VARIATIOENES EN 'E’HE C-MEYGHQ ACYWETY QF ,: ' '

COLCH'ICENE $QLUTIONS

Thesis for the Degree of Ph. D.
MECHEGhN STATE UNIVERSETY
Edward A Greenberg
3965

THESIS

This is to certify that the

thesis entitled

VARIATICIIS IN THE C-MITOTIC ACTIVITY OF

COLCHICINE SOLUTIONS

presented by

Edward Arthur Greenberg

has been accepted towards fulfillment
of the requirements for

Plum—degree inmtolow)
Botany and Plant Pathology

{fig/ea”

Major professor

Date Mia—19.65—

0-169

 

G.
LIBRARY

Michigan State
University

  

 

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ABSERACT

VARIATIONS IN THE C-NITCEIC ACPIVIEY OF

by Edward A. Greenberg

Ehis study sought to determine whether variations
in the c-mitotic activity of colchicine solutions would
occur when stored under certain "standard” laboratory
conditions, and when exoosed to ultraviolet light._It was
hoped that if modifications did occur that these could be
quantified.

fhe experimental materials were: seedlings of Pisum

sativum var. filaska; and the alkaloid colchicine in con-

 

centrations of #00 ppm (1 x 10'3N) for exposure to a fluor-
escent light source and ultraviolet light, and 200 ppm (S x
1O'MM) for treatment.

It was found that solutions exposed to ultraviolet
light lost appreciable c-mitotic activity. Exposure to
fluorescent light did not give conclusive results.

The results obtained suggest that it is -robable that
the beta and gamma photoisoners of colchicine are not very
active and may even be toxic.

It was again observed, as in a previous study (Green-
berg, 1962), that different sarples of the dry powder form

the alkaloid vary in their ability to induce the desired

Ft)

0

effects in our test system.

VARIA 1‘: 378 III 3111*) C-I‘III‘C'Z‘IC ACI‘I‘JIE‘I CF

COLCHICINE SOLUCIONS

3y

Wwfl
Edward A. Greenberg

A "HESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCBOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1965

A ,varvn' v‘.’ 7:7 1":1}""\"”"|S
V t r . ,. ,
llL/A...'L 1' 4—) dd)-.J.I.L

Ky sincere thanks to Dr. G. B. Wilson and Dr. J. C.
Elliott for their guidance and advice throughout my train-
ing program.

I also express my gratitude to Dr. A. F. Yanders, Dr.
w. B. Drew, Dr. E. S. Beneke and Dr. J. R. Shaver for have-
ing reviewed this manuscript. .

To my colleagues in the cytology group at kichigan
State University for their collaboration and counsel during
the course of this investigation.

fo hichigan State University, who made it possible
for me to continue my education through a teaching assist-
antship. And to the American Cancer Society, for partial

defraying of some of the research eXpenses.

1 III ll ‘

Plate I. Plants of the genus Colchicum growing in
the Horticulture Gardens of fiichigan State

University.

 

The Genus Colchicum (Hybrid "The Giant").

Plate I.

TABLE of CONTENTS

INPRODUCTION . . . . . . . . . . . . .
LITERAIURE REVIEW . . . . . . . . . .
KATERIALS AND METHODS
General EXperimental Procedures. . .
Fluorescent Light Experiments.
Ultraviolet Light Experiments.

Preparation of the Biological
Test Organism. . . . . . .

OBSERVATIONS . . . . . . . . . . . . .
DISCUSSION . . . . . . . . . . . . . .
SUMMARY AND COI‘SCLUSIOI‘JS . . . . . . . .
BIBLIOGRAPHY. . . . . . . . . . . . .
APPENDIX. . . . . . . . . . . . . . .

iv

LIST OF TABLES

PAGE

Table 1. Data for the Index of Effect Curves. . . . 29

APPENDIX:
EXPERINENIAL NOTE. . . . . . . . . . . . . . . . . . 55
Table I. Data on the Normal Post-prophases, Clumps,
Scatters and Per Cent Effects of the Fresh
and "Altered" Colchicine Solutions. . . . .56
Table II. Mitotic Indices of the Untreated Controls,
Fresh and "Altered" Colchicine Solutions. .61
Table III.Data and the Indices of Effect for the
Fresh and "Altered" Colchicine Solutions. .66
Table IV. Areas of the Curves of the Indices of
Effect, in Arbitrary Units, Obtained from
the Fresh and "Altered" Colchicine Solu-

tiOI’lS...o...............72

LIST OF PLATES AND FIGURES

PAGE
Plate 1. Plants of the genus Colchicum growing in

the Horticulture Gardens of Michigan State
University. . . . . . . . . . . . . . . . . .iii

Plate II. Mitosis in Pigum sativun. Normal Cytolo-

gical Configurations, and Figures Showing

the Effect of Colchicine. . . . . . . . . . 54

Figure 1. Structure of Colchicine and its Isomers. . 10
Figure 2. Index of Effect Curve for a Fresh Treat-

ment Colchicine Solution. . . . . . . . . . .23
Figure 3. Index of Effect Curve for a Solution Stored

2% Hours in the Dark at 22.5°c. . . . . . . 2a
Figure 4. Index of Effect Curve for a Solution Irradi-

ated 2% Hours with Ultraviolet Light. . . . .25
Figure 5. Index of Effect Curve for a Fresh Treat-

ment Colchicine Solution. . . . . . . . . . .26
Figure 6. Index of Effect for a Solution Stored 3

Weeks in the Dark at O-S°C. . . . . . . . . .27
Figure 7. Index of Effect for a Solution Stored 3 A

Weeks in the Dark at 22.5°C. . . . . . . . . 28

vi

INTRODUCTION

This study has been carried out as a continuation of
a previous study (Greenberg, 1962); both have dealt with
determining the biological activity remaining in colchicine
solutions kept under various "standard" laboratory condi-
tions and after irradiation with ultraviolet light.

The first study (Greenberg, 1962) dealt with colchi-
cine solutions kept in the dark at room temperature (21-23
°C) and under refrigeration (O-5°C), for predetermined
lengths of time.

The present investigation was made to determine
whether modifications occunmfl.in solutions of colchicine
exposed to fluorescent and ultraviolet light sources. If
modifications were observed in c-mitotic activity, it was
hOped that these could be quantified by the number and
kind of specific cytological configurations produced in a
biological test system.

Colchichuasolutions have been reported to be unstable
by several investigators. Eigsti and Dustin (1955) state
that solutions may lose as much as 20 per cent of their
activity after 5 weeks. Powell (1951) mentions that the
alkaloid maintains its activity rather well when refrig-

rated. Epstein (unpublished,1955) reports using a solu-
tion of colchicine that had been kept under refrigeration
for over 7 months, and noted no difficulty in obtaining

the desired results in treated Bradescantia staminal hairs.

 

Wood (1357) found through spectrophotometric and chemical

1

studies, that when colchicine solutions were kept in the
dark at room temperature and tested at regular intervals,
up to seven months, they did not show appreciable changes.
Van't Hof (1931) suggests that colchicine solutions should
be prepared five minutes prior to being utilized, because
the compound deteriorates rather rapidly in aqueous solu-
tions.

Hadder and Wilson (1958) carried out studies to
relate particular concentrations of fresh colchicine solu-
tions to specific effects observed in mitotic cells of
Pisum root tips. They proposed a mathematical model which
could be used to measure the c-mitotic potency of a com-
pound. This model was utilized in part to analyze the data
of this invescigation.

Aside from the report of Eigsti and Dustin (1955),
and the studies of Wood (1957) and Hadder and Wilson (1958),
it appears that no others have been carried out to obtain
qualitative and quantitative comparative data on colchicine
solutions to be employed in cytological work. Our experience
demonstrates that neither the fresh nor the stored colchi-
cine solutions produce a constant effect.

In the previous study (Greenberg, 1962) it was
found that:

a. Colchicine solutions stored in the dark at
O-5°C and at 21-23°C for mlre than #8 hours,
showed a progressive loss of activity.

b. She dry powder form of the alkaloid, once

exposed to light and moisture, also showed a

O

{1}

F]

s of activity over a 3 month period.

c. Different batches of the alkaloid appeared
to differ in their ability to produce the
expected cytological effects.

Our test system was the meristematic portion of

Pisum primary roots; specifically those cells which are

 

'actively engaged in mitosis.

According to Bowen and Wilson (1954), and Hadder and
Wilson (1958), colchicine produces two quite characteristic
cytological configurations in Pisum. All other configura-
tions have been found by Hyppio (1954) to be an expression
of one of these two configurations. fhese are the charac-
teristic "clumps" of Powell (1951) and D'Amato (19M8a) and
"scatter" of Levan's (1938-1939) c-mitosis (colchicine
mitosis), reported extensively in the literature. The
number of "clumps" and/or ”scatters" are a measure of the
alkaloid's biological activity at a given time (Hadder and

Wilson, 1958).

LIEERAFTAE REVIEW

Colchicine is a compound whose biological, pharmaco-
logical and physico-chemical characteristics and activities
are complex.

van Tamelen and co-workers (1961) have been able to
synthesize the compound, but state that colchicine posses-
ses som unusual features which, combined into a single
structure, offer a unique challenge in synthesis.

The substance has been extracted fr m members of the
Liliaceae (Eigsti and Dustin, 1955), especially from the
autumn crocus or meadow saffron, Colchicum autumnale L.

It has also been extracted as a natural product, together
with several other derivatives, from related genera of the
sub-family Wurmbaeoideae (Cross, 1964 and haul, 195%).

The history of the use of colchicine is extensive
and reaches far into ancient times, apparently having been
used by the Egyptians for the treatment of gout. More re-
cently, after Pernice (1889) discovered its action on di-
viding cells (Eigsti, Dustin and Gay-Winn, 19M9L it has
been employed as an antimitotic agent.

Hyppio (195M), Hadder (1957) and Biesele (1958) have
reviewed the different aspects of the effect of the alka-
loid on mitosis. Stetten (1958), Copeman (196%) and Eschner
(1964) have considered the medical aspects of colchicine.
A complete review of the importance of colchicine in agri-
culture, medicine, biology and a discussion of ts chem-
istry, by Loudon, may be found in a book by Eigsti and

u

Dustin (1955).

Since the discovery of Pernice (1889) many observa-
tions have been made on the effects of the drug on mitosis,
but the fact still remains that very little is known as to
how the compound acts biochemically on mitosis. Eigsti and
Dustin (1955) suggest that the compound acts on the spindle
by entering into a chemical combination with an intracellu-
lar receptor. Mazia (1956) suggests that colchicine affects
the "secondary bonding mechanism" of the spindle. Levan and
fistergren (19M3L and Dstergren (19hh) mention that colchi-
cine may act as a narcotic which may affect certain meta-
bolic mechanisms involved in the formation of the Spindle.

The two principal cytological effects of the compound,
which are the most important for this investigation, are
the "balled meta,hases" of Powell (1951), D'Amato (19H8a),
Barber and Callan (19h3) and Berger and Witkus (1943); and
the "scattered” configurations characteristic of Levan's
(1938-1939) c-mitosis. Hadder (1957), and Hadder and Wilson
(1958) have pointed out that the two configurations, which
express different degrees of the same primary effect, are
dependent on time and concentration.

Gaulden and Carlson (19h9-1951), Levan (1938-1939)
and hindmarsh (195?) believe that the aberrations are a

fic affinity of colchicine for the pre-

1-!-

result of the spec
cursors of, and/or the fully formed achroma ic figure. The
same investigators and others (Levan and Ostergren, 1943,

3'.

Ostergren, 19hh, Eigsti and Dustin, 1955, hazia,1956,and

6

horrison and Wilson,1958) are of the Opinion that the action
of colchicine is to change the spindle from a fibrillar to

a corpuscular structure, and this seems to be related to the
compound's physico-chemical activities.

Not only has it been difficult to define the biologi-
cal and pharmacological activity of the alkaloid, but the
chemical synthesis of colchicine has proven to be just as
difficult.

Early investigators of the chemical nature of colchi-
cine were Houde who first isolated the alkaloid in 1887
(Cohen and Cook, 19HO), Windaus (1911-192H), who proposed
a phenanthrene ring system (Cook, 19hh), Cook (19h4), who
established that ring 3 is seven membered, and Dewar (1945),
who proposed that ring C zas also seven membered (see
Figure 1). Thus, with this ’nitial characterization, colchi-
cine, was placed ahong th tropolone compounds.

Early biosynthetic work carried out to determine the
pathways followed in the elaboration of the compound by the
plants was done by Walaszek at al, (1952). These investi-
gators used C1H02 and obtained lahfled colchicine from the
exposed plants.

Leete and Nemeth (1960) administered DL-phen'lalanine-

3-C1L+ to Qolchicum bizantinum and obtained colchicine label-

(D

d in ring B. A year later (1961), these investigators re-
ported using sodium acetate-1-C1L‘L and obtained the label

, ., .. u.
on the M-acetyl group, and with L-mmhionine—methyl-C1' tney

obtained colchicine labeled on the O- and N-methyl groups.

7

Leete (1963) using DL- phenylalanine-Z—C11+ observed
activity in C5 of ring 3.

Battersby_gt_gl, (1934), state that methods using
tracer experinents establish that ring A and carbons 5, 6
and 7 of ring 3 are derived phenylalanine and cinnamic acid.

Leete (1935) proposes a biosynthetic pathway for col-
chicine and also mentions that Battersby (1964) was able to
show that tyrosine—31-C1u is incorporated into C12 cf ring
0. He also found that feeding DL-tyrosine-u-mLt to sprout-
ing Colchicum bizantinum corms labeled colchicine at C9 of
ring C.

Pertinant to this investigation has been the know-
ledge of the apparent instability of colchicine solutions,
especially when exposed to sunlight and ultraviolet light.
There appears to be a correlation between the particular
structure of the isomers and their capacity to act as effec-
tive antimitotics, the configuration of ring C being of
most importance.

Grewe (1946) reported that when a colchicine solution
was irradiated with ultraviolet light, the characteristic
absorption peaks of 250 and 350 mu gradually disappeared
and a new one appeared at 270 mu. He found that the new
peak represented the formation of a photoisomer of colchi-
cine named "Lumicolchicine". In 1951 the same investigator
and W. Wulf exposed a 0.2 per cent colchicine solution to
sunlight, obtaining after 5-7 weeks a crystalhne precipitate.

This precipitate could be separated into three fractions on

8
the basis of differential solubilities in alcohol. The frac-
tions turned out to be the alpha, beta and gamma-lumicolchi-
cine isomers, representing 95 per cent of the original col-
chicine solution.

Forbes (1955), through degradative and spectrosc0pic
studies, has gathered evidence suggesting that beta and
gamma-lumicolchicine are stereoisomers having tetracyclic
structures (Figure 1). This isomerization occurs through
theimmuTangement of ring C into a four carbon C ring and a
five carbon D ring. he also stated that he was able to ob-
tain all three isomers by exposing a solution to sunlight,
having first excluded air from the solution with nitrogen.
The beta form was obtained in the largest amounts.

Gardner §t_g;: (1957), in their investigation, noted
the conversion of beta-lumicolchicine to the gamma form,
thus lending support to the hypothesis of a stereoisoxeric
relation between them. Theyalso reported isolating the alpha
form but were later unable to repeat this (Chapman,1963).

Schenk, (uhn and heumuller (1961) transformed alpha-
lumicolchicine into the the beta and gamma photoisomers.

Chapman gt_§;: (1963) have established the structures
of alpha and beta-lumicolchicine and present good evidence
for the structure of the gamma isomer. Their studies in-
volved use of nuclear magnetic resonance techniques and
deuteration. Most interesting is the establishment of alpha-
lumicolchicine as a dimer of the beta isomer of colchicine.

They also found that the alpha form is not easily obtained

9
by irradiation with a mercury arc lam as previously reported.
A cyanine dye filter is required to obtain at least a 20-30
per cent yield.

Investigations have also been carried out with a
stereoisomer of colchicine, isocolchicine, by Steinegger
and Levan (1947). The only diff;rence structurally is the
exchange of positions of the =0 and -OCH3 groups of ring C.
Such an exchange of positflns has been noted by these inves-
tigators to reduce the biological activity from 70-100 times
in comparison to colchicine. The suggestion is put forth
that the activity of colchicine and related compounds is
regulated by their thermodynamic activity. Colchicine, it
is claimed, looses its specific activity upon being trans-
formed into isocolchicine, which th n acts only by its
physical activity.

Dauben and Cox (1963) photoisomerized isocolehicine
to the lumiisocolchicines:hiorder' to obtain evidence for the
particular structures of beta and gamma-lumicolchicines.

Chapman.g§_al, (1963) have also carried out studies
on the photoisomerizmjon of isocolchicine and obtained +0-
50 per cent yields of lumi-products having ultraviolet light

absorption peaks similar tc those of beta and gamma-lumi-

colchicine.
The synergistic and antagonistic effects of other
compounds on the antimitotic activity of colchicine have

0

been reporte by Eigsti and Dustin (1755), Trezzi and Balin

(1956), horrison and Wilson (1958) and Siesele (1755).

Figure 1. Structures of Colchicine and its Isomers.
I. Colchicine (Chapman _tngl., 1963).
II. Isocolchicine (Steinegger and Levan. 1947).
III. Luniisocolchicine (Chapman at al” 1963 and
Dauben and Cox, 1963).
IV. Gamma-lumicolchicine (Chapman.§; al., 1963).

V. Beta-lumicolchicine (Chapman et al., 1963).

VI. Alpha-lumicolchicine (Chapman at 31,, 1963).

 

 

 

 

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Figure 1.

11
Although of general interest in pro'iding comparative data,
they still are not readily interpretable.

Studies on the mass spectra of colchicine compounds
have been carried out by Wilson gt §l° (1963), and Optical
rotation measurements of the same by Irbek at al. (196%).

Cross (1934) and Kaul (196%) ha e recently isolated
colchicine, lumicolchicines, and other colchicine-like
compounds from some genera of the sub-family Wurmbaeoideae.
They have thus shown that some compounds made:ynthetically
exist as natural products. Forbes (1955) reports that
éantavy isolated beta and gamma-lumicolchicine as a natural
product in 1951-1952.

Physical and chemical studies have contributed much
information as to the particular structures and physico-
chemical activities of colchicine and its isomers. Compara-
tive quantitative studies should be performed to relate spe-
cific isomeric configurations to the ability of inducing
certain effects on biological systems, as has been done
by Steinegger and Levan (19h7). Hadder and Wilson (1958)
have proposed a model system which could possibly be cor-
related with our present knowledge of the compounds and
which could relate specific structures to biological acti—

vity or function.

MATERIALS AND METHODS
GENERAL EXPERIHBXIAL PROCEDURE
The meristematic root tip of the comaon garden pea,
Pisum sativum var. Alaska, was used as the biological test
system in this investigation. The peas were guaranteed to be
free from chemical treatment by the supplier, the Ferry

Morse Seed Company.

.mlm-‘yJ—n .4—1‘1'
..

The chemical, colchicine, was obtained from the Light's

Chemical Company Ltd., Bucks, Colnbrook, England.

I-H‘MM .

The investigation was divided into two parts. The
first was concerned with exposing a MOO ppm (1 x 10'3N)
colchicine solution to a fluorescent light source at room
temperature; the second part consisted of exposing a solu-
tion of the same concentration to ultraviolet light. The
light eXperimental runs were grouped into 12 hour, 3 week
and 15 week exposure periods. She ultraviolet light experi-
ments were carried out for only 2% hour eXposure periods.

When solutions were exposed for as long as 3 to 15
weeks, it was necessary to autoclave all glassware and
Seitz-filter the solutions. Previous experience (Greenberg,
1962) demonstrated that the solutions were capable of sup-
porting bacterial and fungal organisms over a range of temp-
eratures.

The solutions and glassware utilized in the ultra-
violet experihents did not have to be treated in any spe‘
cial manner.

In order to insure that the dry powder stock of col-

12

13
chicine would not be modified in any way, it was stored in

an evacuated desiccator and kept at 0°C.

Eluorescent Light EXperiments:
Each #00 ppm colchicine solution for these experimen-

tal runs was divided into three aliquots. One was used as

.f__.

the fresh control treatment, a second one exposed to the
light source, and the third, when run, was placed in the
dark and kept at 22.5°C for as long as the solution eXposed

to light.

 

Exposure of the solutions was carried out in a lined
orchidarium to insure that the light from another source
would not fall on then.The temperature was kept at 22.5°C.
The solutions exposed to light were placed two feet beneath
two 20 watt cool-white Kenrad Fluorescent Lamps set parallel
and one foot apart.

Three exlosure periods (12 hounn 3 weeks and 15 weeks)
were chosen in accord with previous experience (Greenberg,
1952).

The third aliquot, whenever run, was put in a flask
covered with aluminum foil and placed under a small card-
board box, also lined with foil. This solution was also
kept, under the conditions mentioned, in the orchidarium.

At the end of the exposure period each solution was
mixed with.the appropriate amount of nutrient to give a

\
200 ppm (5 x 1O’Th) solution with which to treat tune peas.

Ultraviolet Ligft Experiments:

m

A ain each #00 ppm colchicine solution was divided in-

032

to three aliquots. These were a fresh treatment control, a
solution to be exposed to ultraviolet light, and one to ser-
ve at times as the dark room temperature control.

Four eXperiments were performed: one at 34°C, and the
other three at 22.5°C.

Three hundred mifliliters of solution to irradiated
were placed in 100 x 50 mm pyrex crystalizing dishes and
eXposed toeulultraviolet source for 2% hours. The source
was placed 1 cm above the free surface of the solution.

Two different energy sources were utilized. The solu-
tions used in the first two runs (one at 34°C and the other
at 22.5°C) were exposed to a short wave, mercury quartz-
type table lamp (Model V #1) manufactured by U V Products

Inc. of San Gabriel, California; who specify that the lamp

0

delivers a peaked beam of 2537 A at two feet. The other
two runs were irradiated with a 17"-15 watt G.E. Champion
Germicidal Lamp which also delivers a beam at 2537 3.

To guarantee that the solutions were evenly irradia-
ted a Lagnetic teflon-covered stirring bar was activated by
a nagnetic stirrer. The variable resistance of the stirrer
was by-passed when it was found that it would heat the

24 hour period. Thus, the

C.)

solutions above 22.5"C over
knob on the stirrer was set at its highest reading (zero
resistance) and the system was connectel to a Powerstat.
This system provided excellent stirring velocity control

and eliminated the heating problem.

15

The whole system was placed in a standard chemical
hood with the glass gate lined with aluminum foil. This was
done to prevent either sunlight or artificial light from
falling on the solutions. A constant stream of air going
through vents in the gate and out through an exhaust fan in
the hood kept the solutions at 22.5°C.

Agitation, temperature and air flow caused some evap-
oration from the surface of the solutions. Since a measured
quantity was irradiated each time, the volumes were easily
adjusted at the end of the exposure period wit} distilled

deionized water.

Preparation pf the Biologicgl Test Organism:

The seeds were soaked in distilled deionized water at
25°C for six hours in a germinator prior to germination.
These were then rolled in moist paper toxéflng and placed up-
right in beakers with an inch of distilled water. Each paper
roll was covered with a layer of waxed paper to prevent
excessive evaporation. The peas were then placed in a germi-
nator at 25°C for from 33 to 40 hours allowing the seeds to
grow primary roots 2.5-3.0 cm in length.

At the end of the germination period the seedlings
having the required root length were suspended on 1/h inch
waxed wire meshes and the roots allowed to remain in a 1/h
strength hoagland nutrient solution for two hours.

After a two hour eqrilibration period one dish of

peas was allowed to remain undisturbed to serve as the un-

16

treated control. The remaining seedlings were transfernfilto
the 200 ppm test colchicine solutions, and maintained in
them for 30 minutes; after which they were removed, washed
with distilled water and returned to a nutrient solution.
All solutions were filter-aerated for the duration of the
experimental run. This was done to aerate and agitate the
solutions so that all the seedlings received the same amount
of oxygen and nutrients.

The concentrations chosen, especially the ones used
in treating the seedlings, are based on past experience and

'7

the suggestion by Van't nof the

C!”

the most successful con-
centration fortreating Pisum is 150 ppm (3.76 x 1O'HK).
Because difficulty was encountered in obtaining the desired
effect at this concentration a 200 ppm solution was chosen.
Good results were obtained and a fairly rapid recovery period
was observed, without evidence of toxicity with the fresh
control solutions.

A 400 ppm (1 x 10'3h) solution was chosen as a stock
and experimental solution, because it was close to the treat-
hen range and easily adjustable for experimental purposes.

Five primary roots were taken from the seedlings in
each culture dish every two hours after the initiation of
treatment for ten hours. The excised primary roots were
placed in Pinaar's fixative, consisting of a 6:3:2 mixture
of atsolute methanol, chloroform and prOpionic acid, and
evacuated for ten minutes. The appropriately coded vials

were then capped and placed in a refrigerator for 24 hours.

17

The primary root neristems were prepared for analysis
by the Feulgen "squash" technique after the roots had been
hydrolyzed in 1H HCL at 50°C for 18 min. fhe prepared slides
werethen dehydrated in 9:1 tertiary-butyl alcohol and ab-
solute ethanol for at least 12 hours, then made permanent
w'th diaphane. These slides were then exanined and scored
for effect.

The characteristic "clump” and "scatter" configurations

(Plate II, Figures A-B) induced in dividing cells of Pisum

 

root meristems (Powell, 1351) were used as the diarnostic

index in this investigation. Each ”altered" solution of col-
chicine was compared to a solution kept in the da h at room

temperature, and to a fresh "unaltered” solution, by recor-

LA

ding the number of "clumps”, ”scatters" and normal mitotic
figures occurnng in the meristematic tissues. Untreated
control seedlings were also examined and scored so as to
have a control index f ritosis.

?he "clumps" and "scatters" were either recorded per
one thousand cells scored at random r per two-hundred post-
prophases (”clumps”, "scatters" and normal post—proxhases).
Four out of every five slides prepared pcr sampling period
were scored :or the particular COHll

J.‘\

and the averages record d.
1' ,0 fl , n '9. ,JC‘, '
Indices of efiect (gander and wilson, 19yo were ob-
tained at two hour intervals up to six hours after the

initiation o; a 30 ninute treatment with colchicine. Curves

were plotted for these points and the area under these

18
curves served as measure of effect. These areas, in arbi-
trary units, were then used to compare fresh treatment solu-

tions with ”altered” colchicine solutions.

Colchicine produces sev ral well known and specific
aberrations in mitosis. Specifically its action seems to be
on thegxecursors of, and/or the achromatic figure. This,
then, places the action of the compound at the prometaphase,
netaphase and anaphase stag es.

A chara cteris ic effect observed in the cells of
tree ated pea seedlings is the "scattered netaphase" of Levan's
c-mitosis. It has been suggested that this is a partial
effect on the spindle (Levan,1938-1939, Barber and Callan,
19h3, D'Aneto, 1948a, 31 gsti an d Dustin, 1955, Hadder, 1957).
Ehe second effect noted and believed to be due to a complete
inactivation of the spindle is the "clumped configuration"
of Powell (1951), Bowen and Wilson (19) +), Hadder and Wilson
(1958), Van‘t hof (1961) and Green berg (1962) for Pi sum,
which eventually leads to polyploidy (Van't Hof, 1960-1961).

Hadder and Wilson (19) 8), based their investigation
upon the degree of effect observed after treatment of pea
seedlings with fresh colchicine solutions of different con-
centrations. 1heir study sug ted that "scatters" appear
first in time and "clumps" later, providing the dose is
high enough to produce them. They emphasized that "sea ters"
can not give ise to "cluzrps", since with continuous low
dose treatments only ”scatters” are observed. fhey conclude
that,"'scatters' and 'clumps‘ express different desrees of
the same effect, and that the degree of effect is dependent

upon dosage, which may be expressed as duration of eXposure

19

20

van concentrationf

H.

to a g
fne activity of tie colchicine solutions was measured
by means of a "Colchicine Index of Effect" devised by these
investigators. The configurations appearing after treatment
had to be distinguished from each other. Since "scatters"
appear before "clumps% and are the only configuration appear-
ing with low doses,and since it was assumed they represent
a less severe cytological effect than "clumpsfl they were
multiplied by a factor of one and "clumps" by a factor of

tVIOo

 

Configuration Svnbol Weight
Clumps Z 2
Scatters Y 1
Normal Post-proph. X 0

The total number of post-pmxhases (n) scored per slide
was approximately 200 in all cases.

Ihe Index of Effect is expressed by the following
formula:

I. E. = 1(Y) + 2(2) + 0(Xl
n

It was shown that this index changes smoothly with
time, and the rate of change is Uependent upon dose, giving
a measure of activity or potency at any given time.

Van't Hof (1960-1961) determined that concentration
and duration of treatment are important in producing the

1

best reaction anc recovery from the effects of the alkaloid.

21

7“}
Cf
Hg
Q)
ct-

fhe reaction time i. ocriod during which chromos ozxe
configurations ("clumps” and/or "scatters") leading to tetra-
‘ploidy are observed in significant numbers, and recovery
period the interval from peak reaction (which occurs at two
hours after the initiation of treatment) to the point where
ten or less per thousand figures si:owing the effect are ob-
served. He concludes,Qum deally the duration of the treatment
should be as short as is consistent with a rapid rise in
and recovery from the effect of the alkaloid

In this investigation, as well as in the previous one
(Greenberg, 1932), a modification of Hadder and Wilson's
(1958) study ant1 the incorpo rc.ti on of Van' t Hof's (1960)
ideas were followed in obtaining measurements of the effect
of the colchicine solutions.

Indices of effect were plotted at in crvals of two
hours from 0-6 hours after the initiation of treatment.
From the curves for these indices areas \srere ontaincc in
arbitrary units. i‘hese areas give a 1:2.sire of the total
reactivity of the colchi cine solutions and were used to
compare fresh solutions with each other and to their res-
‘ective "altered” solutions. Instead of tr eating the seed-
lings continuously for the duration of the eXQerinent as

had been done by Hadder and W.ilson, the seedlings were x-

4

posed to a half hour pulse tree tnent as was st L33c ted by
Van't Hof.

Y

Data and information have 1“ecn included in the tables

in the apucndit of this ranuscript from the previous study

22

(Greenberg, 1962) dealing wit-1 observed variations in the

rs

ffectivezw o colchicine kept under a variety of ”stan-
dard" laboratory conditions. fhey is ve been included in or-
der to present a general picture of observed modifications
of colchicine effectiveness under these co neitions.

The data for the indices of effect and areas under
the curves for these indices are found in fables III and IV
of the amendix.

Representative curves are sh wn in Figures 2-7 for the
data taken from Tables III and IV of the appendix and inclu-

n t ‘5318 t

(D

ded in fable 1. Jhey were chosen because they repres
has been observed for some title by several inmres stors in
our laboratory, but 11ave not been rigidly qua nti :ied and
compared.

Mi lres 2, 3 and h represent data ootained for a fresh

treatx .ent solution, a solution stored 2% hours in the dark

at 22. °C, a 1d a solution irra izeted 24 hours at 3M° C, \.*i h

When the solution which was stored for 24 hours in the
dark at 22.5°C is compared with its fresh counterpart a los
of 13 per cent in activity is observsi. The solution irradia-
ted for 24 hours shows a loss of as much as 7%.5 per cent in
activity.

Fi

C 2
L.
L-j
(D
U)
\n

\D
O\
J
:4.
Q.
\
*1
(D
J
w
t.)
m
(I)
I)
cf
,2.-

3
C
D

° L n . - -. t , ~ u an r0 .: 1

a solution scored 3 teens in the dafn at O-) C, and a solu-
: 4- "“ ‘ —~ ' ’ ' L I) :0 f." V r\ va ~ r.“ I.
tion SCO;;U in tie dark at 22., C is: the sane lbnobh or

time.

23

 

2.0L

/,
///I/
sir- I FAWIIIIIIiltlmfia
o o 0
4|.

Hommmm mo XMDZH

—- —-+.~-—-——--—-

-. “—5-.. .———»—.-

 

 

Figure 2.

X OF EFFECT

INDE

re
0

 

Figure 3.

2
TIME IN HOURS

I D

20 O‘

 

 

N.

TIKE IN HOURS

Figure H.

 

mi-

INDEX or EFFECT

2.Cr

 

Figure 5.

[‘0
O\

[0 y-

TII-IE III HOURS

».

O\..

-.._ -_..—-— J- —-

27

 

2.0?
1.0’

[2.

HOURS

‘V
L"

I

l

I’ E

T

Figure 6.

28

 

 

 

 

>0
.
-tr
\\
\\\\\\
,fl/ -2
//,
/x
//,
/
///
//
x;////
NU » p r /
c (U 0 r)
9. 1n .

Hommmm mo NWOZH

HOURS

IN

TIRE

Figure 7.

CO
0
[\

:.o_

O.FF

N.OF

O.NF

 

mean: smmnpmmme
Ca won4

0.0m

m in

o.ma

mmoq &

 

00
m.F
.o m.mm um game map CH mxoms m ompOpm
m.
m.—
m._
.o mic um xpmc one CH axooz m UmgOpm
m.
0.9
m.—
.smohm
F.
P.
o. -
.o :m we >3 spa? .mp3 rm dmpmweoan
m.
0.9
m.P
.o m.mm pm xpmo mew CH .mg: :m Umnoum
m0
N.P
5..
pommmm
mo Koan .Smogm

 

H mqm<9

RN10 RN30
:11 :11

.me:

30

Again it may be observed that the refrigerated solu-
seens to have lost about 5 per cent activity, while the
solution kept at 22.5°C has lost about 29 per cent of its
activity.

The percentage losses were obtained by using the fol-
lowing formula:

Per cent loss = S] - SQ x 100
So

S0 = area under the fresh solution curve.

S1 area under the "altered" solttion curve.

La general, there is a slight decrease in activity with
an increasing length of time of exposure to higher tempera-
tures and artificial light. When solutions are eXposed to
ultraviolet light source there is a considerable loss of
activity.

Solutions eXposed to artificial light for hree weeks
and fifteen weeks showed no apparent loss of activity, the
solution exposed for twelve hours showed 9 per cent loss
of activity. Since the observed results give conflicting
data they are not explainable at this time.

No fresh control was run for solution (3) eXposed to
light for 3 weeks and for solution (2) exposed for 15 weeks.
Since they were prepared from the same sample of dry powder
and at the same time as their respective solutions (2) and

(1), it is belmved that they are comparable.

‘\
“a
.A

re

shown that different batches

H.
U)
H
:3
<1
(I)
(’3
c r
Ho
0*}
(3

(.1-
p.
O
:5
5

U)

31
of the dry powder do not have the same biological activity.
This may be observed when the activities of the fresh solu-
tions are compared. fhere may be as much as 57 per cent dif-
ference in activity.
Table II of the appendix contains data on the Kitotic

Indices, which represent the nunber of dividing cells

*o

CI‘

113

O~¢

thousand scored at random, for the experixental runs. I
particular measurement serves as an indicator which would

snow-up any discrepancies in the test organism. Any varia-

C7
H.
I...)

ity in the test organism would modiiy the experimenta
results. It has been found and confirned many times in our
laboratory that the average mitotic index of the "Alaska"

variety of Pisum sativum is close to 71.

DISCUSSION

The purpose of this investigation, initiated to deter-
mine in a quantitative manner changes in the biological ac-
tivity of colchicine when kept under certain "standard"
laboratory conditions and when exposed to ultraviolet light,
was attained in part.

Answers were sought to such questions as: "Does the
compound remain stable either in the dry powder form or in
solution?‘VWhat laboratory storage conditions are best suited
for an unstable compound such as colchicine?‘ "How long may
the compound be stored before any detectable changes are
evident or of major significance?"

The action of colchicine on mitosis has been difficult
to assay and even today biochemical evidence for its "singu-
lar" action is conspicuously lacking. Complicating the issue
are the effects of a vast number of chemicals, some related
to colchicine and others not, which seem to behave as mitotic
poisons in a manner similar to colchicine. Ehis has caused
much controversy over when and how the chemicals act on
mitosis. Biesele (1058) points out that mitotic poisons
have been classified not so much on the basis of their chem-
ical structures, but rather on their effects on mitosis or
by the type of aberrations they produce in different phases
of mitosis..This author points out that the difficulty en-
countered in the classification of mitotic poisons consists
in the fact that a given agent may induce more than one type
of aberration, and that these may vary in intensity of ex-

3‘ 2

3

Lu

pression and the time at which they appear. They have been
found to be dependent on the time of exposure and concentra-
tion utilized.

Hyppio and Wilson (1955), interpret mitosis as a pro-
cess composed of a number of cycles of which spindle forma-
tion, function and dissolution comprise a part. It is to
these particular phases of the mitotic process that Bowen
and Wilson (1954) assign the action of colchicine. Zhe pro-
cess of spindle formation and function should not be consi-
dered a single cycle, but rather the integration of several
metabolic cycles which eventuate in spindle formation and
function. Evidence for this comes from Biesele (1958), who
states that there are three possible points of attack on
the spindle formation:

a. Inhibition of the formation of the apparatus

from sulfur containing protein, RNA and poly-

saccharide.

b. Inhibition of disulfide-bridge formation
(Kazia, 1955) by agents which disturb the
oxidation-reduction condition of the cells,
and by sulfhydryl reactants.

c. Inhibition of the "secondary-bonding mecha-
nism" described by Yazia (1956) as essential
in Spindle formation.

Interference with any one of these stages would lead

to inactivation of the spindle, and thus of chromosome

movement in mitosis.

3H

The uniqueness of colchicine may be seen when it is
compared with the many compounds which are known to affect
the spindle. Colchicine is not toxic over the range of con-
centrations and conditions in which other compounds with
alleged c-mitotic activity are very toxic or ineffective
(Eigsti and Dustin,1955). Eigsti and Dustin (1955) also
make it quite clear that when comparing the effects of col-
chicine on the spindle, no general statement may be made
which can be applied to all living organisms. An example is
the finding by Levan and fistergren (1943) that colchicine
is active at lower doses on Pisum than it is on Allium.

Although considerable studies have been made with
many compounds which seem to confuse the issue, much has
been learned from them. Eigsti and Dustin (1955) and Biesele
(1958) point out that studies have been carried out to de-
termine what groups are necessary for the activity of the
colchicine molecule. Phey also report that modifications
of the molecule have been made in order to determine which
ones, if any, lead to an increase in the effectiveness of
the compound in inducing polyploidy or in inhibiting neOplas-
tic growth without being toxic to the organism.

Eigsti and Dustin (1955) summarize the findings of
many investigators in four points characterizing the acti-
vity of colchicine.

‘

a. It is of importan e that the esterified

side chains of rings B and C are at a pro-

per distance from each other.

35

b. At least one methoxy group appears to be
indispensable in ring A.

c. Esterification of the amino-group of ring
B may increase activity, but this is not
necessary for activity.

d. Ring C must be seven membered and the
hydroxy group should be either esterified
or replaced by an amino-group which is
itself esterified.

They present as an example the different activities
of colchicine and its isomer, isocolchicine, observed by
Steinegger and Levan (19%7). The reasons for the differences,
they state, may lie in the formation of hydrogen bonds be-
tween the side chains of ring C and B; in particular the
methoxy groups. Thus, it seems these side chains are chemi-
cally active, and the same investigators proposed that a
chemical combination between the alkaloid and intracellular
receptors leads to Spindle inactivation.

Kazia (1956) gives a probable eXplanation of the
reactions leading to spindle inactivation. He subjected
sea urchin eggs to colchicine and then isolated the "mitotic
apparatus". He found that the contracted chromosomes were
embedmfl.in.a gelplike substance which showed no semblance
of organization. He prOposed that the formation of the spin-
dle from precursors involves two processes; the formation
of a gel and the orientation of the gel into a highly orga-

nized fibrillar structure. Colchicine, according to him,

36
seems to attack the latter process, and in doing so speci-
fically seems to attack the"secondary bonding mechanism”.
The possible targets seem to be the cell centers and kine-
tochores.

It has been known for some time that colchicine iso-
merizes to its alpha, beta and gamma photoisomers when ex-
posed to ultraviolet light and sunlight (Grewe, 19%6, Grewe
and Wulf, 1951 and Forbes, 1955). It has also been found
that if colchicine solutions are left standing and exposed
to air they become toxic. It has been proposed, but not
fully established, that the observed toxicity is due to the
formation of a compound called "oxydicolchicine" resulting
from the chemical combination of two colchicine molecules
and an atom of oxygen (Eigsti and Dustin, 1955).

Chapman at al. (1963) have established the structures

 

of alpha and beta-lumicolchicine and have presented good
evidence for the structure of the gamma isomer. They have
also found that the alpha isomer is not formed when solutions
are exposed to a mercury arc lamp. Alpha-lumicolchicine, a
head to head dimer of beta-lumicolchicine, is formed only
when a cyanine dye filter is interposed between the source
and the solution.

Forbes (1955) found that there was a preponderance
of the beta isomer formed when solutions were exposed to
sunlight. Ehere was also present in the solution a mixture
of the alpha and gamma-lumicolchicne.

The solutions in this investigation were exposed di-

37
rectly to an ultraviolet light source without the cyanine
dye filter. It is quite probable that very little alpha-

lumicolchicine was formed in the irradiated solutions. The

beta and gamma isomers should have formed, and since the
solutions showed losses of biological activity it is very
likely that the two isomers are not as effective as colchi-
cine.

There was evidence of toxicity as illustrated by the
presence of pycnotic nuclei in considerable numbers in the
first 2-4 hours after the initiation of treatment of the
seedlings.

Three possibilities may be considered here to explain
the toxicity observed. Since the solutions were exposed to
air, ultraviolet light irradiation may accelerate the for-
mation of the compound"oxydicolchicine", mentioned by Eigsti
and Dustin (1955); another possibility is that the photo-
isomers themselves are toxic; and lastly it may well be that
a toxic compound, which may or may not be related to col-
chicine, was formed in the solutions through irradiation.

Solutions maintained either under refrigeration or at
room temperature in the dark or exposed to artificial light
showed progressive loss of activity with increase in sto-
rage time. No evidence of toxicity was observed up to three
weeks; but those solutions kept for a longer period of time
under artificial ligh did show some toxicity. Again air
was not excluded from these and the possibiltiy that "oxy-

dicolchicine" or some other toxic agent nay have been pre-

sent cannot be ruled out.

Thus, from these results and those from physico-chemi-
cal observations of Forbes (1955), Gardner at al, (1960),
and especially Chapman and co-workers (1963), the point may
be made that the transformation of colchicine from a tri-
cyclic structure to its tetracyclic isomers has caused a
reduction in c-mitotic activity. This supports Eigsti and
Dustin's (1955) ideas that the prOper distance must be
maintained between the side groups of ring B and C, and that
the integrity of ring C must also be maintained. Jhese re-
lationships are changed when ring C is transformed into a
4 carbon C ring and a 5 carbon D ring, and the molecule of
colchicine takes on new steric configurations. fhese changes
might possibly affect the reactivity of the side groups and
their ability to act as hydrogen acceptors, which would be
in accord with Eigsti and Dustin's (1955) and fiazia's (1956)
hypothesis on the action of colchicine on the spindle.

Although not an integral part of this investigation,
observations were made on whether the "altered" solutions
were capable of inducing the c-tumor (colchicine-tumor).
The results were not quantified, but it was noted that the
irradiated solutions were capable of inducing the c-tumor

in seedling roots. Eigsti and Dustin (1»55), describe the

\

c-tumor formation induced by colchicine and several other
compounds. They state that it has been well established
that c-timor formation and c-nitotic activity are two dif-

feren -ffects of the compound. In this case c-mitotic acti-

39
vity was low and the c-tumor activity was comparable to that
induced by fresh solutions.

The other solutions subjected to various "standard”
laboratory conditions also gave evidence of c-tumor activi-
ty even when their c-mitotic activity was reduced compared
to fresh solutions.

From evidence gathered thus far it may be suggested
that further work should be carried out to determine the
biological activity of the different photoisomers of col-
chichmu With support from the physical sciences it has be-
come evident that the relationship of the side groups to
a molecule and to themselves plus the structural configu-
ration of the whole molecule determines biological activity;
such is probably the case with colchicine.

Supporting evidence for this suggestion comes from
work done in our laboratory in the past by Tsou (195+) and
Nyppio, Tsou and Wilson (1955) on the c-mitotic action of
Technical Lindane.

Eigsti and Dustin (1955) reported that both the gamma
and delta isomers of Technical Lindane (hexachlorocyclohex-
ane) have been found to be active antimitotics by Carpentier
and Fromegeot (1950).

Tsou (unpublished) tested all five isomers of Tech-
nical Lindane and found that only the gamma isomer was ac-
tive. Chis then points to the necessity of knowing which
isomer of colchicine is active and which one(s) are respon-

sible for loss of activity and toxicity.

HO

It would be helpful if, through physico-chemical stu-
dies, a methOd of treating colchicine solutions could be
devised by means of which one could be assured that the
isomer one had in solution was the most active and would
give more or less the same results everytime it was used.
This would be of great importance when using colchicine in
tagging pOpulations of synchronous cells as proposed by
Van't hof, Wilson and Colon (1960); inasmuch as variability
of the colchicine solutions would be eliminated.

Gout, a metaboliccfisease, has been and still is treat-
ed with colchicine (Eigsti and Dustin, 1955, Stetten, 1958
and COpeman, 196%). It would be of interest to know if col-
chicine p§r_§e or one of its isomers is better in the treat-
ment of the metabolic disease.

In conclusion then, it has been found from the pre-
vious study (Greenberg, 1962) and from this investigation
that colchicine solutions begin to show loss of activity
after storage for 2% hours in the dark at room temperature
(22.5°C) and after 48 hours in the dark under refrigeration
(O-5°C). Solutions exposed to a fluorescent light source
gave inconclusive results. Che solution exposed for 12 hours
shows a loss of about 9 per cent activity, while the solu-
tions exposed for from 3 to 15 weeks are slightly more ac-
tive than the fresh treatment solution. Dhe controls them-
selves were not as active as desirable. Solutions CXposed
to ultraviolet light demonstrated considerable loss of ac-

tivity after 24 hours of eXposure.

1+1

It is also suggested that the dry powder form be tested
before use since it was found that it showed significant
variation from batch to batcl.

Increasing concentration and exposure time to colchi-
cine solutions will only increase the probability of en-
countering toxicity. This is very important tocxnsider when
working with cells and tissues of warm blooded animals, since
it is known that colchicine is toxic to them at concentra-

tions which are innocuous to plants (Eigsti and Dustin,

1955).

SUZIZC.’hY AZID CONCLUSIONS
Solutions of colchicine were subjected to incident
irradiation from two radiant energy sources. This was done
with the hope of determining what conditions ordinarily
found in "standard" research laboratories would be most
suited for the storage of an unstable compound such as col-
chicine. The data obtained could then be compared quantita-
tively.
fhe results of the two investigations, Greenberg (1962)
and this one, indicate that:
a. Colchicine solutions may be stored up to
48 hours at O-5°C in the dark without show-
ing appreciable loss of biological activity.
b. Solutions subjected to irradiation by ultra-
violet light showed considerable loss of
activity.
c. The dry powder f rm f the alkaloid obtain-
ed from the suppliers is not constant in
the degree of effect induced,zand that in-
dividual samples of the powder, when stored

time in the dark at 0-

F1)

for long periods 0
5°C, also lose biological activity.

d.

¢od

Vith supporting evidence from he litera-

ture it is possible that the beta and gamma-

(D

lumicolchicines ar not biologically active
and may even be toxic.
s. It is suggested thatfurther studies be

1+2

Lt3

using the

system to
purified photoisomers
derivatives to set up

system, with which to

Raider and Wilson

test the activity of
and other colchicine
a classification

correlate the results

observed with known physico-chemical data.

BIBLIOGRAPHY

Ashley, J. N. and J. 0. Harris. 1944. Purification of
Colchicine by Chromatograpgy. J. Chem. Soc. 677

Barber, H. N. and H. G. Callan. 19h3. The Effects of Cold
and Colchicine on Mitosis in the Newt. Proc. Roy.
Soc..131:258-271.

Battersby, A. R., R. Binks and D.A. Yeowell. 196%.
Biosynthesis of Colchicine. Proc. Chem. Soc. 86.

Battersby, A. R., R. Binks, J. J. Reynolds and D. A.
Yeowell. 196M. Alkaloid Biosynthesis. Part VI.
The Biosynthesis of Colchicine. J. Chem. Soc. 4257

Berger, C. A. and C. R. Witkus. 19h3. A Cytological Study
of C-Mitosis in the Polysomatic Plant Spinacia
oleracea with Comparative Observations on Allium
ggpa. Bull. Torrey Bot. ZQ;H57-h66.

Biesele, J. J.. 1958. Mitotic Poisons and the Cancer
Problem. Elsevier Publishing Company. 214 pages.

Bowen, C. C. and G. B. Wilson. 1954. A Comparison of
Several Antimitotic Agents. J. Hered..&§:2-9.

Buchanan, G. L., J. w. Cook, and J. D. Loudon. 19HH.
Colchicine and Related Compounds. Part IV.
Synthesis of 2:3:4:5-, 2:3:h:6- and 2:3:hz7-Tetra-
methoxy-9-methyphenanthrenes. J. Chem. Soc. 325

Chapman, 0. L. and D. J. Pasto. 1960. Photochemical
Transformations of Simple Troponoid Systems. I.
Photocu-trOpolone Metyl-ethcr. J. Am. Chem. Soc.

fig:3642

L+5
, H. G. Smith, and R. W. King. 1963. The Structure
ofJS-Lumicolchicine. J. Am. Chem. Soc. §§:803-805.
, H. G. Smith and R. w. King. 1963. The Structure
ofoc-Lumicolchicine: Some Examples of Diamagnetic
Schielding by the Carbon-oxygen Double Bond. J.
Am. Chem. Soc. §§:803-812.
, H. G. Smith and P. A. Parks. 1963. Photoisomeri-
zation of Isocolchicine. J. Am. Chem. Soc. fij:
3171-3173-

Cohen, A., J. Cook and E. Roe. 1940. Colchicine and
Related Compounds. J. Chem. Soc. (London) 19H-197.

Cook, J. w., w. Graham, A. Cohen, R. w. Lapsley and C. A.
Lawrence. 194%. Colchicine and Related Compounds
Part III. J. Chem. Soc. (london) 322.

Copeman, W. S. C.. 196%. A Short History of The Gout and
the Rheumatic Diseases. University of California
Press. 227 pages.

Cross, A. D., A. El-Hamidi, J. Hrbek, Jr. and F. Santavy.
1964. Substances from the Plants of the Subfamily
Wurmbaeoideae and Their Derivatives. LVIII. The
Constitution of Cornegerine. Collec. Czech. Chem.
Comm. ,22:1187

D‘Amato, F.. 19h8a. The Effect of Colchicine and Ethylene
Glycol on Sticky Chromosomes in Alliug gepa. Hered.

H;83-103.

, 1959. C-Mitosis and Experimental Polyploidy in

 

Plants. Genetica Agraria 11:17-26.

96

Dauben, w. G. and F. G. Willey. 1962. Photochemical
Transformations. XIII. The Nechanism of the
Reaction ofzfi3'5-cholestadiene. Tetrahedron Letters
893.

, K. Koch, O. L. Chapman and S. L. Smith. 1961.
Photoisomerizations in thecc-Tropolone Series: The
Hechanistic Path of theac-Tropolone to h-oxy-2-
cyclopentenylacetic Acid Conversion. J. Am. Chem.
Soc. ‘83:1768.

, and D. A. Cox. 1963. Photochemical Transforma-
tions. XIV. Isocolchicine. J. Am. Chem. Soc.
85:2130-213h.

Dewar, h. J. S.. 1945. Structure of Colchicine. Nature
155:111-1-150.

Eigsti, O. L. and P. Dustin Jr.. 1955. Colchicine in
Agriculture, Nedicine, Biology and Chemistry. Iowa
State Press. #70 pages.

Eigsti, O. J., P. Dustin Jr. and N. Gay-Winn. 19M9. On
the Discovery of the Action of Colchicine on Mitosis
in 1889. Science 119:692.

Epstein, F.. 1955. Studies of Colchicine Effects upon
Mitosis in Living Stamen Hairs of Tradescantia.
Umpublished, study in partial fullfilment of M. 8.
degree.

Eschner, R.. 196%. Fatal Poisoning by Colchicine. Prakt.
Lekar. 33:369-371.

#7

Forbes, E. J.. 1955. Colchicine and Related Compounds.

Part XIV. Structure offg- and‘X-Lumicolchicine.
J. Chem. Soc. 386%.

Gardner, P. D., R. L. Brandon and G. R. Haynes. 1957.

The Structures of}?- and K-Lumicolchicine. Ring-

D Elaboration Products. J. Am. Chem. Soc. 22:633h.

Gaulden, M. E. and J. G. Carlson. 1949. Action of Colchi-

cine on Spindle Material in the Living Cell. J.

Tenn. Acad. Sci. 23:1OM.
, and J. G. Carlson. 1951. Cytological Effects of

 

Colchicine on the GrasshOpper Neuroblast in vitro

with Special Reference to the Origin of the Spindle.

Exp. Cell Res. ,g:h17-u33.

Greenberg, E. A.. 1962. Changes in the Biological Activity

of the Alkaloid Colchicine, Under Varied Storage
Conditions, as Keasured by two Cytological Effects
on Pisum sativum var, Alaska. (N. S., thesis, Mich-

igan State University).

Grewe, R.. 1946. Ein Umlagerungsproduct des Colchicins

durch Bestralung (Lumicolchicin). Naturwiss,33:187.
, and w. Wulf. 1951. Die Umwandlung des Colchicins

 

Hadder,

durch Sonnenlicht. Chem. Ber. ‘83:621.

J. C.. 1957. Cytological Measurements of C-Hitotic
and Prophase Action. (Ph. D. thesis, Eichigan State
University).

, and G. B. Wilson. 1958. Cytological Assay of

C-Hitotic and Prophase Poison Actions, Chrom. 2:91.

us

Hindmarsh, H. R.. 1952. The Effect of Colchicine on the
Spindle of Root Tip Cells. roc. Linn. Soc. H. S.
N. 303-30”.

Hrbek, J. Jr., J. P. Jennings, w. Kline and F. Santavy.
1964. Substances from the Plants of the Sub-family
Wurmbaeoideae and Their Derivatives. LX. Optical
Rotation and Rotatory Dispersion of the Colchicine
Alkaloids. Coolec. Czech. Chem. Comm. 23:2822-2831.

Hyppio, P.A.. 1954. The Effect of Colchcine upon the Recha-
nism of fiitosis. (Ph. D. thesis, Kichigan State
University).

Hyppio, P. A., T. H. msou and G. B. Wilson. 1955. Some
Notes on the C-hitotic Action of Colchicine and
Technical Lindane. CytolOCia 29:163-176.

Kaul, J. L., B. K. hoza, F. Santavy and P. Vrublovsky.
196%. Substances from the Plants of tie Sub-family
Wurmbaeoidcae and their Derivatives. LIX. Isola-
tion of Alkaloids from some Plants of the Sup-fami-
ly wurmoaeoideae. Coolec. zech. Chem. Comm. 22:
1689-1701.

Leete, E.. 1965. Biosynthesis of the Tropolone Ring of
Colchicine. Tetrahedron Letters. #5:333.

, 1965. Biosynthesis of Alkaloids. New and

 

Unexpected Routes to the Pyridine and Piperdine
Rings have been Discovered. Sci. 1M2:1000.

, 1963. The Biosynthesis of the Alkalcids of

 

Colchicum. III. The Incorporation of Phenyl-

i... 9
alanine-2-C1M into Colchicine and Demecolchicine.
J. Am. Chem. Soc. ,§E:3666.

, and P. E. Hemeth. 1930. The Biogenesis of the

 

Alkaloids of Colchicum. I. Incorporation of Phenyl-
alanine into Colchicine. J. Am. Chem. Soc. 82:6055.

, and P. E. Hemeth. 1961. The Biogenesis of the

 

Alkaloids of Colchicum. II. Tracer Studies with
Acetate-1-C1H and Nethionine-methyl-C1h. J. Am.
Chem. Soc. ‘83z2192-219h.

Levan, A. 195%. Colchicine-Induced C-Kitosis in two House
Ascites Tumours. Hereditas 39:1-6h.
, 1938. The Effect of Colchicine on Root Hitosis
in Allium. Hereditas gfi:%71-M86.

, 1939. The Effect of Colchicine on heiosis in

 

Allium. Hereditas 25:9-26.
, 19h0. The Effect of Acenaphthene and Colchicine
on Eitosis of Allium and Colchicum.

, and G. Ostergren. 1943. The Kechanism of c-

 

hitotic Action. Hereditas 22:381-H43.

Nazia, D.. 1956. The life History of the Cell. Am. Sci.
33:1-32.

Morrison, J. H. and G. 3. Wilson. 1958. Influence of
L-Ascorbic Acid on the Colchicine Reaction. Sci.
121: (3311).

Ostergren, G.. 19M4. Colchicine Mitosis, Chromosome Con-
traction, Narcosis and Protein Chain Folding.

Hereditas. 3Q:h29-M67.

So

Powell, S. S.. 1951. Comparative Effects of Colchicine
and Various Nucleic Acid Salts upon Somatic Hitosis.
(H. S. thesis, Michigan State University).

Schenk, C. 0., H. J. Kuhn and O. A. Neumuller.' 1961.
Photoisomerisierungen der Lumicolchicin und des
Colchicins. Tetrahedron Letters. #1:12-16.

Sorkin, N. 19h6. Iso-colchicicine. Helv. Chim. Acta 29:
2H8.

Steinegger, E. and A. Levan. 19h7. Constitution and C-
Hitotic Activity of Iso-colchicine. Hereditas 33;
38 5-396 .

, and A. Levan. 19MB. The C-Hitotic Qualities

 

of Colchicine, Trimethyl Colchicinic Acid two
Phenanthrene Derivatives. Hereditas 33:193-203.

Stetten, D. Jr.. 1958. Gout and Ketabolism. Sci. Ame.
,12§:73.

Trezzi, F. and L. Balin. 1956. Ricerche Spettrofotometiche
Sull'azione della Colchicina su Estratti Proteici
in £1329. Atti. della Dai Rediconti dell' Accademia
Nazionale dei Lincei. 29:60-70.

Tsou, T. M.. 1954. The Hitotic Effect of Technical Lindane
(Y-Hexachlorocyclohexane). (Ph. D. thesis, Michigan
State University).

Van't Hof, J.. 1951. Rate Changes of the Hitotic Cycle.
(Ph. D. thesis, Hichigan State University).

Van't Hof, J., G. B. Wilson and A. Colon. 1960. Studies

on the Control of Mitotic Activity. The Use of

51

Colchicine in Tagging of a Synchronous POpulation
of Cells in the Heristem of Pisum sativum. Chroma.
11:313-321.
, and G, B. Wilson. 1963. Studies on the Control
of Mitotic Activity. The Effect of Respiratory
Inhibitors on the hitotic Cycle Time in Pisum
sativum. Chromosoma. 13:39-46.

van Tamelen, E. E., T. A. Spencer, Jr., D. S. Allen and R.
L. Orvis. 1961. The Synthesis of Colchicine.
Tetrahedron 13:8-34.

Walaszek, E. J., F. E. {eisey and E. h. K. Geiling. 1952.
Biosynthesis and Isolation of Radioactive Colchicine.
Sci. .ll§:225.

Wilson, G. B. and C. C. Bowen. 1951. Cytological Effects
of Some More Antimitotics. J. of Hered. 32:251.
, and P. Hyppio. 1955. Some Factors Concerned in
the Hechanism. of Mitosis. Cytologia 20:177-18h.

Wilson, J. E., H. OHashi, H. Budzikicwcz, F. sentavy and c.
D. Jerossi. 1963. NassSpectrometry in Structural
and Stereochemical Problems. XXXIII. Colchicine
Alkaloids. Tetrahedron ‘19:2225-2231.

Wood, D. R. 1957. Stability of Colchicine in Pharmaceuti-
cals. Pharm. J. _1__'Z§:188

APPEZ‘FD IX

PLATE II

Figures A - B. Hitosis in Pisum sativum. Hormel cytologi-
cal configurations, and figures showing

effect of colchicine.

Figure A. Normal prometaphase (pm) and metaphase (m).
Figure B. Clumn (cl) scattered metanhase (sm) and
. 3 i

scattered anaphase (sa).

Each division of the scale represents 10

microns.

PLATE II

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SpectrOphotometric measurements were made on solutions
which were stored under various "standard" laboratory con-
ditions in this investigation and the previous one (Green-
berg, 1962). It was hoped that observed modifications in the
biological activity of colchicine solutions could be cor-
relatcd to observable changes in the nltrariolet light ab-
sorption peaks of the solutions.

Aqueous solutins at 16 ppm were measure} with a an
505 spectrophotometer and no variations could be detected
between fresh solutions and those stored under different
conditions for varying lengths of time, even when biological
activity had been modified as measured by the Pisum test.

No measurements were performed on the solutions ex-

posed to ultraviolet light.

-.1

further studies should be carried out in a more
systematic manner to correlate physical and chemical changes
with observed modifications of biological activity.

Special thanks are extended to the BiOphysics Depart-

‘0

Lichigan State University for permitting the use of

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Table IV. Areas of the Curxes of the Indices of Effect
Obtained from the Fresh and ”Altered" Colchicine
Solutlons.

1 x , °. ' 1 OH
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Irrad. 24 hours with uv at 3H°C:
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