h i l i c p h o sp h o ru s atom i s

s u s c e p t i b l e t o n u c l e o p h i l i c a t t a c k , p a r t i c u l a r l y by t h o s e n u c l e o ­

p h i l e s c l a s s i f i e d as h a rd (P e a r s o n and S o n g s ta d , 1967) ( e . g .

hyd roxy g r o u p s , o x y a n i o n s ) , In a d d i t i o n t o a c t i v a t i n g th e

p h o s p h o ry l c e n t r e to w a rd s n u c l e o p h i l i c a t t a c k , t h e d i c h l o r o ­

v i n y l group i s a b e t t e r l e a v i n g g roup th a n th e m e th y l g ro u p .

C o n s e q u e n t ly , such a t t a c k l e a d s t o s c i s s i o n o f t h e P - 0 ( d i c h l o r o ­

v i n y l ) bond and t o t h e d e m e th y lp h o s p h o r y la t io n o f t h e a t t a c k i n g

n u c l e o p h i l e (E q u a t io n I I I , S e c t io n 1 . 3 ) .

The r e a c t i o n m echanism d e p i c t e d i n E q u a t io n I I I i s f u n d a m e n ta l

t o b o th t h e i n s e c t i c i d a l a c t i o n o f d i c h l o r v o s and th e d e t o x i ­

f i c a t i o n o f d i c h l o r v o s by mammals, D ic h lo r v o s e x e r t s i t s

i n s e c t i c i d a l a c t i o n by a t t a c k a t p h o sp h o ru s by t h e s e r i n e h y d ro x y l

o r th e c o r r e s p o n d in g oxyan ion , l o c a t e d a t t h e a c t i v e c e n t r e o f

t h e a c e t y l c h o l i n e e s t e r a s e , l e a d i n g t o t h e d i m e th y l p h o s p h o r y l a t i o n

o f t h i s group and th e l o s s o f enzyme a c t i v i t y . I n t h e c u r r e n t

c o n te x t i t i s e x t r e m e ly im p o r ta n t t o r e c o g n i s e t h a t t h i s m echanism

- 163 -

a l s o a p p l i e s when th e a t t a c k i n g n u c l e o p h i l e i s w a t e r and t h a t ,

i n mammalian t i s s u e s and b lo o d , su c h r e a c t i o n i s g r e a t l y

a c c e l e r a t e d by th e c a t a l y t i c a c t i o n o f e s t e r a s e s (Hodgson

and C a s id a , 1962 ; H utson and H o a d le y , 1972a; H u tson arid

H o a d le y , 1972b; B l a i r e t a l , 1 9 7 5 ) . As a consequence

th e d im e th y l p h o s p h o r y l a t i o n o f w a te r ( h y d r o l y s i s ^ o f

d i c h l o r v o s ) i s e x t r e m e ly e f f i c i e n t and c o n s t i t u t e s t h e p r e ­

dom inan t d e t o x i f i c a t i o n r e a c t i o n u n d e rg o n e by t h i s compound

i n v iv o ( F ig , 7 4 ) .

The m e th y l c a rb o n a to m (s) c o n s t i t u t e a second e l e c t r o p h i l i c

c e n t r e i n t h e d i c h l o r v o s m o le c u le . However, compared w i t h th e

e l e c t r o n - d e f i c i e n t p h o sp h o ru s atom, t h e s e c a rb o n atoms p o s s e s s

a r e l a t i v e l y h ig h e l e c t r o n d e n s i t y . I n p r a c t i c e , t h e i n t r i n s i c

s t r u c t u r a l d i f f e r e n c e s be tw een t h e s e two c e n t r e s l e a d s t o

marked d i f f e r e n c e s i n r e a c t i v i t y to w a rd s d i f f e r e n t n u c l e o p h i l e s .

Whereas th e p h o sp h o ru s atom i s p a r t i c u l a r l y s u s c e p t i b l e t o

a t t a c k by " h a rd " n u c l e o p h i l e s , th e e l e c t r o p h i l i c c a rb o n atom

r e a c t s p r e f e r e n t i a l l y w i t h " s o f t " n u c l e o p h i l e s , i . e . groups^

com bin ing h ig h p o l a r i s a b i l i t y w i th a r a t h e r low b a s i c i t y . A

t y p i c a l example i s t h e s u l p h u r atom o f m e r c a p t a n s . T h u s , u n d e r

s u i t a b l e c o n d i t i o n s , d i c h l o r v o s i s a b l e t o t r a n s f e r one o f

i t s m e th y l g roups t o su ch a r e a c t i o n p a r t n e r . I n mammalian

l i v e r , r e a c t i o n w i t h t h e s u l p h y d r y l group o f g l u t a t h i o n e i s

g r e a t l y a c c e l e r a t e d by th e c a t a l y t i c a c t i o n o f a s o l u b l e

S - a l k y l t r a n s f e r a s e ( H o l l i n g w o r th , 1969; H u tson e t a l , 1971;

Dicowsky and M o re l lo , 197 1 ) ,

[CH

C12

.CH2

OH]

-»

CHC1

2.C

H20-

gluc

•

- 164 -

- 165 -

D ic h lo r v o s can a l s o t r a n s f e r one o f i t s m e th y l g roups t o

t h e n i t r o g e n atoms o f h e t e r o c y c l i c r i n g s y s te m s , e c go W-7

atom o f g u a n in e i n v i t r o . I n th e c u r r e n t c o n te x t t h i s r e a c t i o n

was 5% o f t h a t p ro d u ce d by .[methyl-^C]MM S u n d e r i d e n t i c a l

c o n d i t io n s o But i n c o n t r a s t t o t h e e n z y m e -c a ta ly s e d r e a c t i o n

w i t h g l u t a t h i o n e , mammals do n o t p o s s e s s enzymes c a p a b le o f

a c c e l e r a t i n g th e r e a c t i o n o f an e l e c t r o p h i l i c m e th y l c a rb o n

atom o f d i c h l o r v o s w i t h W -n u c le o p h i l e s ,

D ic h lo r v o s u n d e rg o e s v e ry r a p i d m e t a b o l i c d e g r a d a t i o n i n a

w id e v a r i e t y o f t i s s u e s i n c l u d i n g s k i n , lu n g , i n t e s t i n e , b lo o d

p la s m a , e r y t h r o c y t e s , l i v e r , k i d n e y , h e a r t , s k e l e t a l m u sc le

and b r a i n (Z a ik a , 1 9 7 2 ) . The u b i q u i t y and e f f i c i e n c y o f th e

d i c h l o r v o s d e g ra d in g enzymes a r e su c h t h a t th e p a r e n t compound

c a n n o t b e d e t e c t e d i n t h e t i s s u e s o r b lo o d o f a n im a ls i n h a l i n g

d i c h l o r v o s a t up t o 12 t im e s p r a c t i c a l u s e c o n c e n t r a t i o n s

(Page e t a l , 1972; B l a i r e t a l , 1 9 7 5 ) ,

Of t h e two m a jo r r o u t e s , e s t e r a s e - c a t a l y s e d h y d r o l y s i s i s

q u a n t i t a t i v e l y th e more im p o r ta n t and o c c u rs i n a w id e r a n g e

o f t i s s u e s (Hodgson and C a s id a , 1 9 6 2 ) , The t i s s u e d i s t r i b u t i o n

o f enzymes c a t a l y s i n g th e m e th y l t r a n s f e r from d i c h l o r v o s to

g l u t a t h i o n e i s l e s s w e l l - d e f i n e d . However, such 5 - a l k y l t r a n s ­

f e r a s e s a r e known to b e a c t i v e i n l i v e r and k id n e y ( C l i f t o n

e t a l , 1975; K a p lo w itz e t a l , 1975; H abig e t a l , 1974)«

Both o f t h e s e m a jo r m e t a b o l i c pa thw ays a r e o p e r a t i v e

i n r a t s , p i g s , m ic e , h a m s te r s and man (H utson and H o ad ley ,

1 9 7 2 a ,b ; H utson e t a l , 1971; Page e t a l , 1972) and t h e r e

i s e v id e n c e (H utson and H o ad ley , 1 9 7 2 a ,b ; H u tson e t a l , 1971)

- 166 -

t h a t a t a d e q u a te dose l e v e l s , t h e s e pathw ays a r e fo l lo w e d

r e g a r d l e s s o f t h e r o u t e o f a d m i n i s t r a t i o n , However, an_6

a t y p i c a l l y low K v a lu e ( 3 ,2 x 10 M) has been r e p o r t e d f o r

t h e e s t e r a s e i n human b lo o d p la sm a ( B l a i r e t a l , 1975) and i t

i s v e r y p r o b a b le t h a t t h e em phasis on th e h y d r o l y t i c r o u t e

o r s c i s s i o n o f th e P - 0 ( v i n y l ) bond w ould become even more

p ronounced a t low doses o f d i c h l o r v o s , T hus , on t h e b a s i s o f

t h e i r f i n d i n g s , t h a t d im e th y l p h o s p h a te was t h e m a jo r d e g r a d a t i o n

p r o d u c t and t h a t d e s m e th y ld i c h lo r v o s was a b s e n t from th e t i s s u e s

o f p ig s exposed t o a tm o s p h e r ic c o n c e n t r a t i o n s o f d i c h l o r v o s

r a n g in g from 0 , 0 5 - 0 , 5 y g ,1 \ Page e t a l (1972) s u g g e s t e d t h a t

c le a v a g e o f t h e P - 0 ( v in y l ) bond was t h e o n ly s i g n i f i c a n t

pa thw ay o p e r a t i v e u n d e r su ch c o n d i t i o n s . F u r th e r m o r e , t h e con­

t r i b u t i o n o f h y d r o l y s i s to t h e m e ta b o l i s m o f o rg a n o p h o s p h a te s

r e l a t i v e t o 0- d e a l k y l a t i o n i n c r e a s e s as t h e d o se i s lo w ered

(PLapp and C a s id a , 1958) and H o l l i r ig w o r th (1969) h a s r e p o r t e d

t h a t t h e p r o p o r t i o n s o f r a d i o a c t i v e ca rb o n d i o x id e and d e s m e th y l -

p a ra o x o n d e c r e a s e d m ark e d ly and t h e u r i n a r y e x c r e t i o n o f

d im e th y lp h o s p h a te i n c r e a s e d as th e dose o f th e r e l a t e d compound,

m e th y l p a ra o x o n , was d e c r e a s e d . R e l a t i v e l y h ig h e x p o s u re s t o

m e t h y l - l a b e l l e d d i c h l o r v o s r e s u l t i n t h e g e n e r a t i o n o f c a rb o n

d i o x id e (H u tson and H o ad ley , 1972a) and i n c o r p o r a t i o n o f t h e

l a b e l i n t o n a t u r a l components su c h as a d e n in e , g u a n in e and u r i n a r y

7 -m e th y lg u a n in e (W ennerberg , 1974; W ennerberg and L o f r o t h , 1974)

The g e n e r a t i o n o f c a rb o n d i o x id e and n a t u r a l i n c o r p o r a t i o n

o f th e l a b e l i n t o t h e s e compounds i s a lm o s t c e r t a i n l y d e p e n d e n t

on th e i n i t i a l m e th y l t r a n s f e r to g l u t a t h i o n e (Wooder e t a l , 1 9 7 7 ) ,

- 167 -

I n th e c u r r e n t s tu d y a d m i n i s t r a t i o n o f 5 - m e t h y l g l u t a t h i o n e to

r a t s by t h e i n t r a p e r i t o n e a l r o u t e r e s u l t e d i n t h e g e n e r a t i o n

o f l a r g e amounts o f ^ C O ^ ( a p p ro x . 70%) w i t h i n t h e f i r s t 24 -

48 h o u rs a f t e r d o s in g . T h is r e s u l t was i n good ag reem en t x^ith

t h e r e s u l t s r e p o r t e d by H o l l in g w o r th (1970) and i n d i c a t e s

t h a t o x i d a t i v e d e m e t h y la t i o n > o f S - m e t h y l c y s t e i n e i s a major*

m e t a b o l i c pa thw ay i n t h i s s p e c i e s (K u c h in s k a s , 1 9 6 5 ) . Exam­

i n a t i o n o f t h e s o f t t i s s u e DNA f r a c t i o n from r a t s t r e a t e d w i t h

S - m e t h y l g l u t a t h i o n e r e v e a l e d t h e i n c o r p o r a t i o n o f t h e l a b e l

i n t o a d e n in e , g u a n in e and thym ine b u t as was th e c a s e i n t h e

s t u d i e s w i t h f o rm a te and m e th io n in e no 7 - m e th y lg u a n in e c o u ld

be d e t e c t e d i n th e DNA.

I t was n o t p o s s i b l e t o m easu re t h e o u tp u t o f CO^ i n t h e

c u r r e n t d i c h l o r v o s i n h a l a t i o n s t u d i e s . However, t h e f a i l u r e t o

d e t e c t l a b e l l e d a d e n in e and g u a n in e i n t h e n u c l e i c a c i d s , o r

l a b e l l e d 7 -m e th y g u an in e i n t h e u r i n e o f t h e r a t s employed i n

t h e s e e x p e r im e n ts p o i n t s t o a l i m i t e d p r o d u c t i o n o f S - m e t h y l -

g l u t a t h i o n e , and th u s p r o v id e s a d d i t i o n a l e v id e n c e f o r t h e p r e ’

p o n d e ra n c e o f r e a c t i o n a t t h e p h o s p h o ry l c e n t r e and th e v e r y

weak i n v iv o m e t h y l a t i n g r e a c t i v i t y o f d i c h l o r v o s , even i n t h e

c a s e o f e n z y m e - a c c e le r a t e d r e a c t i o n s w i t h t h e f a v o u re d

n u c l e o p h i l e , g l u t a t h i o n e .

Thus, t h e a l k y l a t i o n d a t a on b o th d i c h l o r v o s and MMS

c o r r e l a t e s w e l l w i th t h e t o x i c p r o p e r t i e s o f t h e s e two

compounds. MMS w hich i s known t o a l k y l a t e DNA i n b a c t e r i a

(Lawley e t a l , 1 9 7 4 ) ; mammalian c e l l s in c u l t u r e (Lawley

e t a l , 1974) and th e i n t a c t an im a l (O’Connor e t a l , 1973;

F r e i and L aw ley , 1 9 7 6 ) , and i s m u ta g e n ic i n a l l t h r e e sy s te m s

- 168 -

(Dean, 1972; R o b e r t s e t a l , 1971; Dean and B l a i r , 1976) and i s

a l s o c a r c in o g e n i c i n t h e mouse (C lapp e t a l , 1968) and i n t h e

r a t (Magee, 196 9 ) . D ic h lo r v o s on t h e o th e r h a n d a l k y l a t e s DNA

i n , and i s m u ta g e n ic t o b a c t e r i a , b u t , a l t h o u g h i t m e t h y la t e s

t h e DNA mammalian c e l l s i n c u l t u r e (Lawley e t a l , 1 9 7 4 ) ,

m u t a g e n i c i t y t e s t s i n t h e s e sy s tem s have b een c o n s i s t e n t l y

n e g a t i v e (Dean, 1972frjGreen e t a l , 1 9 7 4 ) . T h is compound h a s

a l s o g iv e n n e g a t i v e r e s u l t s i n a l l o f t h e mammalian m u t a g e n ic i t y

t e s t s c o n d u c te d t o d a t e ( E p s te in e t a l , 1972; Voogd, 1972;

Dean and T h o rp e , 1972; Dean and B l a i r , 197 6 ) . S t u d ie s con­

d u c te d i n mammals on b o th t h e t e r a t o g e n i c i t y (T horpe e t a l , 1971)

and t h e c a r c i n o g e n i c i t y o f d i c h l o r v o s (W itherup e t a l , 1971; C o l l i n s

e t a l , 1971; B l a i r e t a l , 1976) have a l s o p roved t o be n e g a t i v e .

6 .2 O r i g in s o f u r i n a r y m e th y la t e d p u r i n e s

R a d i o l a b e l l e d 7 -m e th y lg u a n in e h a s been d e t e c t e d i n t h e u r i n e

o f r a t s and m ice a f t e r h i g h i n t r a p e r i t o n e a l d o s e s o f DMN and

d i c h l o r v o s (W ennerberg , 1973; W ennerberg and L o f r o t h , 1 9 7 4 ) .

The r e s u l t s o f s t u d i e s i n t o t h e m e t h y l a t i o n o f DNA by d i c h l o r v o s

i n d i c a t e t h a t , a t l e a s t i n t h e c a s e o f t h i s compound, t h e u r i n a r y

7 - m e th y lg u a n in e d oes n o t o r i g i n a t e i n t h e DNA.

I n t h e c u r r e n t s t u d i e s , r a d i o a c t i v i t y was a s s o c i a t e d w i th

b o th t h e 7 - m e th y lg u a n in e and 3- m e th y la d e n in e f r a c t i o n s from t h e

u r i n e o r r a t s a f t e r i n t r a p e r i t o n e a l i n j e c t i o n o f h ig h d o s e s o fX A X A[m e th y l- C]MMS and [m e th y l - C ] d i c h l o r v o s . I n t h e c a s e o f MMS

t h e o r i g i n o f t h e u r i n a r y 3 -m e th y la d e n in e was i n v e s t i g a t e d u s i n g

a r a t w i th [ ^ C ] f o r i n a t e l a b e l l e d DNA. T h u s , w h i l e e x p o s u re to

u n - l a b e l l e d MMS caused t h e e x c r e t i o n o f r a d i o l a b e l l e d 7 - m e th y l -

g u a n in e no r a d i o l a b e l l e d 3 - m e th y la d e n in e was d e t e c t e d i n d i c a t i n g ,

a l b e i t in a l i m i t e d way, t h a t t h e l a t t e r compound d i d n o t o r i g i n a t e

- 169 -

i n DNA. T h is h y p o t h e s i s i s s u p p o r te d by th e c u r r e n t work and by

t h e work o f W ennerberg (1 9 7 3 ) , who f a i l e d to d e t e c t t h e p r e s e n c e

o f 7 -m e th y lg u a n in e o r 3 - m e th y la d e n in e m o i e t i e s i n DNA, b u t

i d e n t i f i e d b o th o f t h e s e compounds i n t h e u r i n e o f r a t s and m ice

exposed t o m e t h y l - l a b e l l e d d i c h l o r v o s .

The n a t u r a l o c c u r r e n c e o f m e th y la t e d p u r i n e s i n mammalian

u r i n e was f i r s t d e m o n s t r a te d by K ruger and Saloman (1 8 9 8 a ,b ) who

d e s c r i b e d th e p r e s e n c e o f 7 -m e th y lg u a n in e i n norm al human u r i n e .

T h is f i n d i n g h a s s i n c e b een c o n f i rm e d (Weissman e t a l , 1 9 5 7 a ,b )

and a d a i l y e x c r e t i o n o f some 65yg h a s been r e p o r t e d i n t h e r a t

(C raddock and M agee, 1 9 6 7 ) . W h i l s t i t i s p o s s i b l e t h a t some p a r t

o f t h i s u r i n a r y 7 - m e th y lg u a n in e may be o f d i e t a r y o r i g i n i t i s

now c l e a r t h a t a t l e a s t a m ajo r p o r t i o n i s g e n e r a t e d by t h e

a c t i o n o f m e t h y l t r a n s f e r a s e s . From a q u a n t i t a t i v e s t a n d p o i n t ,

t h e m ost im p o r ta n t r o u t e i s l i k e l y t o i n v o lv e t h e e n z y m a tic

m e t h y l a t i o n o f g u a n in e m o i e t i e s i n c e r t a i n s p e c i e s o f RNA and

th e su b s e q u e n t r e l e a s e o f 7 -m e th y lg u a n in e d u r in g t h e c a t a b o l i s m

o f RNA (Scheme 1 ) . T hus , 7 - m e th y lg u a n in e i s a n a t u r a l c o n s t i t u e n t

o f b o th t-RNA (Borek and S r in v a s a n , 1966) and m-RNA (Rottm an

e t a l , 1974; C roner and H u r w i tz , 1975; F u r u ic h and M u ira , 1 9 7 5 ) ,

t h e m e th y l g ro u p s b e in g i n s e r t e d a t s p e c i f i c g u a n in e m o i e t i e s ,

d u r in g t h e p o s t s y n t h e t i c m o d i f i c a t i o n o f t h e s e n u c l e i c a c i d s ,

by t h e a c t i o n o f s p e c i f i c m e t h y l t r a n s f e r a s e s . 5 -A d en o sy l

m e th io n in e i s u t i l i s e d a s t h e m e th y l donor i n t h e s e r e a c t i o n s

(Borek and S r i n i v a s a n , 196 6 ) . Both o f t h e s e RNA s p e c i e s , b u t

p a r t i c u l a r l y t-RNA, a r e t u r n e d o v e r r a p i d l y , and b e c a u s e t h e r e

i s no s a lv a g e pa thw ay f o r 7 - m e th y lg u a n in e (C raddock e t a l , 1968) ,

c i2 c = c h - o - p ;>— ivietnyigiutatmone

I

COOH

L—Homocysteine

O - CH[HCHO]

I— C Pools I— C Pools

C -C -H ^ I

HO WtH

h s - c h 2- c h 2c h - c o o h

L—Homocysteine Inosimcacid .-■*

HC^5—Adenosylmethionine HC-NH

Adenylic acid

NH

HoNC IS] Guanylic acid

R - ©S—Adenosylmethionme

| Adenosine R

P ” GuanosineN triphosphate

R - ® - ® - ®R - ® - ® - ®Adenosinetriphosphate

t-R N Am -RN AInosme

To chain— R —® - R - ® - R — “ R - ® “ R - ® - R -

Xanthylic

Hypoxanthine

Adenine Guanine

HO^ ^ N ^

Xanthine

Hcs ^ c

3—Methyladenine

H0N" ^ N'

7—Methylguanine

Scheme 1 Natural pathways leading, via S — Methyl- glutathione, to the incorporation of the methyl carbons of dichlorvos into the ring and methyl carbon atoms of 7 —Methylguanine and 3—Methyladenine with partial retention of the methyl hydrogen atoms. (Emphasis is placed on the biosynthesis and degradation of ribosyl derivatives of purines. Similar pathways exist for the generation of ring-labelled purines in DNA).

- 171 -

th e m e th y la t e d p u r in e would be e x p e c te d t o be e x c r e t e d unchanged

i n t h e u r i n e . However, w h i l e 7 -m e th y lg u a n in e i s a n a t u r a l

e x c r e t o r y p r o d u c t , 3 - m e th y la d e n in e h a s n e v e r been r e p o r t e d to

o c c u r n a t u r a l l y i n mammalian t i s s u e s o r u r i n e .

The c u r r e n t s t u d i e s have shown t h a t t h e a d m i n i s t r a t i o n

o f b o th r a d i o l a b e l l e d p u r i n e s and compounds t h a t can c o n t r i b u t e

t o t h e 1-C p o o l s l e a d to t h e p r o d u c t i o n o f r a d i o a c t i v e u r i n a r y

m e th y la t e d p u r i n e s . A d m i n i s t r a t i o n o f a d e n in e o r g u a n in e t o a ,

o t h e r w i s e n o rm a l , r a t gave r i s e t o u r i n a r y 7 - m e th y lg u a n in e and

i n t h e fo rm e r c a s e t o " 3 - m e t h y l a d e n i n e " . The i n c o r p o r a t i o n o f

e x o g e n o u s ly formed p u r i n e s i n t o DNA n e c e s s i t a t e s t h e i r c o n v e r s io n

i n t o t h e c o r r e s p o n d in g n u c l e o s i d e t r i p h o s p h a t e and i t f o l l o w s

t h a t t h e p r o d u c t i o n o f 7 -m e th y lg u a n in e , by t h e m echanism o u t l i n e d

a b o v e , r e q u i r e s t h e c o n v e r s io n o f b o th g u a n in e and a d e n in e i n t o

g u a n o s in e t r i p h o s p h a t e (GTP). The m e t a b o l i c r e a c t i o n s l e a d i n g to

t h e s y n t h e s i s o f GTP from t h e s e p u r i n e s a r e w e l l e s t a b l i s h e d

and a r e i l l u s t r a t e d i n Scheme 1. S i m i l a r r e s u l t s t o t h o s e o b t a i n e d

w i th a d e n in e were found when sodium f o rm a te was i n j e c t e d i n t o

b o t h p a r t i a l l y h e p a te c to m is e d a d u l t r a t s , and n e o n a t a l r a t s .

The i m p l i c a t i o n o f such f i n d i n g s i s t h a t any r a d i o a c t i v e l y

l a b e l l e d m a t e r i a l , i r r e s p e c t i v e o f i t s a l k y l a t i n g p r o p e r t i e s ,

t h a t i s c a p a b le o f d o n a t in g c a rb o n f r a g m e n t s . t o t h e 1-C p o o l s

w i l l c a u se l a b e l l i n g o f u r i n a r y p u r i n e s , A d m i n i s t r a t i o n o f

m e t h io n i n e , t h e p r e c u s o r o f t h e p ro p o se d m e th y l d o n o r , to

p a r t i a l l y h e p a te c to m is e d r a t s a l s o r e s u l t s i n t h e l a b e l l i n g

o f 7 -m e th y lg u a n in e and " 3 - m e th y la d e n in e " .

172 -

E x a m in a t io n o f t h e s o f t t i s s u e s o r l i v e r DNA f r a c t i o n s from

t h e r a t s u se d i n t h e s e e x p e r im e n ts r e v e a l e d , i n e v e ry c a s e ,

r a d i o a c t i v i t y a s s o c i a t e d w i t h a d e n in e and g u a n in e 0 I n a d d i t i o n

DNA from t h e m e th io n in e t r e a t e d r a t showed th e p r e s e n c e o f r a d io - ­

a c t i v i t y a s s o c i a t e d xtfith t h e 5 - m e t h y lc y to s i n e m a rk e r , i n d i c a t i n g

q u i t e c l e a r l y t h a t u n d e r t h e s e c o n d i t i o n s ' DNA m e t h y l t r a n s f e r a s e

was o p e r a t i v e o I n no c a s e was d e t e c t a b l e r a d i o a c t i v i t y a s s o c i a t e d

w i t h t h e 7 -m e th y lg u a n in e o r 3 - m e th y la d e n in e f r a c t i o n s 0 Based on

t h i s d a t a i t was c a l c u l a t e d t h a t 7 -m e th y lg u a n in e a c c o u n t s f o r

l e s s thar i 0 o00004% o f t h e g u a n in e m o i e t i e s i n n o rm a l mammalian

DNA0

A lth o u g h a p r o p o r t i o n o f th e u r i n a r y m e th y la t e d p u r i n e s

p r e s e n t a f t e r e x p o s u re t o p ro v en a l k y l a t i n g a g e n ts may w e l l be

d e r i v e d from th e a l k y l a t i o n o f DNA and RNA th e e x i s t e n c e o f

n a t u r a l pa thw ays t o 7 -m e th y lg u a n in e and 3 -m e th y la d e n in e c a s t s

s e r i o u s d o u b ts as t o t h e v a l i d i t y o f u t i l i s i n g such m ea su re ­

m ents i n t h e a s s e s s m e n t o f th e i n v iv o a l k y l a t i n g r e a c t i v i t y o f

such compoundso I n t h i s c o n t e x t , i t h a s b een p ro p o s e d , on th e

b a s i s o f d e t e c t i o n o f m e th y la t e d p u r in e s i n t h e u r i n e o r r a t s and

m ice exposed to m e t h y l - l a b e l l e d d i c h l o r v o s , b u t o n a t e and t r i c h l o r -

p hone , t h a t t h e s e o rg a n o p h o sp h o ru s e s t e r s d i r e c t l y m e t h y la t e

g u a n in e and i n some i n s t a n c e s a d e n in e m o i e t i e s i n v iv o (W ennerberg

and L b f r o t h , 1974; L d f r o t h e t a l , 1974; Dedek e t a l , 1 9 7 6 ) 0

These a u th o r s have f u r t h e r s u g g e s t e d t h a t su ch r e a c t i o n o c c u rs

a t t h e l e v e l o f n ,u c le ic a c id s 0

L ik e d i c h l o r v o s t h e m e ta b o l i s m o f MMS a l s o g iv e s r i s e to

CO2 as an e x c r e t i o n p r o d u c t ( P i l l i n g e r e t a l , 1968; Cummings

and W alton , 1970) 0 M e ta b o l ism o f MMS i s com plex , p re su m a b ly as

173 -

a c o n seq u e n c e o f i t s h i g h r e a c t i v i t y tow ards t i s s u e n u c l e o p h i l e s

b u t a p p e a r s to i n v o lv e r e a c t i o n w i t h g l u t a t h i o n e , as ju d g e d by

t h e e x c r e t i o n o f S - m e t h y l c y s t e i n e and r e l a t e d compounds i n th e

u r i n e D A lth o u g h i t i s p r o b a b le t h a t a t l e a s t p a r t i s form ed by

t h e o x i d a t i v e d e m e th y la t io n o f 5 - m e t h y l c y s t e i n e (H o rn er and

K u c h in o k a s , 1959) i t has been s u g g e s t e d t h a t m ost o f t h e e x p i r e d

CO^ i s d e r i v e d from th e h y d r o l y t i c c le a v a g e o f t h e p a r e n t compound

( P i l l i n g e r e t a l , 1970: Cummings and W a lto n , 1 9 6 8 )0 However,

i r r e s p e c t i v e o f th e m echanism th e n e t r e s u l t would be l a b e l l i n g

o f t h e p o o l o f 1-C f ra g m e n ts ( f o r m a ld e h y d e ) „ Thus i n t h e c a s e

o f b o th MMS and d i c h l o r v o s , t h e c a rb o n atom o f t h e 0 -m e th y l

group can t h e r e f o r e be d o n a te d to t h e 1-C p o o l , p a r t o f

w hich a p p e a r s as CC^o On p u r e l y t h e o r e t i c a l g rounds b o th

compounds w ould be e x p e c te d t o g iv e r i s e t o l a b e l l e d u r i n a r y

p u r i n e s (Scheme 1 ) 0 T h is t h e o r e t i c a l e x p e c t a t i o n i s b o rn e o u t by

t h e e x p e r im e n ta l f i n d i n g s „ The d a t a p r e s e n t e d by W ennerberg

and L b f r o t h (1974) shows s i g n i f i c a n t amounts o f r a d i o a c t i v i t y

a s s o c i a t e d w i t h u r i n a r y x a n t h i n e , h y p o x a n th in e , g u a n in e a nd ,

p o s s i b l y a d en ineo The r a d i o a c t i v i t y a s s o c i a t e d w i t h t h i s l a t t e r

group o f compounds c o u ld o n ly have b een in t r o d u c e d d u r in g th e

de novo s y n t h e s i s o f t h e s e p u r i n e m o i e t i e s „ I t i s t h e r e f o r e

n o t s u r p r i s i n g t h a t s i g n i f i c a n t amounts o f r a d i o a c t i v i t y w ere

a s s o c i a t e d w i t h t h e a d e n in e and g u a n in e m o i e t i e s o f b o t h DNA

and RNA o f mouse t i s s u e s a f t e r e x p o s u re to [ m e t h y l - ^ C ] d i c h l o r v o s

(W ennerberg , 1973)„ In d ee d i n th e c u r r e n t u r i n a r y p u r i n e

s t u d i e s , e x a m in a t io n o f th e s o f t - t i s s u e DNA f o l l o w i n g e x p o s u re

- 174 -

t o b o th MMS and d i c h l o r v o s r e v e a l e d e x t e n s i v e l a b e l l i n g o f a d e n in e

and g u a n in e m o i e t i e s . The p r o b a b le in v o lv e m e n t o f S - m e th y l -

g l u t a t h i o n e i n t h i s p r o c e s s was d e m o n s t ra te d f o l l o w i n g th e

i n t r a p e r i t o n e a l i n j e c t i o n o f r a t s w i th S - [ m e t h y l - ^ C j g l u t a t h i o n e .

S i m i l a r p a t t e r n s o f l a b e l l i n g t o t h o s e o b s e rv e d f o l l o w i n g

fo rm a te and m e th io n in e w ere s e e n i n b o th u r i n e and s o f t t i s s u e

DNA. R e -e x a m in a t io n o f t h e u r i n a r y 7 -m e th y lg u a n in e f r a c t i o n ,

w h i l s t n o t a t t h i s s t a g e e x h a u s t i v e , f a i l e d to p r o v id e e v id e n c e

f o r t h e p r e s e n c e o f " 3 - m e t h y la d e n in e " . I n t h i s p a r t i c u l a r

e x p e r im e n t t h e l a b e l l i n g o f t h e 'p u r in e r i n g s y s t e m s was low er t h a n

o b se rv e d w i th f o rm a te o r m e th io n in e . Thus t h e f a i l u r e to

d e m o n s t r a t e t h e p r e s e n c e o f M3 - m e th y la d e n in e " may be a con­

se q u en ce o f t h e low e x t e n t o f l a b e l l i n g . C l e a r l y i t i s im p o r ta n t

t o c o n d u c t a d d i t i o n a l s t u d i e s t o e s t a b l i s h w h e th e r 3 - m e th y l ­

a d e n in e i s a n a t u r a l e x c r e t i o n p r o d u c t .

The c u r r e n t d a t a d e m o n s t r a t e s t h a t t h e d e t e c t i o n o f

r a d i o l a b e l l e d 7 -m e th y lg u a n in e , p e r s e , i n t h e u r i n e o f r o d e n t s

exposed t o m e th y l l a b e l l e d jm e th y la t in g a g e n t s does n o t c o n s t i t u t e

e v id e n c e f o r t h e s p o n ta n e o u s m e t h y l a t i o n o f g u a n in e m o i e t i e s

a t any l e v e l o f o r g a n i s a t i o n e . g . a t t h e nu c l e o t i d e o r n u c l e i c

a c id l e v e l s . I t would be p re m a tu re to d raw a s i m i l a r c o n c lu s i o n

w i th r e s p e c t to t h e d e t e c t i o n o f u r i n a r y 3 - m e th y la d e n in e

u n t i l u n e q u iv o c a l e v id e n c e h a s b een o b t a i n e d f o r t h e n a t u r a l

o c c u r r e n c e o f t h i s b a s e i n mammalian u r i n e . However, t h e c u r r e n t

e v id e n c e s t r o n g l y i n d i c a t e s a need f o r c a u t i o n i n t h e i n t e r ­

p r e t a t i o n o f such d a t a , and i t i s a l r e a d y c l e a r t h a t t h e

e x c r e t i o n o f 3 - m e th y la d e n in e c a n n o t be employed as e v id e n c e

f o r t h e m e t h y la t i o n o f t h e a d e n in e m o i e t i e s o f DNA.

- 175 -

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