Polyamine mutagenesis : evidence for a unique mechanism
by Janis Elaine Hulla
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in BIOCHEMISTRY
Montana State University
© Copyright by Janis Elaine Hulla (1981)
Abstract:
A chemically distinct class of polyamines has been found to have genetic toxicity in the Ames bio
assay, Ethylenediamine (ene), diethylenetriamine (diene), triethylenetetramine (triene), and
tetraethylenepentamine (tetraene) were tested for mutagenicity on strain TA100 of Salmonella
typhimurium. Triene showed substantial mutagenic activity of 0.052 His+ revertants per nanomole.
Ene, diene and tetraene showed activity of 0.0048, 0.0057 and 0.0058 His+ revertants per nanomole,
respectively. It has been suggested that activity of triene is due to an alklating impurity such as
ethyleneimine (A. Hedsnstedt, 1978). My results indicate that ethyleneimine is not responsible for the
activity shown by triene, Triene retained constant mutagenicity through repeated recrystallizations.
Triene also retained its mutagenicity in the presence of excess Cleland's reagent which eliminates the
mutagenicity of ethyleneimine and other alkylating agents. These results and structural considerations
suggest that triene is a mutagen which interacts with DMA solely by an noncovalent, eletrostatic
association. STATEMENT OP PERMISSION TO COPY
In p r e s e n tin g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f th e
re q u ire m e n ts f o r a n advanced d e g re e a t M ontana S t a t e U n iv e rs ity , I
a g re e t h a t t h e L ib ra r y s h a l l make i t f r e e l y a v a ila b a le f o r in sp ectio n =
I f u r t h e r a g re e t h a t p e rm is s io n f o r e x te n s iv e copying o f t h i s t h e s i s
f o r s c h o la r ly p u rp o s e s may be g ra n te d by my m ajo r p r o f e s s o r , o r , i n h i s
a b se n c e, by th e D ir e c to r o f L ib ra rie s =
I t i s u n d e rsto o d t h a t any
copying o r p u b lic a tio n o f t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l n o t be
a llo w e d w ith o u t my w r i t t e n prem ission=
S ig n a tu re
T
POLYAMINE MQTffiENESIS:
EVIDENCE EOR A UNIQUE MECHANISE
JANIS ELAINE HULLA
A t h e s i s su b m itte d in p a r t i a l f u l f i l ln e n t
o f t h e re q u ire m e n ts f o r th e d egree
of
MASTER OF SCIENCE
.
in
BIOCHEMISTRY
Approved:
I
C h a irp e rso n , G rad u ate C % n itte e
f /
G rad u ate D e ar/
MONTANA STATE UNIVERSITY
Bozeman, Montana
December
iii
ACKNCBgLEDQlENT
I would l i k e t o e x p re s s my g r a t i t u d e t o th e fo llo w in g peoples
Dr» Sam R ogers f o r f a r i n g h i s know ledge, g u id a n c e and
d e d ic a tio n i n th e p u r s u i t o f t h i s m a s te r 's degree=
He i s g r e a tly
adm ired n o t o n ly a s a s c i e n t i s t , b u t a s a t r u l y s in c e r e p e rs o n .
Dr= G iy ly n W arren f o r h e r g en ero u s c o n tr ib u tio n s o f tim e ,
re s o u rc e s and knowledge t h a t have so g r e a t l y f a c i l i t a t e d my le a r n in g .
P a u le tte J a k a n o sk i and Cindy Kosso f o r th e c o n tr ib u tio n o f t h e i r
te c h n i c a l s k i l l s and m oral s u p p o r t.
Dan B a n c ro ft fo p h i s e x c e lle n t te c h n i c a l a s s i s t a n c e .
P eg g ie R ile y f o r rem in d in g me o f d a te s and d e a d lin e s .
Her h e lp i n
t h e p r e p a r a tio n o f t h i s t h e s i s i s g r e a t l y a p p re c ia te d .
T h is t h e s i s was s u p p o rte d by th e M ontana S t a t e A g r ic u ltu r a l
E x p erim en t S ta tio n , th e D epartm ent o f C h em istry , M ontana S t a t e
U n iv e rs ity and D r. and M rs. G rover H u lla .
F in a l l y , I w ould l i k e t o d e d ic a te t h i s t h e s i s t o th e memory o f
H arry and Libby S c h o lte n , how lu c k y I was t o have known th e n .
TABLE OF CONTENTS
Page
V ita o . o o o . o . ,
.................. o . o o . . . , o . o o . . O i i
Acknowledgment
.o .
T ab le o f C o n te n ts
ill
.................................
L i s t o f T a b le s . . . . . . . .
L i s t o f F ig u re s
^
. . .
iv
................................... . . . . . . .
vi
.................. . . . . . . . . . . . . . . .
v ii
A b s tra c t . . . . . . . . . . . . . . .
............................... . . . .
ix
I n tr o d u c tio n . . . . . . . . . . . . . . . . . . . . . . . . .
I
C h en ica l mechanisms o f m u ta tio n
................................... ....
I
The r o l e o f r e p a i r . . . ............................................
B ie n e ig h b o r e f f e c t
.......................... ....
. . . . . . . .
19
.
25
V a r ia tio n s o f m u ta tio n a l f r e q u e n c i e s .................. ....
26
The Ames b io a s s a y
28
................................... ....
The t e s t e d p o l y a m i n e s ......................
30
S ta te n e n t o f th e P urpose . . . . . . . . . . . . . . . . . . . 3 5
M a te ria ls an d Methods
............................................................. . . . . .
36
M utagenesis p l a t e a s s a y on S a lm o n e lla typhim urium
b a c te ria l s tra in s . . . . . . . . . .
.................. . . . . .
36
P r e p a r a tio n o f th e polyam ine s o l u t i o n s ............................................ 36
P r e p a ra tio n o f e th y Ie n e h n in e and C le la n d 1s re a g e n t
s o lu tio n s . . . . . . ...................... . . . . . . .......................
36
P r e p a r a tio n o f th e m ixed s o lu tio n s . . . . . . . . . . . . .
37
P r e p a r a tio n o f m edia . . . . . . . . . . . . . . . . . . . .
38
A ssay o f a c t i v i t y
38
.......................................................................... ....
V
£sge
Dose re s p o n se a s s a y p ro c e d u re
S p o t t e s t a ss a y p ro c e d u re
o . . . . . . . . . . .
. . . .
38
. . . . . . . . . . . . . . . . .
T rie n e r e c r y s t a l l i z a t i o n . . . . . . . . . . . .
R e s u lts . . . . .
0 .
. . . . . . . . . . . . .
i . . . .
39
.
. . . . o ' .
Q u a n tita tio n o f th e m u ta g e n ic ity o f e n e , d ie n e , t r i e n e ,
and t e t r a e n e i n TAlOO . . . . . . ............................... . . . .
40
41
.
41
Q u a n tita tio n o f th e m u ta g e n ic ity o f t r i e n e and
e tty le n e in d n e w ith and w ith o u t C le la n d ’ s re a g e n t . . . . .
41
P a tte r n s o f m u ta g e n ic ity o f e tty le n e im in e and t r i e n e
i n TAl535, TAlOO and TA98 . . . . . .................. . . . . . . .
46
Minor r e s u l t s
47
...........................................
DlSCUSSlOn o e . e e e e e e e o e e e e e e e e e e e e e . e e
51
The m u ta g e n ic ity o f t r i e n e ...............................................
51
A p o s s ib le mechanism
54
R e fe re n c e s ................................... .... . . ...................... ....
.
61
vi
LIST OF TABLES
T a b le
. Ie
Page
Background c o u n t l i m i t s f o r th e s t r a i n s o f
Salm O nella t y ^Ammur ium e e e o o e e o e e e e e
e e e *
39
2 0 K ie m u ta g e n ic itie s o f C le la n d 1s r e a g e n t, t r i e n e w ith
and w ith o u t C le la n d 1s r e a g e n t, and e th y le n e im in e
w ith and w ith o u t C le la n d 1s r e a g e n t ................................... .... ,
46
3e
4.
5.
6.
P a tte r n s o f a c t i v i t y o f e th y le n e im in e and t r i e n e i n
TA1535, TAlOO and TA98
........................... .
47
The m u ta g e n ic ity o f th e m ix tu r e s , e th y le n e im m e and
t r i e n e , e th y le n e im in e , t r i e n e and C le la n d 1s r e a g e n t,
i n te rm s o f t h e in d iv id u a l m utagens
49
„
H is+ r e v e r t a n t c o u n ts f o r e t t y le n e im in e w ith and
w ith o u t c y s te in e , g lu ta th io n e and C le la n d ' s re a g e n t . . .
50
The p K 's o f s e le c te d polyam in es . . . . . . . . . . . . .
56-57
v ii
LIST OF FIGURES
FJlciure
Page
1.
D ie norm al p u r in e and p y rim id in e p a i r s .................. ....
2
2.
The l e s s fa v o re d ta u to m e rs o f th e b a s e s „ = „ . . . » » .
3
3»
A com parison o f th e b a se p a ir in g o f amino and im ino
c o n f ig u r a tio n s o f a d e n i n e .................. ......................... ....
4
4 0 A com parison o f th e b a se p a ir in g o f k e to and e n d
c o n f ig u r a tio n s o f g u an in e
............................................ «. .
5»
60
70
Se
9=
ID ,
l i e
12»
13o
5
The d eam in atio n o f g u a n in e , a d e n in e and c y to s in e by
n i t r o u s a c id . . . 0 0 . 0 0 . 0 0 . . . . . . . . 0 0 .
7
The in d u c tio n o f OG— »TA and AT—-»GC m u ta tio n s by
n i t r o u s a c i d ........................... »
...........................
8
The m is p a ir in g s o f a d en in e t o u r a c i l and c y to s in e
t o h y p o x a n th in e ................................... ......................... .... . . . . »
9
The m is p a ir o f th e e n d form o f 7 - e tly lg u a n in e and
th y m in e .................. ................................................................ .... » » . .
10
The m isp a i r o f th e a m jh o ly te form o f 7 - e th y l guanine
and thym ine e e e e e e e e e e e e e e o e e e e e e
11
e
e
The m is p a ir o f 3 - e t t y la d e n in e and c y to s in e
12
The c o n v e rsio n o f c y to s in e t o 5 ,6 -d ih y d r o -6 hydroxyla m in o c y to sin e by hydroxylam ine
14
The amino and c a ti o n form s o f 5 , 6- d i t y d r - 6-h y d ro x y l
am ln o c y to sm e e e e e e e e e e e e e e e e e e e e e
e
15
The m is p a irin g s o f th e amino and c a t i o n form s o f 5 , 6 d ih y ro - 6-h y d ro x y la m in o c y to sin e w ith a d e n in e . . . . . . .
16
14.
The s t r u c t u r e o f th e thym ine dim er
15.
The p ro c e s s o f p o s t - r e p l i c a t i v e r e p a i r
16.
The re c o m b in a tio n a l r e p a i r p ro c e s s
e
. . . ............................... 20
. . . . . . . . .
.......................... ....
23
24
v iii
P igure
Paap
17.
The s t m c t u r e s o f th e t e s t e d poly am in es = . . . . . .
. . 31
18.
Ohe s t r u c t u r e s o f th e n a t u r a l l y o c c u rrin g poly am in es
. . 32
19.
The m u ta g e n ic ity o f th e s e r i e s o f t e s t e d polyam ines . . .
2 0.
M u ta g e n ic ity o f t r i e n e w ith and w ith o u t C le la n d 1s
re a g e n t i n TA100 . ................................... . . . . . . . . . .
21»
42
.
M u ta g e n ic ity o f e t t y le n e im in e w ith and w ith o u t
C lelandV s re a g e n t i n TA100 . ...................... ....
. . . . .
43
44
22.
M u ta g e n ic ity o f C le la n d 's re a g e n t i n TA100
. . 45
23.
M u tag e n ic ity o f e tty le n e im in e and t r i e n e w ith and
w ith o u t C Leland1s re a g e n t i n TAL00 . . . . . . . . . . .
48
24.
An exam ple o f a l k y l a t i o n o f a b a se by e t t y le n e im in e . . .
51
25.
The r e a c t i o n o f e t t y le n eim in e w ith C le la n d 's r e a g e n t
53
. .
Ix
ABSTRACT
A c h e m ic a lly d i s t i n c t c l a s s o f p o ly am in es h a s b een found t o have
g e n e tic t o x i c i t y i n t h e Ames b Io a ssa y e E tly Je n e d ia m in e ( e n e ) /
d ie th y le n e tr ia m in e (d ie n e ), t r i e t l y l e n e t e t r am ine ( t r i e n e ) , and
t e t r a e th y le n e p e n tam ine (te tra e n e ) w ere t e s t e d f o r m u ta g e n ic ity on
s t r a i n TAlOO o f S a lm o n e lla typhdm uriunu T rie n e showed s u b s ta n tia l
m utagenic a c t i v i t y o f 0.052 H is+ r e v e r t a n t s p e r nanom ole. Ene, d ie n e
and t e t r a e n e showed a c t i v i t y o f 0.0048, 0.0057 and 0.0058 H is+
r e v e r t a n t s p e r nanom ole, r e s p e c tiv e ly . I t h a s been su g g e s te d t h a t
a c t i v i t y o f t r i e n e i s due t o a n a l k l a t i n g im p u rity such, a s
e tly le n e im in e (A. H e d an ste d t, 1978). My r e s u l t s in d ic a te t h a t
e t t y le n e im in e i s n o t r e s p o n s ib le f o r th e a c t i v i t y showh by t r i e n e .
T rie n e r e t a i n e d c o n s ta n t m u ta g e n ic ity th ro u g h re p e a te d
r e c r y s t a l l i z a t i o n s . T rie n e a l s o r e ta in e d i t s m u ta g e n ic ity in th e
p re se n c e o f e x c e s s C le la n d 's r e a g e n t w hich e lim in a te s t h e m u ta g e n ic ity
o f e t l y le n e im in e and o th e r a lk y la tin g a g e n ts . These r e s u l t s and
s t r u c t u r a l c o n s id e r a tio n s s u g g e s t t h a t t r i e n e i s a m utagen w hich
i n t e r a c t s w ith DMA s o l e l y by an nonioovalent, e l e t r o s t a t i c a s s o c ia tio n .
INTRODUCTION
Chem ical mechanisms o f m u ta tio n
A m u ta tio n i s d e fin e d by W e b s te r's New C o lle g ia te D ic tio n a ry a s :
" . . . a r e l a t i v e l y perm anent change i n h e r e d ita r y m a te r ia l
in v o lv in g e i t h e r a p h y s ic a l change in chromosome
r e l a t i o n s o r a b io c h em ica l change i n th e cocbns t h a t make
up g e n e s ,"
I n 1953, a decade b e fo re th e g e n e tic code was e lu c id a te d , a mechanism
f o r m u ta tio n t h a t in v o lv e d a b io c h e m ic a l change i n th e n u c le o tid e
sequence o f DNA w as proposed (Watson and C ric k , 1953).
The new model
f o r th e s t r u c t i r e o f DM. su g g e s te d t h a t t h e n u c le o tid e b a s e s , a d en in e ,
th y m in e, g u a n in e and c y to s in e a r e n o rm a lly p r e s e n t in th e m o st p ro b a b le
ta u to m e ric form .
When t h i s i s t r u e , th y m in e, g u a n in e, c y to s in e and
a d e n in e a r e i n th e k e to and am ino c o n f ig u r a tio n s .
In th e s e
c o n f ig u r a tio n s , hydrogen bonding betw een p u r in e s and p y rim id in e s a r e
r e s t r i c t e d a s p re s e n te d i n F ig u re I .
When t h e b a se s ta u tc m e r iz e , th e
l e s s fa v o re d e n o l and im in o form s o c cu r.
They a r e shown i n F ig u re 2,
These l e s s fa v o re d c o n f ig u r a tio n s a r e c a p a b le of m is p a irin g , a llo w in g a
change in b a se sequence a f t e r r e p l i c a t i o n o f th e d au g h ter s tr a n d s and a
r e s u l t i n g m u ta tio n (Watson and C riC k, 1953),
The c o r r e c t and p o s s ib le
in c o r r e c t p a i r i n g s a r e compared i n F ig u re s 3 and 4,
D uring t h e l a t e 1950's and e a r l y 1960 's th e mechanism o f in d u c tio n
by ch em ical m utagens was in v e s tig a te d (F re e se , 1959; L o v e le ss , 1959;
F re e s e e t a l „ 1961; Champe and B en zer, 1962; K rie g , 1963; V ie lm e tte r
ADENINE
THYMINE
GUANINE
CYTOSINE
F ig u re I .
The norm al p u rin e and p y rim id in e p a i r s .
3
g u a n in e
thym ine
ENOL FORM
ENOL FORM
ADENINE
IMINO F O R M
CYTOSINE
F ig u re 2 .
IMINO F O R M
The l e s s fa v o re d ta u to m e rs o f th e b a s e s .
AMINO FORM
ADENINE
IMINO FORM
CORRECTF^MR
CYTOSINE
INCORRECT PAIR
F ig u re 3. A com parison o f th e b a se p a ir in g of am ino and im ino
c o n f ig u r a tio n s of a d e n in e .
5
g u a n in e
KETO FORM
CYTOSINE
CORRECT PAIR
GUANINE
ENOL FORM
THYMINE
INCORRECT PAIR
F ig u re 4.
A com parison o f b a se p a ir in g o f k e to and e n d c o n f ig u r a tio n s
o f g u a n in e .
6
an d S c h u s te r, 1960; C ooper, 1964).
N itro u s a c id was one o f th e
c h em ica l a g e n ts t h a t was in te n s iv e ly in v e s tig a te d d u rin g t h i s p e rio d ,
U sing to b a c c o m osaic v i r u s CTMV), a n RNA c o n ta in in g v i r u s , m u tan ts w ere
found t o be produced by c h em ica l r e a c tio n s betw een th e n i t r o u s and
r ib o n u c le ic a c id s , r a t h e r th a n by changes in c o a t p r o te in s o r
a g g re g a tio n phenomena (G ie re r and Mundry, 195® .
N itro u s a c id
c h e m ic a lly a l t e r e d th e in d iv id u a l b a se s o f t h e r ib o n u c le ic a c id
m o lecu le.
D ie r e s u l t o f such change i s a b a se w ith in th e n u c le ic a c id
m o lecu le t h a t i s c h e m ic a lly a l t e r e d fro m th e o r i g i n a l and can r e s u l t i n
m u ta tio n f i x a t i o n a f t e r r e p l i c a t i o n .
N itro u s a c id r e a c t i o n c a u se s th e
i n s i t u c o n v e rsio n o f g u a n in e t o x a n th in e , ad en in e t o hy p o x an th in e and
c y to s in e t o u r a c i l .
The 6 ' am ino g ro u p o f a d en in e and c y to s in e a r e
d eam in ated w h ile i t i s t h e 2 ’ am ino gro u p o f g u an in e t h a t i s l o s t
(S ch u ste r and Schamm, 1958; R e in e r and Zamenhof, 1957; B rooks and
C aw ley, I9 6 0 ; G ie re r and Mundry, 1958).
D ie d e am in atio n r e a c tio n s a r e
shown i n F ig u re 5.
Normal hydrogen bonding o f th e b a s e s i s p r im a r i ly s p e c if ie d by
th e 6 ' k e to g roups of thym ine and g u a n in e and th e 6 ' am ino groups o f
a d e n in e and c y to sin e^
For t h i s re a so n , d e a m in a tio n a t th e 2'
p o s i t i o n o f g u a n in e i s n o t r e s p o n s ib le f o r th e in d u c tio n o f m u ta tio n s ,
though i n a c t i v a t i o n may r e s u l t (V ie lm e tte r and S c h u s te r, 1960).
Two
ty p e s of m u ta tio n s r e s u l t from n itr o u s a c id tr e a tm e n t OG------ PTh and
AT— » GC
How th e s e occur a f t e r th e f i r s t and second r e p l i c a t i o n s o f
7
ADENINE
GUANINE
F ig u re 5.
HYPOXANTHINE
XANTHINE
The d e a m in a tio n o f g u a n in e, a d e n in e and c y to s in e by n itr o u s
a c id .
8
TMV i s shown i n F ig u re 6 =
ZrGG-V
% - U G MJAJj
tTPHj J -b6^ oV htaI
v t a J,
-i s ^ 4-HC4 -2^
vgcj7
C -C y to sin e f G -G uaninef U -U ra c ilf A-Adeninef T-Biym inef H-Hypoxanthine
F ig u re 6= The in d u c tio n o f CG— TA and AT— c>GC m u ta tio n s by n itr o u s
a c id .
T hese m u ta tio n s a r e th e r e s u l t o f m is p a irin g s t h a t occur a s shown
i n F ig u re 7 .
In a d d itio n t o m utagens t h a t d e am in ate b a s e s , a lk y l a t i n g a g e n ts
can a l s o induce m u ta tio n .
A lk y la tin g a g e n ts can be i n d i r e c t a c tin g f
i n h i b i t i n g DNA r e p l i c a t i o n b u t n o t c a u sin g a m is p a ir.
D ir e c t a c tin g
a lk y la tin g a g e n ts such a s m u sta rd s, e th y Ie n e im in e f e p o x i e s and a lk y l
a lk a n e s u lf o n a te s c a u se a t y p i c a l b a se p a ir in g a t g u a n in e s i t e s having
7 (N) a lk y la tio n s (L o v ele ss, 1969)-
For exam ple, tw o m echanism s fo r th e
occurence o f m u ta tio n ^ due t o e t t y !m eth an e s u lf o n a te in d u c tio n hare
been proposed (Laewley an d B rooks, 1961; K rie g f 1962).
A m is p a ir may
occur when t h e new ly form ed a n alo g h a s a more fa v o ra b le e q u ilib riu m
f o r th e r a r e r e n o l ta u to m e r.
A gain, e n d s a r e c a p a b le o f m is p a irin g .
To i l l u s t r a t e t h i s ty p e o f m is p a ir , 7 -e th y lg u a n in e , a n a d e n in e a n alo g ,
i s shown i n F ig u re 8 m is p a ir e d w ith th y m in e.
A m is p a ir may a ls o be th e
r e s u l t o f an a n a lo g having a new pK v a lu e f o r th e d i s s o c i a t i o n o f
9
URACIL
ADENINE
HYPOXANTHINE
CYTOSINE
F ig u re 7.
The m is p a ir in g s o f ad en in e t o u r a c i l and c y to s in e to
by poxan th in e .
10
7- ETHYLGUA NIIME
THYMINE
ENOL FORM
INCORRECT PAIR
F ig u re 8 .
The m is p a ir o f th e e n d form o f 7 -e h ty I guanine and thym ine.
a hydrogen io n so t h a t th e io n iz a tio n o c c u rs a t p h y s io lo g ic a l pH.
exam ple, g u a n in e h a s a pK f o r i t s I(N) hydrogen o f 9.2.
For
The pK of th e
same hydrogen on a 7 ,9 -s u b s t i t u t e d g u an in e i s changed t o around 7.
r e s u l t i s p o s tu la te d t o be a m is p a ir in g d u rin g r e p l i c a t i o n .
The
If 7-
e th y lg u a n in e w ere t o fo rm an am p h o ly tic m o lecu le due t o a s h i f t of pK,
i t can p a i r w ith thym ine a s shown i n F ig u re 9 and r e s u l t i n a base
s u b s t i t u t i o n m u ta tio n .
a n a lo g s a r e p o s s ib le .
I t sh o u ld be m entioned t h a t tw o o th e r base
7 -E tty lg u a n in e i s th e m ost f r e q u e n tly form ed
a n a lo g , b u t 3 -e th y la d e n in e h a s a ls o b een found.
K rieg s u g g e s ts th e
g u an in e a n a lo g , 3 -e t t y la d e n in e when i n th e e n d c o n f ig u r a tio n
11
P zh S
7- ETHYLGU AN IN E
THYMINE
AMPHOLYTE
INCORRECT PAIR
F ig u re 9 .
The m is p a ir o f am pholyte form o f 7 - e t t y lg u a n in e and thym ine.
m is p a i r s w ith c y to s in e (K rieg, 1963).
The m is p a ir i s shown i n F ig u re
10 .
Not o n ly i s th e
In situ
c r e a tio n o f b a se a n alo g s p o s s ib le , b u t th e
s u b s t i t u t i o n o f a n a n a lo g d u rin g r e p l i c a t i o n can a l s o le a d t o m u tatio n
due t o m is p a ir in g d u rin g su b se q u e n t r e p l i c a t i o n .
guan in e a t 7 (N) c r e a te s a q u a te rn a ry ammonium.
The a l k y l a t i o n o f
The p o s i t i v e charge i s
sh a re d by 9 (N) th u s w eakening th e g ly c o s id ic lin k a g e and a llo w in g
th e h y d ro ly s is o f 7 - e t t y lg u a n in e from t h e DNA m olecule.
The gap
r e s u l t i n g from such a depur ! n a tio n may le a d t o r e p l i c a t i o n e r r o r s such
a s b ase p a i r d e le t io n s , s u b s t i t u t i o n s and fram e s h i f t s .
r e p l i c a t i v e e r r o r s i n r e p a i r o f such a gap c an a ls o
B o str
12
3- e t h y l a d e n i n e
F ig u re 1 0 .
cy to sin e
The m is p a ir o f 3 -e tity la d e n in e and c y to s in e .
le a d t o m u ta tio n .
E rro r-p ro n e r e p a i r i s d is c u s s e d l a t e r .
Two q u a l i t a t i v e l y d i f f e r e n t b ase p a i r s u b s t i t u t i o n n u ta tio n
m echanism s have been proposed (F re e s e , 1959).
Base a n a lo g s such a s 7 -
e tty lg u a n in e and 3 - e t i y la d e n in e r e s u l t i n t r a n s i t i o n a l n u ta tio n s .
T r a n s itio n s a r e th o s e m u ta tio n s in w hich a p u rin e i s s u b s tit u te d f o r
a n o th e r p u r in e o r a p y rim id in e i s s u b s t i t u t e d f o r a n o th e r p y rim id in e .
Purine Transitions
Pyrimidine Transitions
a d en in e
guanine
thym ine
c y to s in e
g u an in e
a d en in e
c y to s in e
thym ine
B ase a n a lo g s r e s u l t i n t r a n s i t i o n e r r o r s because s te r e o c h e m ic a lI y , th e
b e s t f i t i s s t i l l a p u rin e w ith a p y rim id in e and v i s a v e rs a .
These
13
e r r o r s a r e l e s s l i k e l y t o be re c o g n iz e d by r e p a ir sy ste m s.
ty p e o f m u ta tio n d e s c rib e d i s c a l l e d a tr a n s v e r s io n .
The second
T ta n s v e rs io n s
r e s u l t from a p u rin e r e p la c in g a p y rim id in e o r a p y rim id in e re p la c in g
a p u r in e .
Purine — +Pyrimidine Transversions
Pyrimidine — »Purine Tranversions
a d e n in e ----» thym ine
th y m in e ------ ►a d en in e
a d e n in e ---- » c y to s in e
c y to s in e ------ » a d en in e
g u a n in e ---- » thym ine
th y m in e-------►guanine
g u a n in e ---- » c y to s in e
c y to s in e -------►a d en in e
These n u ta tio n s o ccu r sp o n ta n e o u sly and because of a p o o r f i t t i n g p a i r ,
th e d o u b le h e lix o f th e grow ing c h a in may be b e n t.
cau se f u r t h e r e r r o r s i n r e p l i c a t i o n .
The bending may
T ta n s v e rs io n s may be induced by a
g r e a t im balance o f th e n u c le o tid e r a t i o d u rin g r e p l i c a t i o n , o r by
p h y s io lo g ic f a c t o r s t h a t s e p a r a te th e DNA s tr a n d s , t h i s a llo w in g room
f o r th e m is p a irin g t o o c cu r.
I t h a s been su g g ested t h a t i n t e r c a l a t i n g
a g e n ts s e p a r a te th e DMA s tr a n d and a l s o cau se tr a n s v e r s io n s (L em an,
1 9 6 3 ).
The i n t e r c a l a t i n g c l a s s o f compounds w i l l be d is c u s s e d l a t e r .
N itro u s a c id m u tag en esis i s a b i d i r e c t i o n a l t r a n s i t i o n .
T hat i s ,
i t p ro d u ces t r a n s i t i o n s i n b o th d i r e c t i o n s , P u rin e 1 ----- ^ p u rin e 2 o r
P y rim id in e 1------» P y rim id in e 2.
The d is c o v e ry o f a u n id ir e c tio n a l mutagen
h a s p ro v id e d a v a lu a b le te c h n ic a l to o l f o r g e n e tic a n a ly s is .
Hydroxylam ine com bines w ith c y to s in e p ro ducing 5 ,6 -d i by d r 0- 6bydr oxy la m in o c y to sin e a s shown i n F ig u re 11.
I t h a s been shown t h a t
14
CYTOSINE
AM INO FORM
CYTOSINE
IMINO FORM
NH2OH
NHOH
5.6-DIHYDRO-6HYDROX YL AMINOCYTOSINE
F ig u re I L
\
The c o n v e rsio n o f c y to s in e t o 5 f6 -d ih y d ro -6 -h y d ro x y la m in o c y to s in e by hydroxylam ine.
15
th e g e n e tic s p e c i f i c i t y o f t h i s compound i s u n i d i r e c t i o n a l , r e s u l t i n g
i n o n ly g u an in e: c y to s in e ——» a d en in e: th y m in e t r a n s i t i o n s (F reese,
B autz and F r e e s e , 1961).
Two m echanism s s im ila r t o th o s e f o r 7 -
a lk y lg u a n in e have been proposed f o r hydroxyla m in e m u ta g e n esis ( P h illip s
and Brown, 1966).
5 -6 -d ih y d r o - 6 -h y dro x y la m in o c y to sin e can e x i s t a s a n
im in o ta u to m e r a s shown in F ig u re 11, a n amino ta u to m e r, o r a s a c a tio n
a s shown i n F ig u re 1 2.
NHOH _H
NHOH
5,&DlHYDRO-6-
5.&D IH Y D R O -6 -
HYDROXYL AMINOCYTOSINE
H YDROX YL AMINOCYTOSINE
AMINO FORM
CATION
F ig u re 1 2 . The amino and c a ti o n form s o f 5 , 6- d i t y d r o - 6- t y d r oxy lam ino­
c y to s in e .
The am ino ta u to m e r i s a n alo g o u s t o th e p a r e n t c y to s in e and p a i r s
c o r r e c t l y w ith guanine.
The im in o and c a tio n form s o f d i t y d ro - 6-h y ­
droxy la m in o c y to sin e a r e c a p a b le of m is p a ir w ith a d en in e a s shown in
F ig u re 13.
The u n i d ir e c tio n a l c h a r a c te r o f hydroxy la m in e m u tag en esis
IMINO FORM
CATION
F ig u re 13.
ADENINE
ADENINE
The m is p a ir in g s o f th e am ino and c a tio n fo rm s o f 5 ,6 d ih y d r o - 6-h y droxyla m in o c y to sin e w ith a d e n in e .
17
makes i t a u s e f u l to o l i n c l a s s i f y i n g m utagens s p e c i f i c a l l y f o r th e DNA
b a se p a i r lo c a te d a t th e m u ta tio n s i t e .
The s ta n d a rd te c h n iq u e i s t o
s t a r t w ith a s in g le w ild ty p e b a c t e r i a and s e l e c t a s e r i e s o f m u tan ts
from i t u s in g a p a r t i c u l a r mutagen.
T e s t m utagens a r e th e n a s s ig n e d t o
s e e w hich w i l l b a c k -m u ta te th e m u ta n ts t o w ild ty p e .
F o r example*
i f a m u tan t i s r e v e r te d by a b i d i r e c t i o n a l m utagen, b u t n o t by
hydroxylam ine, i t would c o n ta in a n ad eninezthym ine b a se p a i r a t th e
m u ta tio n s i t e b ecau se hydroxylam ine c a u s e s o n ly guanines c y to s in e t o
a d e n in e : thym ine t r a n s i t i o n s .
In 1961, t h e com parison o f m u ta tio n s i t e s on s e t s o f 5 -b rc m o u ra c il
m u ta n ts and p r o f la v in e m u ta n ts le d t o a p ro p o sa l f o r a new mechanism o f
m u ta g e n e sis (B renner, B u rn e tt, C ric k and O rg e l, 1961).
I t had been
shown t h a t t h e r e w ere no s i t e s in t h e r l l re g io n o f T4 genome common t o
b o th s e t s o f m u ta n ts (B re n n e r, B enzer and B a r n e tt, 1958).
The 1961
g r a ip com pared m u ta n ts h a v in g n u ta tio n s a t d i f f e r e n t l o c i .
The h lo c u s
c o d es f o r a p r o te in t h a t i s e s s e n t i a l f o r a f in is h e d phage.
W ithout a t
l e a s t a n a l t e r e d p r o te in from t h i s lo c u s , t h e phage i s n o t v i a b le .
Ih e
second lo c u s , r , p ro d u c e s a p r o te in t h a t i s n o n e s s e n tia l f o r a v ia b le
phage.
I t was found t h a t t h e number o f p r o f la v in e produced m u tan ts
h av in g m u ta tio n s a t th e h lo c u s was f a r l e s s th e n e x p ec te d i f one
assum es th e p ro d u c tio n of h m u tan ts w ith 5 -b ro m o u ra c il and p r o f la v in e
i s s i m i l a r t o m u ta tio n a t th e r l o c i produced by th e same tw o m utagens.
I t was s u g g e s te d t h e m echanism fo r p r o f la v in e m u tag en esis d i f f e r e d fro m
18
th e p re v io u s ly d e s c rib e d b a s e - p a ir s u b s t i t u t i o n mechanism o f
5-bram oura c i l m u ta g e n esis.
B renner proposed t h a t p r o f la v in e , a n
a c r id in e compound, c au se s m u ta tio n s d ue t o i n s e r t i n g o f d e le tin g base
p a i r s d u rin g DNA r e p l i c a t i o n .
The r e s u l t would be t o s h i f t th e r e e lin g
fra m e s o t h a t each codon t r i p l e t t h a t fo llo w e d th e s i t e o f m u ta tio n ,
w ould code f o r a n i n c o r r e c t am ino a c id .
The r e s u l t a t th e h lo c u s
would be p ro d u c tio n o f a d e f e c tiv e p r o te in an d t h e T4 v i r u s would n o t
be d e te c te d .
B renner based h is p ro p o s a l mechanism on t h e 1951 re s e a rc h
o f Lerman show ing a c id in e m o le c u le s com plex w ith DM su ch t h a t t h e f l a t
a c r id in e and b o th b a s e s l i e p e rp e n d ic u la r t o th e m o le c u la r a x is o f DNA,
By s l i d i n g betw een a d ja c e n t b a se p a i r s , a c r id in e s e p a r a te s them
.
a p p ro x im a te ly 6 . 8A r a t h e r th a n th e norm al 3.4iL
s i z e o f one b a se p a i r (Lerman, 1951).
The d if f e r e n c e i s th e
From t h i s , B renner s u g g e s ts t h a t
one o f th e DNA s tr a n d s adds o r d e l e t e s a b a s e d u rin g su b se q u e n t
re p lic a tio n s .
In 1953, i t was n o te d t h a t s in c e th e m utagenic e f f e c t o f
p r o f la v in e was se e n i n T4 phage b u t n o t Eu. c o l i , th e mechanism m ust .
in c lu d e a m ajo r r o l e f o r re c o m b in a tio n .
R ecom bination i s t h e o n ly
s i g n i f i c a n t d if f e r e n c e b etw een phage and S b. c o l i ENA r e p l i c a t i o n
(Lerman, 1 9 5 3 ).
The r o l e o f re c o m b in a tio n began t o be e lu c id a te d w ith th e
reE in em en t of t h e fram e s h i f t m echanism t o in c lu d e o p e r a tio n o f a r e p a ir
sy stem d u rin g re c o m b in a tio n .
R e se a rc h e rs t e s t i n g a c r id in e s , h av in g
a lk y la tin g s id e c h a in s , f o r r e v e r s io n o f S a lm o n e lla typhim urium found
19
a c t i v i t y n o t shown by unsubs t i tu b ed a c r id in e s ,
Ohe fra m e s h i f t
m u ta g e n ic ity o f th e new g ro u p o f s u b s tit u te d a c r id in e s w as a t t r i b u t e d
t o t h e p re se n c e o f t h e a lk y la tin g s id e c h a in s , even th ough a lk y la tio n
o f b a s e s h a s been shown t o r e s u l t i n b a s e s u b s t i t u t i o n m utagenesis=
It
was s u g g e s te d t h a t a lk y la tin g g roups c au se s u b s t i t u t i o n i n t h e DNA t h a t
r e s u l t e d i n in c re a s e d a c t i v i t y o f DNA r e p a i r sy stem s (Ames and
W h itf ie ld , ISS 6).
I n o rd e r f o r th e a c ti o n o f r e p a i r sy ste m s t o r e s u l t
i n a n in c re a s e d fre q u e n c y o f f r a m e s h if t m u ta tio n , a s ev id en ced by th e
s u b s t i t u t e d a c r id in e s , t h e r e p a i r sy ste m s m ust be e r r o r - p r o n e .
C o n cu rre n t t o t h i s re s e a r c h , e r r o r p ro n e sy stem s o f r e p a i r w ere bein g
im p lic a te d a s f a c t o r s in a c r id in e 'p m echanism of m u tag en esis.
S t r e i s i n g e r su g g e ste d a r o l e f o r m is r e p a ir i n e x p la in in g h i s f in d in g s
t h a t f r a m e s h i f t m u ta tio n s a r i s e m ost o f te n i n a r e a s o f t h e DNA m olecule
h av in g b a s e p a i r re d u n d a n c ie s ( S tr e is i n g e r , Okada, E m rich, Newton,
T s u g ita , T e rz a g h i and Inouye, 1956).
.
I t i s in te re s tin g th a t
S t r e i s i n g e r h a s su g g este d t h a t th e m is a lig n m e n t i s n o t a r e s u l t cf a
s e p a r a tio n o f t h e DM s tr a n d s by a c r id in e a s su g g e ste d by L e h re r, b u t
r a t h e r caused by a s t a b i l i z a t i o n o f th e DMA by th e i n t e r c a l a t i n g
a c r id in e .
The a r e a of s t a b i l i z a t i o n re m a in s c o n n ec te d a f t e r hydrogen
bonds have been broken.
The c o n n e c tio n s r e s u l t in r e p l i c a t i o n e r r o r s
t h a t a r e e rro n e o u s ly r e p a ir e d .
The r o l e o f r e p a i r
Ey th e l a t e 1960's i t was becoming in c r e a s in g ly a p p a re n t t h a t a
20
number of m utagens, m ost n o ta b ly UV l i g h t , were in d u cin g n u ta tio n s by
y e t a n o th e r mechanism.
R e se a rc h e rs (Beukers and B eren d s, 1963; W acker,
1963; S m ith , 1963; S e tlo w , C a r r ie r and B ollum , 1965) have shown t h a t
ex p o su re o f Ec c o l i t o u l t r a v i o l e t (UV) l i g h t r e s u l t s i n p ro d u c tio n o f
p y rim id in e d im e rs w ith in th e DNA m o lecu le.
D im ers a r e th e ph o to ­
p ro d u c ts w hich form when a c o v a le n t bond i s made betw een n eig h b o rin g
p y rim id in e s on th e sam e DNA s tr a n d .
C y to s in e -c y to s in e and c y to s in e -
thym ine d im e rs a r e form ed, b u t th y m in e-th y m in e shown in F ig u re 14 a r e
by f a r th e m ost f r e q u e n t. S t r a i n s o f Em. c o l i may d i f f e r i n t h e i r
THYMINE DIMER
F ig u re 1 4 .
Ih e s t r u c t u r e o f th e thym ine d im er.
s e n s i t i v i t y t o UV f o r two re a so n s .
They can d i f f e r i n t h e i r a b i l t y t o
r e p a i r th e d im er o r i n t h e i r a b i l i t y t o t o l e r a t e th e d im er (W itkin,
1967).
T h is c o n c lu s io n was re a ch e d when i t was observ ed t h a t c e r t a i n
r e p a ir d e f i c i e n t m u ta n ts w ere more s e n s i t i v e to UV i n a c t i v a t i o n and a t
th e same tim e , l e s s s e n s i t i v e t o UV induced m u tag en esis.
T hat i s , i t
appeared t h a t i f a m u tan t was more d e f i c i e n t in some a s p e c t t h a t
21
r e p a i r s UV damage, th e o rg a n !a n was n o t a s a p t t o m u tate u n d er UV
i r r a d i a t i o n , A t l e a s t th r e e r e p a i r sy stem s have been i d e n t i f i e d i n Sn.
c o l l ; p h o to r e a c ti v ity , e x c is io n r e p a i r and p o s t - r e p l i c a t i o n a l r e p a ir .
P h o to re a c tiv e r e p a i r ta k e s p la c e o n ly i n th e p re sen c e o f v is a b l e l i g h t
O
c f aro u n d 400OA wave le n g th s . Die p y rim id in e d im e rs a r e s p l i t jja s i t u
by an enzyme t h a t i s p h o to a c tiv e te d by th e l i g h t .
i s th o u g h t t o in v o lv e o n ly one enzyme.
P h o to re a c tiv e r e p a i r
S in ce p h o to re a c tiv e r e p a ir
r e s t o r e s t h e DNA d i r e c t l y t o th e p r e - i r r a 3 i a t i o t i s t r u c t u r e , i t should
be a n a c c u ra te system t h a t does n o t , i t s e l f , cau se e r r o r s i n t h e DM.
By com paring norm al s t r a i n s t o r e p a i r d e f i c i e n t s t r a i n s , W itk in found
t h a t i f phot © re a c tiv e r e p a i r o f a d im er l e d t o a m u ta tio n , n o n -r e p a ir
o f t h a t d im e r was a t l e a s t t e n tim e s m ore l i k e l y t o r e s u l t i n a
m u ta tio n .
B iis means t h a t p h o to re a c tiv e r e p a i r in c r e a s e s t h e f i d e l i t y
te n fo ld .
A ccording t o th e " c u t and p a tch " m odel of th e e x c is io n r e p a ir
sy ste m , t h e s e c tio n o f DM s tr a n d c o n ta in in g a p y rim id in e d in e r i s
f i r s t in c is e d by an en d o n u cle a se o r g ly c o s y la s e th e n e x c is e d and th e
gap i s e n la rg e d by e x o n u cle a se d e g ra d a tio n .
r e p a i r s y n th e s is and s e a le d by DNA l i g a s e .
The gap i s th e n f i l l e d by
The co m p le te e x c is io n
r e p a i r p ro c e s s in v o lv e s a s e r i e s o f e n z y m a tic r e a c tio n s .
Ety com paring
u l t r a v i o l e t l i g h t m u ta b ility o f s t r a i n s la c k in g e x c is io n r e p a i r t o
s t r a i n s h a v in g th e sy ste m , W itk in found t h a t i f a n e x c is io n r e p a ir
22
c a u s e s a m u ta tio n , i t i s one hundred tim e s l e s s l i k e l y t o r e s u l t i n a
m u ta tio n th a n i f no e x c is io n r e p a i r o f d im e rs o c c u rre d .
E x c isio n
r e p a i r to o , i s f i d e l i t o u s (W itk in , 1969).
W itk in d id f i n d a n e r r o r - p r o n e r e p a i r sy stem .
In an E. c o l i
s t r a i n r e f e r r e d t o a s uyg~„ e x r+ , w hich h a s th e a b i l i t y t o s u rv iv e
u n re p a ire d p y rim id in e d im e rs , a p o s t - r e p l i c a t i o n r e p a i r sy stem c o r r e c t s
gaps i n progeny DKfi.
W itk in s u g g e s ts t h a t such a p o s t - r e p l i c a t i o n
p ro c e s s i s slow and r e s u l t s i n d a u g h te r DNA h av in g one s tr a n d w hich
c o n ta in s p y rim id in e d im e rs.
o u tlin e d i n F ig u re 15.
The p ro c e s s o f p o s t - r e p l i c a t i v e r e p a i r i s
The X1s in d i c a t e d im e rs.
I t h a s been proposed t h a t p o s t - r e p l i c a t i v e r e p a i r o p e r a te s by
a s e r i e s o f re c o m b in a tio n s, c r e a tin g te m p la te s f o r th e s in g le s tra n d
gaps t h a t have r e s u l t e d from th e p y rim id in e d im ers.
The p ro c e s s i s
shown i n F ig u re 16 w here th e s o l i d l i n e s a r e p a r e n t DNA, t h e dashed
l i n e s d a u g h te r s tr a n d s , wavy l i n e s a r e r e p a i r s y n th e s iz e d DNA and X i s
a m u ta tio n .
The m u ta tio n i s a r e s u l t o f e r r o r s i n p o s t - r e p l i c a t i v e
r e p a i r s y n th e s is and l i k e l y t o o c c u r i n e rr o r - p r o n e s t r a i n s su ch a s
u v r~ , e x r+ s t r a i n s (Rupp and H ow ard-Flanders, 19 6 8 ).
I n a n e f f o r t t o e x p la in how th e re c o m b in a tio n p ro c e s s can cau se a n
e r r o r , i t h a s been su g g e ste d t h a t b a se p a ir in g b etw een o v e rla p p in g
s in g le - s tr a n d e d en d s i s a n e a r ly s t e p i n th e p ro c e ss w ith gaps being
r e p a ir e d a s a f i n a l s t e p (H ow ard-Flanders and Boyce, 1966).
I t is
im p o rta n t t o n o te t h a t e x c is io n r e p a i r o f DKA (ferriage d o e s n o t in c re a s e
REPLIC AT IO N
REPAIR O F
THE G A P S
F ig u re 1 5.
The p ro c e s s o f p o s t - r e p l i c a t i v e r e p a i r .
REPLICATION
1» f
V
--------------- -------------------------- %
PAIRING OF DAUGHTER
ST R A N D S
V
* ♦!
y \
< ■ - -------- ---
RECOMBINATION
«■-------------------------------------------------------------- >
*
- Y
r —
RECOMBINATION
ERROR
F ig u re 1 6.
■Ayvv
NO MUTATION
The re c c m b in a tio n a l r e p a ir p ro c e s s
25
th e fre q u e n c y o f m u ta tio n s .
I t i s u n lik e ly , th e r e f o r e , t h a t r e p a i r
s y n th e s is common t o e x c is io n r e p a i r and p o s t - r e p l i c a t i v e r e p a i r would
be r e s p o n s ib le f o r m u ta tio n s i n t h e re c o m b in a tio n m echanism i f th e same
enzymes a r e common t o b o th r e p a i r m echanism s.
The ty p e o f m u ta tio n s
r e s u l t i n g from re c o m b in a tio n e r r o r s have been found t o be m o stly s in g le
b a se s u b s t i t u t i o n s , b o th t r a n s i t i o n s and tra n s /e r s io n s =
A n a ly sis o f UV
induced m u ta tio n s in try p to p h a n s y n th e ta s e A gene h a s shown t h a t
a p p ro x im a te ly 20% o f th e m u ta tio n s a r e o f t h e f r a m e s h if t ty p e .
A n a ly sis s u g g e s t t h a t t h e f r a m e s h i f t m u ta tio n s a r e s in g le b a se
d e le t io n s (W itk in , 1969; Yanofsk y , I t o and Horn, 1966; Brammer, B erger
and Yanofsk y , 1967).
T h is f u r t h e r s u g g e s ts t h a t re c o m b in a tio n d o e s n 't
c a u se m u ta tio n s by unequal exchange w hich would in v o lv e l a r g e r segm ents
o f t h e DNA m o lecule.
The n e ig h b o r e f f e c t
In 1960 B enzer r e p o rte d t h a t th e m u ta tio n a l freq u en cy o f th e
d i f f e r e n t l o c i i n th e r l l re g io n o f T4 phage v a ry su ch t h a t ‘h o ts p o ts "
and " c o ld sp o ts " occur (B enzer, 1960).
The n o n rrandom m u ta b ility o f th e
v a r io u s s i t e s i s e x p e c te d t o be a r e s u l t o f i n t e r a c t i o n s betw een
n e ig h b o rin g b a s e p a i r s .
For exam ple, ad en in e :th y m in e p a i r s a r e
hydrogen bonded l e s s s tr o n g ly th a n g u a n in e :c y to s in e p a i r s .
A segm ent
o f DNA w ith a s e r i e s o f a d en in e :th y m in e p a i r s may be m ore s u s c e p tib le
t o a n i l l i c i t b a se p a ir in g d u rin g r e p l i c a t i o n .
As m en tio n ed e a r l i e r ,
S t r e i s i n g e r , proposed t h a t a r e a s o f b a se redundancy a l s o a llo w a
26
m isa lig n m e n t t h a t le a d s t o fra m e s h i f t m u ta tio n v i a m is r e p a ir
( S tr e is i n g e r e t a L , 1966)s
S t r e i s i n g e r co n firm ed h is i n i t i a l
h y p o th e s is w ith l a t e r r e s e a r c h , showing t h a t s i t e s o f h ig h m u ta tio n
fre q u e n c y w ith in t h e lysozym e gene o f
r e s id u e s (Okada e t a l . , 1972).
T4
c o n ta in re p e a tin g
a d e n in e
Koch showed t h a t i t was in d e ed a
n e ig h b o rin g b a s e p a i r e f f e c t and n o t e x t r i n s i c f a c t o r s t h a t in flu e n c e d
m u ta tio n fr e q u e n c ie s by d e m o n stra tin g t h e a l t e r n a t i o n o f r a t e s of
m u ta tio n .
T h is w as shown e x p e rim e n ta lly by in c o rp o ra tin g b a se
s u b s t i t u t i o n s n e a r a m u ta tio n s i t e (Koch, 1971).
V a r ia tio n o f m u ta tio n a l fr e q u e n c ie s
The fequency o f m u ta tio n v a r i e s from s i t e t o s i t e on t h e DNA
m o lecule,
T h is id e a o r ig i n a te d w ith th e d is c o v e ry o f th e “h o t s p o ts "
o b serv ed by B enzer (B enzer, 1961) and r e i t e r a t e d i n su b se q u e n t p a p e rs
(Koch a n d D ra k e , 1 9 7 0 ; K och, 1 9 7 1 ; O kada e t a l . , 1 9 7 2 ).
The r a t e o f
m u ta tio n i s dependent n o t o n ly on th e b a se p a i r a t th e m u ta tio n s i t e
b u t a l s o on t h e m o le c u la r e n v iro n m en t o f th e s i t e .
t h e sequence o f DNA,. i s g e n e t i c a l l y d e te rm in e d .
T h is e n v iro n m en t,
I t th e r e f o r e fo llo w s
(
t h a t m u ta tio n r a t e s a r e i n p a r t g e n e ti c a lly d eterm in ed .
The freq u e n cy
o f m u ta tio n s i s a l s o dependent on th e p r o b a b ility o f r e p a i r and e r r o r
av o id an ce by t h e DNA r e p l i c a t i o n a p p a ra tu s .
Enzymes v a ry i n t h e i r
■
a b i l i t y t o m a in ta in DNA f i d e l i t y .
For exam ple, a m u ta n t DNA p o ly m erase
h a s been found t h a t i s i t s e l f m utagenic.
gene" p ro d u c t (Speyer, 1965).
T h is enzyme i s a "m utator
M u tato r g e n es a r e m u ta tio n s t h a t
27
r e s u l t i n a d e f e c tiv e gene p ro d u c t w hich in c r e a s e s m u ta tio n a l
fr e q u e n c ie s o f o th e r g en es.
A n ti m u ta to r g en es have a l s o b een found,
D ie gene p ro d u c t o f su ch a gene lo w e rs th e m u ta tio n a l fr e q u e n c ie s ,
A
DNA p o ly m erase o f TA h a s been fo u n d t h a t h a s a n t i n u t a t o r p r o p e r tie s .
I t lo w e rs t h e m u ta tio n fr e q u e n c ie s o f th e phage (Drake an d A lle n ,
ISS8) o
M u ta tio n a l fr e q u e n c ie s a r e f u r t h e r d ependent on t h e p r o b a b ility
o f d e te c tio n o f any g iv e n m u ta tio n .
I t m ust be co n clu d ed t h a t
m u ta tio n a l f r e q u e n c ie s m ost a s s u r e d ly v ary among s p e c ie s and p ro b a b ly
v a ry in d iv id u a l t o in d iv id u a l.
For t h i s re a so n i t may be t h a t l i t t l e
c a n be s a id a b o u t th e e f f e c t s i n man o f th o s e c h e m ic a ls found t o be.
m u tagenic i n non-human sy stem s.
I t re m a in s, how ever, t h a t a c lo s e
a s s o c ia ti o n o f m u ta g e n esis w ith c a n c e r e x i s t s .
For exam ple, th e lo s s
o f e f f e c t i v e e x c is io n r e p a i r o f th y m in e d im e rs , a s se e n i n c e l l s from
p a t i e n t s h av in g xeroderm a pigm entosum , le a d s t o a g r e a t l y in c re a s e d
r a t e o f s u n l i g h t in d u ced s k in c an c e r.
E x c is io n r e p a ir c a p a b i l i t i e s
a ls o a c t t o d e c re a s e o r e lim in a te th e consequence of c h e m ic a lly induced
DNA damage (Maker an d McCormick, 1 9 7 9 ).
The N a tio n a l I n s t i t u t e
o f E nvironm ental H e a lth S c ie n c s h a s i d e n t i f i e d a p p ro x im a te ly 26
c h e m ic a ls t o be f ir m ly a s s o c ia te d w ith human can cer.
An a d d itio n a l 56
s u b s ta n c e s have l e s s d e f i n i t e l y been a s s o c ia te d w ith c a n c e r through
e p id e m io lo g ic a l s tu d ie s (Maugh, 1978),
Ames h a s r e p o r te d t h a t o f 158
c a rc in o g e n s t e s t e d , 138 have p ro v en t o b e m u tag en ic,
D iis f u r th e r
s tr e n g th e n s t h e m u ta g e n e s is -c a rc in o g e n e s is a s s o c ia ti o n (Ames, McCann
28
and Y am asaskiz 1975)„
Mie Mes Moassay
S in ce a n im al t e s t s a r e Ie n g th y ,e x p e n s iv e and sp ac e consuming th e
n eed f o r s h o r t- te r m s c re e n in g t e s t s t h a t a r e b o th s e n s i t i v e and
econom ical h a s le a d t o t h e developm ent o f an e s tim a te d 80 d i f f e r e n t
te s ts .
Four g e n e ra l c la s s e s have been d e sc rib e d ,
They in c lu d e
m u ta g e n e sis and DNA r e p a i r t e s t s w ith m ic ro o rg a n ism s, t e s t s w ith i n t a c t ,
m u l t i c e l l u l a r o rg a n ism s su ch a s D rosoghi l i a , t e s t s f o r m u ta g e n esis w ith
c u ltu r e d mammalian c e l l and t e s t s f o r i n v i t r o c e l l tra n s fo rm a tio n .
The p u rp o se o f th e s e t e s t s i s t o i d e n t i f y th o s e compounds t h a t a r e
p o t e n t i a l h e a l t h h a z a rd s t o humans.
C h em icals shown t o be p o s itiv e i n
one o r more o f th e s c r e e n in g t e s t s become c a n d id a te s f o r e x te n s iv e
t e s t i n g i n a n im al b io a s s a y s .
The e x tr a p o la t io n o f th e i n v i t r o t e s t i n g
a s w e ll a s t h e a n im a l b io a s s a y r e s u l t s t o th e e f f e c t s i n humans h a s m et
w ith s tro n g r e s i s t a n c e .
The c o n tro v e rs y c e n te r s around n o t o n ly th o s e
g e n e tic c o n s id e r a tio n s m en tioned u n e r th e s u b t i t l e ‘,V a ria tio n o f . . . .
M u ta tio n a l F re q u e n c ie s" , b u t a l s o bn dosage le v e l s .
I t i s argued t h a t
h ig h d o s e s o f ch em ica l may o v e rlo a d d e t o x i f i c a t i o n m echanism s o r induce
m e ta b o lic p athw ays t h a t do n o t e x i s t a t l e v e l s o f human ex p o su re.
In
a d d itio n t o th e s e , c o n s id e r a tio n s o f r i s k v e r s e s b e n e f i t a n a ly s e s m ust
be conducted on e ac h s u b sta n c e found t o be c a rc in o g e n ic ,
i s n o t a r i s k - f r e e s o c ie ty .
Our s o c ie ty
A c ce p ta b le r i s k s should be e s ta b lis h e d b u t
29
t h i s to o i s a n e x tre m e ly c o n tr o v e r s ia l to p ic betw een w ould-be
r e g u la to r s and th e b e n e f i c i a r i e s o f th e u se o f chem icals=
The r e s e a r c h con d u cted f o r t h i s t h e s i s h a s em ployed a r a p id and
econom ical s c re e n in g t e s t f o r c h em ica l mutagens=
The Ames t e s t was
developed a b o u t f i f t e e n y e a r s ago by Dr. B ruce Anes o f t h e U n iv e rs ity
o f C a l i f o r n i a a t B erk ele y .
I t u s e s h i s t i d i n e r e q u ir in g m u ta n ts o f
S a lm o n e lla bYphiinurium s e l e c t e d f o r s e n s i t i v i t y and s p e c i f i c i t y t o
r e v e r t t h e h i s t i d i n e r e q u ir in g m u ta n ts t o th e w ild ty p e w hich i s
c a p a b le o f s y n th e s iz in g h i s t i d i n e .
The r e v e r s io n fre q u e n c y can be
c o n sid e re d t o be a m easure o f g e n e tic a c t i v i t y o f th e compound b ein g
te s te d .
Some o f t h e s ta n d a rd s t r a i n s em ployed i n Ames t e s t i n g p o s s e s s
m u ta tio n s a t th e uvrB and r f a l o c i .
These m u ta tio n s r e s u l t in th e l o s s
o f th e e x c is io n r e p a ir system and th e b a c t e r i a l Iip o p c d y s a c c h a rid e
s u r f a c e c o a tin g , re s p e c tiv e ly =
These m u ta tio n s in c r e a s e th e
s e n s i t i v i t y o f th e b a c t e r i a t o m utagens by fo r c in g e r r o r - p r o n e r e p a i r
t o be used and by c a u s in g le a k y c e l l s u r f a c e , r e s p e c tiv e ly .
In
a d d itio n , t h e s ta n d a rd t e s t e r s t r a i n s v a ry i n th e ty p e o f h i s t i d i n e
m u ta tio n s th e y p o s s e s s .
s u b s titu tio n s =
S tr a in TA1535 i s used t o d e t e c t b a se p a i r
I t s H is” m u ta tio n i s a b ase p a i r s u b s t i t u t i o n .
and TA1538 a r e b o th used t o d e te c t f r a m e s h if t m u ta tio n s .
TAl537
F r a m e d iif t
m u ta n ts v a ry a s t o th e sequence o f b a se p a i r s th e y hav e a t th e m u ta tio n
s ite .
TA1537 h a s a r e p e a tin g c y to s in e sequence a t th e l o c a t io n o f i t s
h i s t i d i n e m u ta tio n .
TAl538 h a s a r e p e t i t i v e -C-G-C-G--G-G- sequence a t
30
t h e m u ta tio n s i t e .
Prom th e s ta n d a rd t e s t e r s t r a i n s TM.535 and TM538
tw o a d d itio n a l s t r a i n s have been developed,
TMOO and TA98 were
c o n s tr u c te d by adding a p la sm id w hich i s r e f e r r e d t o a s a n R f a c t o r and
d e s ig n a te d pKHLOl.
T h is p la sm id c a r r i e s drug r e s i s t a n c e m a rk e rs a s
w e ll a s a n e r r o r - p r o n e DNA r e p a i r sy stem and g r e a t l y in c r e a s e s th e
s e n s i t i v i t y o f th e t e s t e r s t r a i n s by in c r e a s in g m utagenic re s p o n se t o
many g e n e t i c a l l y to x ic c h e m ic a ls .
Through uvr~ and pKMIOl
m o d if ic a tio n s Ames h a s produced a r e p a ir - m u ta tio n c o u p le t h a t g r e a tly
in c r e a s e s t h e i n c o r r e c t r e p a i r o f induced m u ta tio n s .
Q ie o th e r
a d d itio n t o th e t e s t i s th e u se o f l i v e r hom ogenates (human, r a t ,
m ouse, e tc .) s o t h a t m e ta b o lite s o f a t e s t ch em ica l may a l s o be
d e te c te d
The in c o rp o ra tio n of t h i s a s p e c t of mammalian m e ta b o lism ,
t h e u v r, r f a and pKMIOl m o d if ic a tio n s have in c re a s e d th e s e n s i t i v i t y
su ch t h a t a b o u t 90% o f th e known c a rc in o g e n s can now be d e te c te d
(W a lk e r, 1979).
The t e s t e d p o ly am in es
The compounds t e s t e d f o r t h i s t h e s i s a r e p o ly am in es,
s t r u c t u r e s a r e shown i n F ig u re 17.
T ie ir
T iey a r e u sed i n d u s t r i a l l y a s
c h em ica l lig a n d s and a r e found i n polym er g lu e s .
They s t r u c t u r a l l y
re se m b le th e n a t u r a l o c c u rrin g p o ly a m in e s, sp erm in e, sp e rm id in e and
p u tr e s c in e shown i n F ig u re 18.
I n i t i a l t e s t s in d ic a te d t h a t th e
polyam ine s e r i e s , e th y le n e d ia m in e (ene), d ie t t y le n e t r am in e (d ie n e ),
t r i e t h y Ie n e t e tra m in e ( tr ie n e ) and te tr a e tiy le n e p e n ta m in e (te tra e n e )
NH2(CH2 )2 NH2
- ENE
N H 2(C H 2)2 N H (C H 2 )2 N H 2
D IE N E
NHa (C H 2) a NH (C H 2 )2 N H (C H 2 )SNHa
T R IE N E
N HgtCHs^S NH(CHa)aN H (C HaJa N H(CHa)a N H
.
F ig u re 17»
■T E T R A E N E
The s t r u c t u r e s o f th e t e s t e d p o ly am in es»
NH2(C H 2)4 N H 2
P U tR E S C S N E
. . N H ^Csy4 NHCC Hg)gN Hg
,
S P E R M ID IN E
N H2CCH2J3 N H(CH2)4 NH (CH2)3 N Hg
SPERMINE . .
F ig u re 1 8 .
The s t r u c t u r e s o f th e n a t u r a l l y o c c u rrin g polyam ines
33
have m u tag en ic a c t i v i t y i n th e Ames te s t»
T rie n e showed t e n tim e s th e
l e v e l o f a c t i v i t y a s t h e o th e r compounds o f t h e s e rie s =
H ed an sted t
(H ed en sted tr 1978) re p o rte d t h a t e n e r d ie n e r t r i e n e and Ne th y lm o p h o lin e h ad a d e te c ta b le d i r e c t m u tag en ic e f f e c t u s in g TAl535
and TAlOO t e s t e r s t r a i n s .
A d d itio n a lly / a t l e a s t tw o o f th e compounds
w ere t e s t e d f o r a lk y la tin g p r o p e r tie s by means o f a r e a c tio n w ith 4 -(p n itro -b e n z y D p y r id in e .
D iene and N -e tiy lm o rp h o l in e gave p o s i t i v e t e s t
f o r a l l y l a tin g p r o p e r tie s .
S in c e th e p o ly a m in e s th e m s e lv e s a r e n o t
a lk y la tin g , H ed en sted t s u g g e s ts th e p re se n c e o f an im p u r ity , such a s
e tly le n e im in e , i s p r e s e n t and r e s p o n s ib le f o r th e m utagenic e f f e c t .
F olyam ines c an be p o ly v a le n t c a tio n s .
The n a tu r a l o c c u rrin g
p o ly am in es a r e m u ltif u n c tio n a l and th e s u b je c t p f e x te n s iv e re s e a rc h .
P u tr e s c in e , sp erm in e and s p e rm id in e a r e v i r t u a l l y u b iq u ito u s in
m u ltip ly in g c e l l s (Cohn, 1971? B ach rach , 1973? Heby e t a l . , 1975? R aina
and J a n n e , 1975).
The f u n c tio n s o f th e n a tu r a l p o ly a m in e s in c lu d e
g ro w th s tim u la tio n (Cohn, 1977).
S p erm id in e h a s been found t o in c re a s e
g ro w th r a t e s by a id in g t h e a s s o c ia ti o n o f rib o so m a l s u b u n its (Cohn and
L ic h te n s te in , 1950).
The a cc u m u la tio n o f p o ly am in es i n t i s s u e s w hich
a r e undergoing r a p id g ro w th , h a s l e d t o th e s u g g e s tio n t h a t th e n a tu r a l
p o ly am in es have p o t e n t i a l a s m a rk e rs o f can cero u s c o n d itio n s
(B achrach, 1976).
w ith DNA.
The n a tu r a l p o ly am in es have been fo u n d a s s o c ia te d
I n DNA phage, th e p o ly am in es condense t h e DNA m o lecu le
c r e a tin g a com pact m ass t h a t i s m ore e a s i l y in c o rp o ra te d i n t o a c a p s id .
34
Sparm ine and s p e rm id in e have been found a s s o c ia te d w ith th e a l t e r e d DNA
c o n fo rm a tio n s i n w hich th e s u g a r -p h o s jh a te backbone f o llo w s a z ig za g
c o u rs e (Wang, Q u igly and K olpak, 1981)«
The s t r u c t u r a l
s i m i l a r i t i e s of th e t e s t e d p o ly a m in e s t o th e n a tu r a l o c c u rrin g
p o ly a m in e s, t h e m utagenic a c t i v i t y o f th e t e s t e d p o ly am in es and
a s s o c ia ti o n o f th e n a t u r a l p o ly am in es w ith DM s u g g e s ts t h a t a u nique
mechanism o f m u ta g e n esis may e x i s t .
STA!IEMENT OF THE PURPOSE
To c o n firm and q u a n t i t a t e th e m u tag en ic a c t i v i t y o f t h e
p o ly a m in e s e th y le n e d ia m in e , d ie th y le n e tr ia m in e , t r i e t h y l e n e t e t r amine
and te tra e th y le n e p e n ta m in e =
To show th e m utagenic a c t i v i t y o f t r i e t t y l e n e t e t r amin e i s due t o
t h e p o lyam ine i t s e l f and n o t e tiy le n e im in e o r s im ila r a lk y la tin g
c o n ta m in a n t.
To s u g g e s t a p o s s ib le mechanism f o r th e m utagenic a c t i v i t y o f
t r i e t h y l e n e t e t r am ine.
MATERIALS AND MEIHQD
The p o ly a m in e s w ere a ssa y e d a s t o t h e i r d o se re s p o n se u sin g th e
Ames t e s t (Amets, McCann and Y am asaski, 1975).
T h is b a c t e r i a l system i s
a S a lm o n e lla t yphim urium h i s t i d i n e r e v is io n a ss a y .
Ih e fiv e s tr a in s
u sed f o r th e s e a s s a y s , TA98, TALOO, TAl535, TAl538 and TA1537, were
g e n e ro u sly s u p p lie d by Dr. Ames.
The g ro w th m edia used w as V ogel-
Bonner (VB) made w ith D ifc o a g a r.
Ih e b a c t e r i a w ere added t o th e p e t r i
p l a t e v i a a n o v e rla y a g a r a s d e s c rib e d by Ames.
The p a t t e r n s o f
m u ta g e n ic ity f o r t r i e n e and e th y le n e im in e w ere e s t a b l i s h e d by th e s p o t
t e s t v a r i a t i o n o f th e Ames t e s t a ssa y .
P r e p a r a tio n o f th e polyam ine s o lu tio n s
S o lu tio n s o f e n e, d ie n e , t r i e n e and t e t r a e n e w ere p re p a re d a t a n
i n i t i a l c o n c e n tr a tio n o f 100 nanom oles (nM) p e r m i c r o l i t e r (A) o r
0.1M.
Each compound was added t o th e to p a g a r i n a l i q u o t s o f te n ,
t h i r t y and f i f t y m i c r o l i t e r s .
u sed d i r e c t l y from th e b o t t l e .
For th e s p o t t e s t s , th e compounds w ere
F i l t e r p a p er d i s c s w ere s a tu r a te d w ith
f i f t e e n m i c r o l i t e r s o f each compound.
Ene, d ie n e , t r i e n e and t e t r a e n e
w e re o b ta in e d from A ld ric h C hem ical Company and w ere o f te c h n ic a l
g ra d e .
P r e p a ra tio n o f e th y le n e im in e and C le la n d 1S re a g e n t s o lu tio n s
E th y ie n e im in e s o lu tio n was p re p a re d a t a c o n c e n tra tio n o f 3.09 x
IO**3 m oles p e r l i t e r .
A liq u o ts o f te n , t h i r t y and f i f t y m i c r o l i t e r s o f
t h i s s o l u tio n w ere added t o t h e to p a g a r b e fo re p la tin g .
A dose
37
re s p o n s e f o r C le la n d 's r e a g e n t was d e te rm in e d u s in g a n i n i t i a l
c o n c e n tr a tio n tw e n ty tim e s t h a t o f th e e t t y le n e im in e or# 6.18 x ItH 2
m oles p e r l i t e r .
T h is s o l u tio n w as th e n added t o th e t o p a g a r i n te n ,
tw e n ty , t h i r t y , f o r t y and f i f t y m i c r o l i t e r a l i q u o t s .
P r e p a ra tio n o f t h e m ixed s o lu tio n s
Four m ixed s o lu tio n s w ere p re p a re d a s fo llo w s :
1) T rie n e and C le la n d 's re a g e n t:
t r i e n e a t a c o n c e n tr a tio n o f
O=IM w as added i n e q u a l volum e t o a s o lu tio n o f 6 J.8 x IO-r2M C le la n d 's
re a g e n t.
I h e m ix tu re was a llo w ed t o s ta n d t h i r t y m in u te s b e fo re
a l i q u o t s o f te n , t h i r t y and f i f t y m i c r o l i t e r s w ere added t o t h e to p
ag ar.
2) E tty le n e im in e and C le la n d 's re a g e n t:
e t t y le n e im in e a t a
c o n c e n tr a tio n o f 3=09 x IO- 3 M was added t o a 6J.8 x IO- 2 M s o lu tio n o f
C le la n d 's re a g e n t.
The m ix tu re w as a llo w e d t o s ta n d t h i r t y m in u te s
b e fo re a l i q u o t s o f te n , t h i r t y and f i f t y m i c r o l i t e r s w ere added t o th e
to p a g a r.
3) T rie n e and e tty le n e im in e :
0.1M o f t r i e n e w as added t o 3,09
x IO- 3 M e t t y le n e im in e i n e q u al volumes=
The m ix tu re was a llo w e d t o
s ta n d f o r t h i r t y m in u te s b e fo re i t w as added t o th e to p a g a r , i n te n ,
t h i r t y and f i f t y m i c r o l i t e r a l i q u o t s .
4) T rie n e , e tty le n e im in e and C le la n d s 's re a g e n t:
0.1M 3.09 x IO-3
M e tty le n e im in e and 6.18 x IO- 2 M C le la n d 's re a g e n t w ere m ixed i n eq u al
volum es.
The m ix tu re w as a llo w e d t o s ta n d t h i r t y m in u te s b e fo re i t w as
38
added t o th e to p a g a r , i n t e n , t h i r t y and f i f t y m i c r o l i t e r a liq u o ts .
P r e p a r a tio n o f m edia
The to p a g a r and p e t r i p l a t e s w ere p re p a re d a s d e s c rib e d by Ames
(Ames, McCann and Y am asaki, 1975).
The g ro w th m edia f o r t h e i n i t i a l
c u l t u r e was s ta n d a rd D ifc o n u t r i e n t b ro th .
A ssay o f a c t i v i t y
The t e s t p l a t e s w e re in c u b a te d a t 37°C f o r t h r e e days.
H is tid in e
in d ep en d en t c o lo n ie s on th e d o se re s p o n se t e s t p l a t e s w ere co u n ted
u s in g a B io tr a n I I a u to m a tic c o lo ry c o u n te r.
S pot t e s t p l a t e s w ere
v i s u a l l y a ss a y e d f o r r e v e r t a n t c o lo n ie s su rro u n d in g th e s a tu r a te d
d is c s
T e s t c u l t u r e s w ere p re p a re d from one m ili l i t e r o f s to c k c u l t u r e in
n in e m i l i l i t e r s o f n u t r i e n t b ro th .
The c u l t u r e was. in c u b a te d i n a 370C
sh a k e r b a th f o r tw o t o tw o and one h a l f h o u rs.
The v ia b le c o u n t was
d e term in e d u s in g a s p e c tro m e te r and s ta n d a rd c u rv e t o a s s u r e c o u n ts
o v e r I x 10 8.
P ro ced u re f o r com bining t h e b a c t e r i a and c h em ica l a s
w e ll a s th e p l a t i n g te c h n iq u e s fo llo w e d , a r e d e s c rib e d by Ames (Ames,
McCann and Y am asaki, 1975).
Each t e s t d o se was p la te d i n t r i p l i c a t e
and re p e a te d on th r e e s e p a r a te d ay s t o in s u r e accu racy and
r e p r o d u c ib ility .
Background l e v e l s o f sp o n tan eo u s r e v e r t i o n t o
p r o to tr o p ly w ere d e te rm in e d by in c lu d in g c o n tr o l p l a t e s c o n ta in in g n o
39
m utagen.
H is tid in e in d e p en d e n t co lo n y c o u n ts on th e background p l a t e s
w ere r e q u ir e d t o be w ith in t h e l i m i t s l i s t e d i n T ab le I b e fo re th e t e s t
was c o n sid e re d v a lid .
T ab le I ,
Background c o u n t l i m i t s f o r t h e s t r a i n s o f S a lm o n e lla
t yphimurium.
S tra in
Count
TAl535
15-25
TAl537
10-20
TA1538
15-30
TA98
25-60
TAlOO
110-180
A ll p l a t e s w ere exam ined f o r t o x i c i t y by v i s u a l l y a s s u rin g a c o n flu e n t
lawn o f m ic ro c o lo n ie s b e n e a th t h e h i s t i d i n e in d ep en d en t c o lo n ie s .
Dose re s p o n se g ra p h s w ere c o n s tr u c te d from th e l i n e a r p o r tio n of
th e d o se re s p o n se c u rv e .
Each p o i n t r e p r e s e n ts c o u n ts fro m n in e p l a t e s
i n t e s t s conducted on t h r e e s e p a r a te d a y s.
P l a t e s w ere p re p a re d th e same way a s f o r th e d o se re s p o n se t e s t
w ith th e e x c e p tio n o f th e a d d itio n o f t h e mutagen.
/i
S te rile f i l t e r
p a p er d i s c s w ere s a tu r a te d w ith f i f t e e n m i c r o l i t e r s o f t e s t compound.
The d i s c s w ere th e n p la c e d i n th e c e n te r o f th e p l a t e a f t e r th e to p
a g a r c o n ta in in g b a c t e r i a had s o l i d i f i e d .
A fte r th r e e d ay s of
40
in c u b a tio n a t 37 °C th e p l a t e s w e r e .v is u a lly a ssa y e d f o r zo n es o f
in c re a s e d r e v e r s io n t o prototroEhy®
I h e s p o t t e s t p l a t e s w ere exam ined
f o r t o x i c i t y th e sam e way a s th e d o se re sp o n se p l a te s .
One gram o f t r i e n e t e t r a c h l o r i d e s a l t w as d is s o lv e d i n seven
m i l i l i t e r s o f 10% KCl a t 25°C,
100 m il ! l i t e r s o f 100% e th a n o l was
added t o p r e c i p i t a t e th e t e t r a c h l o r i d e s a l t .
The p r e c i p i t a t e was
f i l t e r e d and washed w ith 20 m i l i l i t e r s o f 100% a lc o h o l,
was re p e a te d tw ic e .
Ih e p ro c e ss
RESULTS
Q uantifcation o f th e m u ta g e n ic ity o f e n e . d ie n e » t r i e n e and te tr a e n e i n
TAlOO
U sing s t r a i n TAlOO7 w hich d e t e c t s b a se p a i r s u b s t i t u t i o n s , en e,
d ie n e , t r i e n e an d t e t r a e n e w ere found t o have a c t i v i t y o f 0JD048,
0.0057, 0.052 and 0.0058 H is+ re v e rta rits /n a n c m o le , r e s p e c tiv e ly .
The
in d iv id u a l a c t i v i t y l e v e l s a r e shown g r a p h ic a lly i n F ig u re 19.
When t r i e n e was t e s t e d on s t r a i n s TA1535, TA1537, TA1538, TA98 and
TAlOO a c t i v i t y was found i n TAl535, TA98 and TA100 w ith t h e m ost
a c t i v i t y shown i n TAl00.
T rie n e was tc& ic t o TM537 and TA1538 a t a
dose o f f o r t y nonom oles and g r e a te r a s was in d ic a te d by a la c k of
m ic ro c o lo ry c o n flu e n c e a t th e s e d o sa g e s.
Q u a n tita tio n o f th e m u ta g e n c ity o f t r i e n e and efchy len eim in e* w ith and
w ith o u t C le la n d 8S re a g e n t
I h e a c t i v i t y o f t r i e n e was d e te rm in e d t o be 0.052
re v e rta n ts /n a n o m o le i n TA100.
When C le la h d 1s r e g e n t was m ixed w ith th e
t r i e n e , t h e m u tagenic a c t i v i t y rem ain ed a p p ro x im a te ly unchanged a t
0=048 re v e rta n ts /n a n o m o le o f t r i e n e (F ig u re 20).
a c t i v i t y o f 13 re v e rta n ts /n a n o m o le i n TAl 00.
E tly le n e im in e showed
When Cl e la n d 's r e c e n t
was added t o t h e im in e , t h e a c t i v i t y o f t h i s mutagen was c o m p le te ly
quenched and d e te rm in e d t o c au se no r e v e r s io n above th e background
l e v e l (F ig u re 21).
Cl e la n d 's re a g e n t showed no m utagenic a c t i v i t y
above th e background l e v e l over th e ran g e o f c o n c e n tr a tio n used in
m ix tu re s w ith t r i e n e a n d /o r im in e (F ig u re 22).
The above r e s u l t s a r e
42
HIS+ REVERTANTS / NANOMOLE
TRIENE
.0 3 0
DIENE
.0 0 4 8
TETRAENE
.0 0 5 7
.0 0 5 8
REVERTANTS / NANOMOLES
INDIVIDUAL COMPOUNDS
F ig u re 19.
The m u ta g e n ic ity o f th e s e r i e s o f t e s t e d p o ly am in es.
43
(5,383)
L.SD=67
(5,353)
SD=58
3 4 0
_J 3 0 0
H
73,288)
SD =75
2 6 0
TRlENE
AND CLELANDS REAGENT
IOO--
NAN0M0LES OF TRIENE x IO3
F ig u re 20.
M u ta g e n ic ity o f t r i e n e w ith and w ith o u t Cl e la n d 's re a g e n t
in TM 00.
44
1927,257)
SD-23
IMINE
IMINE AND CLELAND'S
REAGENT
'(.309,157)
SD=SO
(1.54,130)
SD=30
(.927,137)
SD=33
0 .3
0 .6
0 .8
1 .2
1 .5
1 .8
NANOMOLES OF IMINE
F ig u re 21.
M u ta g e n ic ity o f e th y le n e im in e w ith and w ith o u t C le la n d 1s
re a g e n t i n TA100.
45
TAIOO: MUTAGENICITY OF
CLELAND'S REAGENT
'(1.86,154)
rSD=17
.(.0618,140)
SD=9
IO G --
(4.64,149)
SD=ZO
(6.18,125)
SD=IS
(3.09,108)
SD=40
MOLES OF CLELAND'S REAGENT x IO''
F ig u re 2 2 .
D ie m u ta g e n ic ity o f C le l a n d 's re a g e n t i n TAlOO
46
sum m arized i n T ab le 2,
d e g re e s o f freedom ,
S ta n d a rd d e v ia tio n was d e te rm in e d u s in g n -1
t o e s lo p e o f th e l i n e b e s t f i t t i n g t h e d a ta p o in ts
i n F ig u re 2 0 , 2 1, and 22 r e p r e s e n ts th e m u ta g e n ic ity of. th e compound
and i s e q u a l t o r e v e r t a n t s p e r nanom ole o f mutagen.
How w e ll th e d a ta
p o in ts f i t t h e s t r a i g h t l i n e i s in d ic a te d by th e c o r r e l a t i o n
d e te rm in a tio n i n T ab le 2 ,
T ab le 2. M u ta g e n ic itie s o f C le la n d eS re a g e n t t r i e n e w ith and w ith o u t
C le la n d 's r e a g e n t, and e th y le n e im in e w ith and w ith o u t C le la h d 's
r e a g e n t.
Commund
His+ Rever t a n t s/N ananol e
C o r r e la tio n
C le la n d 's R eagent
0
0.32
T rie n e
0.052
0.9 9
T rie n e and
C le la n d 's R eagent
0.048
0.97
E th y len eim in e
E th y len eim in e and
C le la n d 's R eagent
13
0 .9 9
0
-0 .7 3
P a tte r n s o f m u ta g e n ic ity o f e t fyyleneim ine and t r i e n e i n T&1535, TAlOO
and TA98
To e s t a b l i s h a d i f f e r e n t i a l b etw een e th y le n e im in e m u ta g e n esis and
t r i e n e m u ta g e n e sis, th e compounds w are in d iv id u a lly t e s t e d u sin g
s t r a i n s o f TAl535, TA100 and TA98,
I t was found t h a t t h e tw o m utagens,
e th y le n e im in e and t r i e n e , d id n o t d e m o n stra te th e same p a t t e r n of
a c t i v i t y i n th e s e s t r a i n s a s shown i n T ab le 3 ,
47
T ab le 3. E b tte m s of a c t i v i t y of e t t y le n e im in e and t r i e n e i n TM 535,
TMOO and TA98.
TM535
TM00
E tiy le n e im in e
+
+ '
T rie n e
+
+
TA98
+
Minor r e s u l t s
To d e m o n stra te th e a d d itiv e e f f e c t o f th e two m utagens,
e t t y le n e im in e and t r i e n e w ere m ixed i n e q u al volum es a t c o n c e n tra tio n s
o f 3«09 x 10“ % and OoOlM, r e s p e c tiv e ly .
The m ix tu re c o n ta in in g th e
tw o m utagens was t e s t e d f o r dose re sp o n se u s in g TMOO=
A second
m ix tu re was made t o c o n ta in Cl e la n d 's re a g e n t a t 6=18 x 10“ % and,
e t t y le n e im in e and t r i e n e a t th e above c o n c e n tr a tio n s a l l i n eq u al
volumes=
TMOO=
T h is second m ix tu re was a l s o t e s t e d f o r d o se re s p o n s e u sin g
The r e s u l t s a r e g r a p h ic a lly p re s e n te d i n F ig u re 23=
M u ta g e n ic ity i n te rm s o f H is+ r e v e r ta n t s p e r nanom ole was
a g a in c a lc u la te d from th e l i n e a r p o r tio n o f each d o se re s p o n se curve=
Those d a ta a r e re p o rte d i n T ab le 4 a lo n g w ith th e c a lc u la te d s t r a i g h t
l i n e c o rre la tio n =
The in d iv id u a l m u ta g e n ic ity of t h e t r i e n e and
'
e t t y le n e im in e a r e a l s o in c lu d e d f o r comparison=
S in ce th e t e s t
s u b s ta n c e s in c lu d e m ix tu r e s , i t i s n e c e s s a ry t o r e p o r t t h e i r
m u ta g e n ic ity i n te rm s o f th e in d iv id u a l component mutagens=
Sperm ine and p u tr e s c in e w ere d o se re s p o n se a ssa y e d u s in g TMOO=
The d o sag es ranged from IO"6 t o 2 x 10” 5 m o les p e r p l a t e f o r b o th th e
48
(5,459)
50=60
HIS+ REVERTANTS / PLATE
(3,383)
50 =51
(1,158)
50=65
(5,455)
50=92
(3,369)
50=86
IMINE AND TRIENE
!MINE, TRIENE AND
CLELANDS REAGENT
NANOMOLES OF TRIETHYLENETETRAMINE X IO'3
I
I
I
.3 0 9
.9 2 7
1 .5 4
NANOMOLES OF ETHYLENEIMINE
F ig u re 23.
M u ta g e n ic ity o f e t± y le n e im in e and t r i e n e w ith and w ith o u t
C le la n d 1S re a g e n t i n TA100.
49
T ab le 4. The m u ta g e n ic ity o f th e m ix tu r e s , e th y le n e im in e and t r i e n e ,
e th y le n e im in e , t r i e n e and C le la n d 1s r e a g e n t, i n term s o f th e in d iv id u a l
mutagens=
T est
S u b stan ce
R e v e r ta n ts /
Nanomole
o f !m ine
R e v e r ta n ts /
Nanomole
c£ T rie n e
C o r r e la tio n
E th y len e im in e & T rie n e
24
0.072
0 .9 8
E th y len e im in e & T rie n e
and C le la n d 1S re a g e n t
20
0.063
0.99
E th y len e im in e a lo n e
13
NA
0.9 9
T rie n e a lo n e
NA
0.052
0=99
sp erm in e and p u tr e s c ih e t e s t s .
The r e s u l t s showed no m utagenic
a c t i v i t y above th e background l e v e l o f sp o n tan eo u s r e v e r s io n t o
p ro to tro p h y .
Two compounds o th e r th a n C le la n d ss r e g e n t w ere t e s t e d f o r
t h e i r re d u c tio n o f e th y le n e im in e i n t h e Antes a ssa y .
C y ste in e and
g lu ta th io n e w ere t e s t e d in d iv id u a lly by making m ix tu r e s w ith
e th y le n e im in e .
C le la n d 1s r e a g e n t, c y s te in e and g lu ta th io n e were t e s t e d
a t tw o c o n c e n tr a tio n s , 1.85 x 10“ % and 6.18 x 10” ^M.
E th y len eim in e
was m ixed w ith th e compounds i n e q u al volum e a t a c o n c e n tr a tio n o f 3.09
x 10“ % .
p e r p la te .
The m ix tu r e s w e re M ies t e s t e d u s in g TA100 and 100 m i c r o l i t e r s
The r e s u l t s a r e sum m arized i n T ab le 5.
Each His+
r e v e r t a n t s c o u n t i s th e a v erag e c o u n t o f t h r e e t e s t p l a t e s .
50
T ab le 5« H is+ r e v e r t a n t c o u n ts f o r e th y le n e im in e w ith and w ith o u t
c y s te in e ? g lu ta th io n e and C l e l a n d 's reag en t=
09 x 10“ % E th y len e im in e an d :
1=85 x 10“ % C y ste in e .
. 112 .
6=18 x 10“ % C y ste in e
no t e s t
1 .8 5 x 1 0 " % G lu ta th io n e
;
371
6=18 x 1 0 " % G lu ta th io n e
323
1 .8 5 x 1 0 "% C lelaiya1s re a g e n t
197
6 .1 8 x 10“ % C le la n d 1S re a g e n t
93
09 x 10“ % E th y len e im in e
M icrocolony
Lawn C onfluence
H is+ R e v e rta n ts
346
y
, '
y:
+
+
.+ .
DISCUSSION
Hie mutagenicity of trien e
The m u ta g e n ic itie s o f en e, d ie n e , t r i e n e and t e t r a e n e w ere
e s ta b lis h e d , u s in g TAlO0, t o be 0 4 0 4 8 , 0 4 0 5 7 , 0 4 5 2 and 0 4 058 H is+
r e v e r t a n t s p e r nanom ole, r e s p e c tiv e ly .
From th e s e d a ta , i t c an be see n
t h a t t r i e n e h a s a c t i v i t y t e n tim e s t h a t o f any o f th e o th e r th r e e
compounds th e s e r i e s .
H edensted t (H ed en sted t, 1978) p r e v io u s ly
o b serv ed t h e m u ta g e n ic ity o£ e n e , d ie n e and t r i e n e i n TA100 and TAl535.
She su g g este d th e e f f e c t i s due t o th e p re sen c e of an a l l y l a t i n g
compound su ch a s e th y len eim in e .
p o ly am in es.
i_
I t i s an a lk y la tin g a g e n t and known m utagen (F ig u re 24).
I I I I In »
!MINE
F ig u re 2 4 .
E tJy lem eim in e i s th e monomer of th e s e
I tm—
'
GUANINE
ALKYL-GUAN IN E
An example o f a lk y la tio n o f a base by e tiy le n e im in e .
To e s t a b l i s h th e m utagenic a c t i v i t y o f t r i e n e , i t s e l f , t h r e e l i n e s o f
evidence w ere p u rsu e d .
te tra c h lo rid e s a lt.
re s p o n se a s s a y s .
I) T rie n e was re p e a te d ly r e c r y s t a l l i z e d a s th e
The r e c r y s t a l l i z e d s a l t was used i n a l l d o se
2) C le la n d 's r e a g e n t, D ,L - d i th io th r e ito l, was added
52
a s a s a c r i f i c i a l n u c le o p h ile t o r e a c t w ith any a lk y la tin g c o n ta m in a n t
such a s e th y le n e im in e a s shewn i n F ig u re 25»
The a d d itio n o f C ie la n d 8S
re a g e n t would a l s o rem ove any o th e r s im ila r a lk y la tin g s p e c ie s t h a t may
be p r e s e n t a s c o n ta m in a tio n ,
3) P a tte r n s o f m u ta g e n ic ity f o r t r i e n e
and e th y le n e im in e w ere e s t a b l i s h e d u s in g TA98, TALOO an d TAL535,
T h is
w as done t o d i f f e r e n t i a t e th e a c t i v i t y o f t r i e n e from e th y le n e im in e .
Upon re p e a te d r e c r y s t a l l i z a t i o n s , t r i e n e r e ta in e d i t s m utagenic
a c t i v i t y o f 0.052 H is+ r e v e r t a n t s p e r nanom ole.
When C le la n d 8s
re a g e n t was added t o th e t r i e n e , th e a c t i v i t y rem ain ed r e l a t i v e l y
unchanged (F ig u re 20).
C le la n d 8S re a g e n t was shown t o e lim in a te th e
m utagenic a c t i v i t y o f e th y le n e im in e a s shown i n F ig u re 21.
I f th e
a c t i v i t y o f t r i e n e was i n f a c t a r e s u l t of ethy le n e im in e o r seme o th e r
a lk y la tin g c o n ta m in a n t, t h e a d d itio n o f C le la n d 8S r e a g e n t t o th e t r i e n e
sh o u ld e lim in a te t h a t a c t i v i t y .
A gain, t h e a d d itio n o f C le la n d 8s
re a g e n t d id n o t s i g n i f i c a n t l y change t h e m u ta g e n ic ity of t r i e n e ,
T rie n e d e m o n stra te d d i f f e r e n t i a l m u ta g e n ic ity from e th y le n e im in e when
b o th w ere a ss a y e d u s in g TA98, TAL00 an d TM 535.
E th y len e im in e d id n o t
show m u tagenic s p e c i f i c i t y f o r TA98 w here a s t r i e n e d id (Table 2 ).
can be con clu d ed from th e s e e x p e rim e n ts t h a t t r i e n e i t s e l f i s
m u tag en ic.
I t i s im p o rta n t t o n o te t h a t C le la n d 8S re a g e n t i s n o t m utagenic
(F ig u re 22).
I t was s e l e c t e d a s a s a c r i f i c i a l n u c le o p h ile over
It
53
E T H Y .L E N E
[M INE
C L E L A N D 'S R E A G E N T
H^NCCH2^SCH2(CH O H ^ 29H2S(C H 2 \ NH2
MOMMUTAGENSC T O D U O T
F ig u re 2 5 .
The r e a c tio n o f e th y le n e im in e w ith C le l a n d 's re a g e n t.
54
g lu ta th io n e and c y s te in e b ecau se i t d id n o t show t o x i c i t y a t 6,18 x
10
c o n c e n tr a tio n and was e f f e c t i v e a t e lim in a tin g a lk y la tin g
a c t i v i t y of e th y le n e im in e p r e p a r a tio n s a f t e r p re in c u b a tio n (Table 5),
An a d d itiv e e f f e c t was e x h ib ite d when t r i e n e and e th y le n e im in e
w ere a ss a y e d i n m ix tu re (T able 4),
I n te rm s of tr i e n e " s a c t i v i t y , a n
in c r e a s e from 0,052 H is^ re v e rta n ts /n a n o m o le f o r th e in d iv id u a l
m utagen, t o 0.072 H is+ re v e rta n ts /n a n o m o le f o r th e m ix tu re was shown.
I n te rm s o f e th y le n e im in e , an in c r e a s e from 13 t o 24 H is+
re v e rta n ts /n a n o m o le was shown f o r t h e in d iv id u a l m utagen an d m ix tu r e ,
r e s p e c tiv e ly .
The a d d itio n o f C le la n d 1s re a g e n t red u ced t h e a c t i v i t y
o f th e m ix tu re b u t n o t t o t h e l e v e l o f t h e t r i e n e a lo n e .
I t is fe lt
t h a t by in c r e a s in g th e c o n c e n tra tio n o f th e Cl e la n d 's r e a g e n t, th e
a c t i v i t y o f t h e m ix tu re w ould ap p ro ach t h a t l e v e l shown by t r i e n e ,
0 ,0 5 2 His+ re v e rta n ts /n a n o m o le .
The n a t u r a l l y o c c u rrin g p o ly a m in e s, s p e rm id in e , sp erm in e and
p u tr e s c in e , w ere t e s t e d u s in g TAlOO and found t o be nonm utagenic.
T hese r e s u l t s a r e n o t unexpected when one r a t i o n a l i z e s t h e e v o lu tio n a ry
b a s i s and c o n s id e r s t h e im p o rtan c e o f m a in ta in in g th e f i d e l i t y o f th e
r e p l i c a t i o n p ro c e s s .
A P o s s ib le mechanism
The e s ta b lis h m e n t o f m utag en ic a c t i v i t y a t 0.0048, 0.0057 , 0,052
and 0.0058 H is+ re v e rta n ts /n a n o m o le f o r e n e , d ie n e , t r i e n e and
t e t r a e n e , r e s p e c tiv e ly , le a d s t o t h e q u e s tio n o f why t r i e n e shows te n
55
tim e s th e . a c t i v i t y o f th e o th e r compounds i n th e s e r i e s .
d i f f e r s from d ie n e and t e t r a e n e b y o n ly a
TTiene
g ro u p .
M ahler
an d M tiiro tro showed t h a t d ia m in e s o f th e g e n e ra l fo rm u la , HgN(CHg)nNHg
2^n<10, a s s o c ia te d w ith DNA (M ahler and M eh ro tra , ISS2).
The
a s s o c ia ti o n was in v e s t ig a te d by means o f th e t r a n s i t i o n fro m
h e l i x — » c o i l and by a b so rb a n c y -te m p e ra tu re p r o f i l e s .
DNfiramine a s s o c ia ti o n t o depend on f i v e p a ra m e te rs .
t h e io n ic S tre n g th ,
io n ic s tr e n g th ,
They found th e
They in c lu d e , I)
The a s s o c ia ti o n i s s tre n g th e n e d by a d e c re a s e i n
2) The c o n c e n tr a tio n o f am ine i s a p a ra m e te r.
A t low
c o n c e n tr a tio n th e te m p e ra tu re o f th e m id p o in t cdE t r a n s i t i o n , Tm, i s
dependent on t h e am ine c o n c e n tra tio n ,
A t h ig h e r c o n c e n tr a tio n s , t h e
change i n Tm re a c h e s a maximum v a lu e t h a t c o rre sp o n d s t o s a t u r a t i o n o f
th e MiA w ith am in e.
3) The c h a in le n g th o f t h e am ine was found t o
e f f e c t th e DNA-amine a s s o c ia tio n .
t o h av e th e g r e a t e s t
HgN(CHg) ^NHg, c ad a v e rin e , was found
Tm f o r h e l i x — -> c o il.
4) The b a se c o m p o sitio n
o f th e DNA, t h a t i s , t h e % thym in e and a d e n in e , a l s o v a r ie d th e
t r a n s i t i o n te m p e ra tu re .
F in a l l y , 5) th e DNA t o w hich th e d ia m in e s
a s s o c i a t e had t o be d ouble s tra n d e d .
P a r a l l e l s betw een t h e d ia m in e s and t h e p o ly am in es c an be used t o
a tte m p t a n e x p la n a tio n o f th e d if f e r e n c e i n m u ta g e n ic ity o f th e
p o ly am in es.
The pH v a lu e s o f s e le c te d p o ly am in es ap p ear i n T ab le 6. ,
A t p h y s io lo g ic pH, o n ly th e end am ines o f each o f t h e t e s t e d p o ly am in es
a r e io n iz e d .
T h e re fo re , a t p h y s io lo g ic pH? t h e p o ly am in es p a r a l l e l
56
T ab le 6«
V o l. 2)
B ie pK's o f s e le c te d p o ly am in es ( C r i t i c a l S t a b i l i t y C o n sta n ts
Ene
^H3HCH2CH2NHg
pK a t
2 5 0C
and
Io
H2LZHLeH
7o08±0o03
2o
HLZL0H
9o89±Q o07
+H3NCH2CH2NH2CH2CH2NH3
D iene
pK a t 25°C and OolM
I,
H3LZH2L0H
4 .2 3 ± 0 o 0 3
2o
H2LZHL0H
9„02±Po06
3o
H L Z L 0H
.9,84±0.05
T rie n e
. +H3 HCH2CH2NH2CH2CH2NH2CH2CH2N ^
pK
at
25°C
and
I,
H4LZH3 L0H
3o25±0o03
2.
H3LZH2L0H
6 .5 6 ± p o 0 2
3.
H2LZHL0H
9=08±0.02
4„
HLZL0H
9=74±P.06
Ttetraene +H3 NCH2 CH2 NH2 Oi2 OJ2 NH2 CH2 CH2 NH2 CH2 CH2 NH3
at
2 5 0C
and
I,
H5LZH4L0H
2o
H
3=
H3LZH2L0H
8o05±0o03
4»
H2LZHL0H
9 o l4 ± 0 o 0 4
5o
HLZL0H
9»70±0o04
4L Z H 3 L 0 H
O o lM
2o97±0o07
4o7Q ±0oO 2'
O o lM
O o lM
57
T^Dle Se (co n tin u ed )
D ip ro p y le n e tria m in e
"}"H3 r a 2a i 2<2*2I® 2a i 2a i 2a i 2i®3
pK a t 25°C and O6IM
I0
H3LZH2 LeH
7 ,7 2
2=
H2LZLHeH
9 ,5 7
3»
HtZLeH
2 ,3 ,2 T etram ine
1 0 ,6 5
+H3rai2 (2 i2 <:E2^H2CH2CH2a i 2NK$
pK a t 2 5°;C and 0,5M
1,
H4IZH3 L0H
6,02
2,
H3IZH2L0H
7 ,2 8
3,
H2IZLH0H
9 ,5 0
4,
LHZL0H
1 0 ,2 5
58
th e diamines=,
B inding o f th e d ia m in e s i s presum ed t o o ccu r through one
-"1NHg gro u p a t a p rim a ry s i t e on t h e DM* t h a t th e n f a c i l i t a t e s
b in d in g o f th e o th e r am ine o f some seco n d ary s ite =
The d e c re a s e i n th e
amount o f am ine b in d in g w ith in c re a s e d io n ic s tr e n g th i s e x p ec te d
b e h a v io r i f th e b in d in g , a t l e a s t a t th e p rim ary s i t e , i s io n ic ,
The
p h o sp h ate r e s id u e s i n th e ESiA back bone a r e th e m o st l i k e l y p rim ary
b in d in g s i t e s .
I t h a s been shown t h a t m u ltiv a le n t c a ti o n s , in c lu d in g
sp e rm in e , b in d a t th e phosphate oxygen s i t e s (M ahler an d M eh ro tfa,
ISS 2) o C o n sid e rin g th e b a s e c o m p o sitio n in flu e n c e on t h e d iam in e
a s s o c i a t i o n , M ahler and M eh ro tra p ro p o se t h a t th e seco n d ary s i t e of
-NHg b in d in g i s lo c a te d on a n a d e n in e o r thym ine r e s id u e .
The
sec o n d a ry s i t e m ust a l s o o c cu r o n th e s tr a n d o p p o s ite th e p rim ary
b in d in g s i t e s in c e o n ly d ouble s tra n d e d DNA e x h ib its t h e t r a n s i t i o n
e ffe c t.
The seco n d ary b in d in g s i t e f o r th e t e s t e d p o ly a m in e s may o r
may n o t be th e b a s e s .
S in ce no c o r r e l a t i o n betw een b a se c o m p o sitio n
and p o ly am in e m utagenic a c t i v i t y have been made, g h o s p ia te r e s id u e s
sh o u ld a l s o be c o n sid e re d a s p o s s ib le secondary b in d in g s i t e s .
An a d d itio n a l a s p e c t o f t h e io n i z a t i o n s t a t e s o f t h e am ines i s t h e
p o s s ib le e f f e c t s o f a c id i c o r f r e e b a se am ines.
The p re se n c e o f th r e e
o r more m eth y len e carb o n s betw een am ine groups d e c re a se t h e a c i d i t y o f
t h e c e n t r a l am ine s u f f i c i e n t l y such t h a t a l l am ines rem ain p ro to n a te d
a t p h y s io lo g ic pH,
The pK"s fo r d i p ro p y len et r i amin e a r e in c lu d e d i n
T ab le 6 f o r co m parison.
The n a tu r a l o c c u rrin g p o ly a m in e s, sp erm in e and.
59
sp erm id in e? th e r e f o r e would n o t r e t a i n p r o to n a tio n o f th e i n t e r i o r
amines=
T h is i s i n c o n t r a s t w ith th e m u tag en ic polyam ines= ' One o r
more i n t e r i o r am ines o f d ie n e , t r i e n e and t e t r a e n e a r e a l l i n t h e f r e e
b a s e s t a t e a t pH 7=
2,3 ,2 T e tra m in e i s a l s o in c lu d e d i n T ab le 6=
It
h a s been shown t h a t t h i s compound h a s m utagenic a c t i v i t y , b u t a t a
l e v e l lo w e r th e n t h a t shown by t r i e n e (VonHeinz and S c h ro d e r, 1981) =
When com paring th e m u ta g e n ic ity o f th o s e compounds h a v in g f r e e b ase
am ines t o th o s e h av in g p ro to n a te d a m in e s, t h e q u e s tio n a r i s e s w h eth er
th e f r e e b a se s t a t e c o n tr ib u te s t o t h e m u ta g e n e tic a c t i v i t y o f th e
compound=
W hereas th e f r e e b a s e may c o n tr ib u te t o th e a c t i v i t y , i t i s
f e l t t h a t i t i s n o t t h e m a jo r p a ra m e te r f o r m u ta g e n ic ity s in c e t e t r a e n e
c o n ta in s one m ore f r e e b a se th a n t r i e n e and i s one t e n t h a s a c ti v e .
F u rth e rm o re , p u tr e s c in e can a l s o be c i t e d a s a n a t u r a l l y o c c u rrin g
polyam ine t h a t h a s been shown t o have no m u tag en ic a c tiv ity =
I t la c k s
th e i n t e r i o r am ine so th e p re se n c e o f a p ro to n a te d am ine i s n o t th e
d if f e r e n c e betw een m u tagenic and non-muta g e n ic compounds=
I t h a s been proposed t h a t in c re a s e d s t a b i l i z a t i o n o f DNA m o lecu le
may c a u s e m u ta g e n ic ity ( S tr e is i n g e r e t al= , 1966)=
The m utagenic
a c r id in e s have been found t o in c r e a s e th e rm a l d e n a tu r a tio n te m p e ra tu re s
(Lem an? 1964)=
T h is i s a n alo g o u s t o th e in c re a s e o f
a s s o c ia ti o n o f d ia m in e s w ith d o u b le s tra n d e d DNfi=
Tm shown by th e
I p ro p o se t h a t
t r i e n e 's t e n f o l d m utag en ic a c t i v i t y o v er th e o th e r p o ly a m in e s i n th e
s e r i e s i s a r e s u l t o f a n in c re a s e d s t a b i l i z a t i o n o f t h e DNA molecule=
60
T cien e b in d s th e b a c t e r i a l DM i n a n i o n i c a s s o c ia tio n =
s i t e i s m o st l i k e l y a Phosgfoate resid u e=
Ohe p rim a ry
The seco n d ary b in d in g s i t e i s
lo c a te d on th e o p p o s ite s tr a n d and a t some d is ta n c e su ch t h a t t r i e n e
makes t h e o p tim a l f it=
S in ce t r i e n e i s somewhat lo n g e r th a n c ad a v e rin e
(th e d iam in e t h a t showed o p tim a l a s s o c i a t i o n ) , i t may be t h a t th e
seco n d ary b in d in g s i t e f o r t r i e n e i s a n o p p o s ite s tr a n d phosgfoate
residue=
As a r e s u l t o f t r i e n e s a s s o c i a t i o n , DM s y n th e s is may o c cu r
b e fo re th e re g io n m e lts o u t c a u s in g e r r o r s i n f i d e l i t y and th e
m u tagenic a c t i v i t y displayed=
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Pojyamme mutagenesis: evidence for a u
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Polyamine Mutagenesis:
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