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•S7 Dlebor, Samuel, 1926-
1952 effects of various ontimetaboliles
on the growth and development of F.nna
plpiens.
70p. illu s ., diegrs., tables.
Thesic (:h ,1.; - N.Y.lr. , Graduate
School, 19^2.
Bibliography: p,/j3~-'r0«
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THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
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THE EFFECTS OF VARIOUS ANTIMETABOLITES ON THE
GROWTH AND DEVELOPMENT OF RANA PIPIENS
Samuel Bieber
A pril 1958
A D issertation in: the Department of Biology Submitted
in P a rtia l F ulfillm ent of the Requirements fo r
the Degree of Doctor of Philosophy at
New York U niversity
TABLE OF CONTENTS
Acknowledgments.
Introduction Pages 1-5
M aterials and Methods Pages 6-10
Results:
Introduction Pages 11-13
I.. Comparative A ctivity
of the Analogues Pages 13-16
II* Growth Patterns as Re
vealed by A ntim etabolites Pages 16-19
III* Reversal Experiments Pages 19-31
m . M orphological R esults
A* Gross Pages 21-23
B. H istological Pages 23-27
D iscussion Pages 28-39
Summary Pages 40-42
Bibliography Pages 43-48
Appendix I
Tables I-III Pages 49-51
Plates 1-IX Pages 52-60
(30 Figures)
Appendix II
Data used in constructing
graphs (Figures 1-8) Pages 61-68
Appendix III
Source® o f H feterials Pages 69-70
3.0 I ^
ACKNOWLEDGMENTS
I would lik e to express my sincere appreciation
to Dr. Ross. F. N ig relli, under whose sponsorship th is
in v estig atio n was carried out.
The major portion of th is investig atio n was carried
out under a Burroughs-Wellcome Research Fellowship jo in tly
sponsored by New York U niversity and the New York Zoolo
g ical Society. I am g ratefu l to Dr. Harry A. Charipper
fo r making the fa c ilitie s of the Biology Department of
Washington Square College available to me and fo r acting
as adm inistrator of the Research Fellow ship.
I am indebted to Dr. George H. H itchings of the
Wellcome Research L aboratories fo r h is many suggestions
concerning the problem and the m anuscript and fo r sup
plying generous q u an tities of the analogues and metabo
lite s used in these stu d ies.
The am inopterin, fo lic acid and leucovorin used in
th is investigation were graciously supplied by Dr.
Ruegsegger of the Lederle L aboratories a t P earl River,
New York.
I would also lik e to thank Dr. Seymour Hutner of
the Haskins L aboratories and Dr* E li Goldsmith of the
College of D entistry for th e ir encouragement and con
stru ctiv e criticism .
The photomicrographs were prepared by Mr.. A lbert
Stenger of the Biology Department of Washington Square
College.
Last but not le a st I would lik e to thank my wife
Rosalyn fo r her aid in the preparation of th is manu
sc rip t.
- I -
INTRODUCTION
E hrlich, in the Harben Lectures of 1907 (13), was
the f ir s t to present ideas sim ilar to the present-day
concept of antim etabolites. While E h rlich 's views were
speculative, subsequent studies of enzyme actio n , bacter
ia l and animal n u tritio n , and studies of the pharmaco
lo g ical antagonisms of drugs have allowed the form ula
tio n of a concept of antim etabolites in more precise
term s. The a b ility of a substance, stru c tu ra lly resemb
ling one of the components of a reactiv e system , to in
h ib it the reaction was recognized by enzym ologists in ■
the early 1930's. The lite ra tu re of the related field s
of biochem istry and biology* in the period from 1920
to 1940, revealed several instances of chance observa
tions th at stru ctu ral analogues of a m etabolite In te r
fe re d w ith its biological a c tiv ity . Such findings,
remarkable as they were, were frequently branded as
erro rs. I t w asn't u n til the rep o rt of Woods in 1940 (53)
th at the biochemical im plications were realized . He
reported th at the b a cterio static actio n of sulfanilam ide
and related sulfonamide drugs was com petitively reversed
by p-aminabenzoic acid (PABA). PABA had not previously
been recognized as a bio lo g ically im portant compound, but
Woods' prediction was soon v e rified . The sulfonamide
drugs were thus viewed as stru ctu ral analogues of th is,
m etabolite and were said to owe th e ir b a cterio static
* 2 •
properties to th is fa c t. I t was in ferred th a t they acted
by producing a deficiency, in the b acterial c e ll, of th is
esse n tia l su b strate, w ithout which the organism was un
able to m ultiply.
The rep o rt of Woods prompted a widespread search fo r
chem otherapeutic agents among compounds stru c tu ra lly
related to m etabolites.. Since then the fie ld of in h ib i
tio n analysis has received tremendous impetus from the
synthesis of many such compounds. I t has also come to
include those compounds which, though not stru ctu rally
analogous to m etabolites, com petitively in h ib it th eir
u tiliz a tio n by an organism* Almost a ll of the pioneer
work in the fie ld has been carried .o u t on b acteria.
Interference w ith the u tiliz a tio n of th e ir m etabolites
was read ily reflected in an in h ib itio n of growth which
could be measured. Such studies have shed lig h t on many
of the in tric a c ie s of biochem ical mechanisms. Studies
carried out on higher forms have been prim arily concerned
w ith the e ffe cts on organ system s. Recently a few re
ports appeared concerning investigations carried out on
the developing chick (1, 8, 9, 31,; 48,, 52, 54). Aside
from the gross e ffe cts on survival and attem pts a t meta
bolic rev ersal of in h ib itio n , these in v estig ato rs did not
attem pt to observe the more subtle effects on growth and
on the d iffe re n tia tio n of the various organ system a, or
possibly on the in itia tio n of the various physiological
or biochem ical phenomena. Except fo r a b rie f attem pt by
Brachet (6) and a more recent prelim inary report by
Rosenbaum and Verlardo (40), amphibia have not been u ti
lized fo r such stu d ies. The amphibian egg lends its e lf
read ily to experim ental and biochem ical observations on
early morphogenesis. The chick embryo is not easily ac
cessible for chemical studies before the second or th ird
day of incubation, by which time early m orphogenesis is
ended.
H itchings and his collaborators have made an in ten
sive study of the in h ib itio n of the growth of Lacto
b acillu s easel and other systems by analogues of purine
and pyrim idine bases (5, 7, 14-19, 25-28, 42, 48). Over
a period of ten years several hundred such analogues were
synthesized and tested . They were found to have a v ariety
of biological effects which were attrib u ted to various
types and degrees of interference w ith the biosynthesis
of nucleic acids and related compounds. The key posi
tio n of nucleic acids in the fundamental processes of
growth and d iffe re n tia tio n has been w ell established.
Thus the d e sira b ility of testin g such antim etabolites on
a developing form is obvious. Such studies might co n tri
bute to the comparative biochem istry of nucleic acid
metabolism and to the knowledge of the mode of action of
the antim etabolites. These compounds might also provide
some knowledge as to the relatio n sh ip of sp ecific compounds
- 4 -
to morphologic development.
I t was the consideration of the preceding facts th at
led to undertaking the follow ing study of antim etabolites
and amphibian development. The leopard frog, Rana p iu ien s.
was chosen because its re la tiv e ly short period of develop-*
ment affords the opportunity to study the aforem entioned
phenomena. A prelim inary attem pt at such studies was
undertaken by B ieber, N ig relli and H itchings (4 ). This
study revealed th at despite the d iffic u ltie s inherent in
immersion techniques careful experim ental m anipulation
makes the approach fe a sib le . Along w ith the purine and
pyrim idine analogues, am lnopterin (4-am inopteroylglutam ic
acid) was used as a reference compound. The action of
am inopterin in in te rfe rrin g w ith nucleic acid synthesis
has been established in recent years (81, 45). This com
pound had previously been tested on the developing chick
(9, 31, 46, 58) and in a prelim inary fashion on the
developing frog (40).
As a re su lt of the prelim inary experim ent, the fo l
lowing long range p ro ject fo r the study of antim etabolites
on amphibian development was. se t up in conjunction w ith
the Wellcome Research Foundation. In cases where success
fu l In h ib itio n of development was obtained the follow ing
experim ental procedures were to be applied.
1. In itia tio n of treatm ent a t various stages of
development from fe rtiliz a tio n , through metamorphosis.
