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This is to certifg that the

thesis entitled

Cytological Effects of Certain Organic
Chemicals

presented bq

Marilyn J. Huston

has been accepted towards fulfillment

of the requirements for

M.S. dmyméh.Botanx(Cytology)

W
G.B.Wilaon

Major professor

[hm 27 Feb.1952.

 

0-169

 

 

 

 

my

. 1

a

CYTOLOGICAL EFFECT 13 OF BEHI'tIH

OPXCI IIIC CIIEL'I CAL S

by

Harilyn Janet Huston

A TI 3313

Submitted to tr c School of G aduate Studies of Iichigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of
EZAST A F SC

Department of jotanv and Plant Pathology
School of Science and Arts.

1952

THEStS

y/‘i/‘l "I'll 5 '3‘

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t

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5 M4 a r',
31&a15

Thesis Abstract

AA Arc GET -,...H.. )mA ,7". “Am ~ " A AA -"Tpx m’r": “An A— r:
- -. '* - , ‘: : .v ‘ -' l '
CY}: ULUH II ‘32:. ‘J .LJ) «bl “ILL-45:- é'l L"-L'\.J.F‘..Lu —— U J‘¢J':“>—b.bfiwu

”D 3'

Larilyn Janet Huston

It was the purpose of this study to investigate the
cytological effects of two insecticides, 0-1014 and Svstox,
and three antibiotics, Rimocidin, Thiolutin and P.A.-96.

he neristems of Allium cepa were treated with solu-

 

tions of the five organic substances in a continuous
treatment up to 12 hours and ranging from ccncentre tions
1,000 to 500,000 parts per million for 0-1014 and Svstox

per million for Rimocidin,

.L

and from 2(30 to 1,000 parts
Thiolu tin and P.A.-96. Th root tips were collected at
hourly intervals and the smear technique used for slide
preparation.

In the study of the slide material the occurrence of
five things was looked for: (l) pindle abn01nali ties
and c-mitosis, (2) change in the relative numbers of

pronhase, metaphase and post—metaphase fi3u res, (3) over-

contraction of the chromosomes, (4) an increase in the

percentage of reductional groupings, and (5) necrosis.
P.A.-96 was found to have no evident cytological

effects while the only effects of Rimocidin and Systox

'1 O N

were found to be toxi . In material tree so with Thiolutin

Karilyn Janet H‘ston

there was observed an over— —contra cticn of the chromosomes
at retap hase which was accompanied by an increase in the
relative number of metapha see as comnared t pr passes

and post-metaphases. A correlation vm 3 suggested between
these two effects. 0-1014 also showed evidence of an
over- ~contraction of the chromosomes. At me taphase there
was Spindle disruption w: liCh resulted in a disor .anization

cal of c-mitosis. Io cyto-

}—.o

of the metagehase figures tgrp
logical effects were recovered from either Thiolutin or
0-1014 ar Id these substances were therefore conc‘udsd to

be toxic at anv level of ccrIcentra tion capable of pro-

ducing mitotic abnormalities.

#22:.» «(42" 1,. arm
Dr. G. B. Wilson

Botaly and Plant Pathology

 

Introduction . . . . . . . .
Material and Iethods . . . .
Observations . . . . . . . .
Controls . . . . . . .
Insecticides
0-1014 . . . . . . .

systox o o o o o o o

Antibiotics .

ThiOlUtln o o o o o
Rimodicin . . . . .

P 0A. '96 o o o o o 0
Table l o o o o o o o o
2 O O O C C C O C

5 o o o o o o o o

4 0 O O C O O C .

5 o o o o o o o 0

Text Figure

CAMP
O

DiSCUSSion o o o o o o o o o

Spindle abnormalities and c-mitosis

Change in the relative numbers of prophase

metaphase and post-metaphase figures .

Over-contraction of the
Reductional groupings .
Necrosis . . . . . . .
Summary . . . . . . . . . .
Literature Cited . . . . . .

Acknowled3uents . . . . . .

Description of Plates . . .

chromosomes

I O O O U 0

IKTRGDUCTICN

 

One goal of an investigation of the cytological effects
of different organic chemicals is the formulation of new
concepts involved in chromosome reproduction and the me-
chanics of the mitotic process. many investigators have
made such studies and consequently there is a large amount
of literature dealing with the effects of organic substances
upon mitosis. Effects similar to those produced by such
treatments are to be found under naturally occurring con-
ditions and should be considered, as pointed out by Huskins
(1948), as the result of a natural but infrequent process
which the influence of chemical treatment makes more evident.
The study of the effects, whether naturally or artificially
induced, of such mitotic inhibitors is important in the
matter of somatic segregation and reduction, he mechanisms
of cell division and its cytochemical processes, mutation,
elucidation of the nature of cancer or tumor cells and the
discovery of useful growth inhibitors.

The discovery by Blakeslee and Avery in 1937 of the
polyploidizing action of the alkaloid colchicine has lead
to the study of numerous substances with similar properties,
with the investigation of the cytological mechanism.causing
the polyploidy being of prime interest. Since the initial

1

work with colchicine it has been found that the progress of
cell division and the mitotic mechanisms may be suppressed
or modified by a variety of physical and chemical means,
but colchicine has proven to be one of the most effective
of the chemical substances in this reapect. This is partly
due to the fact hat the action of colchicine is specific
with regard to the inactivation of the spindle apparatus
and partly due to the fact that the inactivation process of
the spindle is reversible and not toxic, at least if the
exposure to the colchicine is not overly prolonged and the
concentrations erployed are not too strong (Levan 1943).
After r moval of the plant from the influence of colchicine,
the spindle will recover and the cell, with its increased
chromosome complement, will continue to function no “all".
Cells recovering from colchicine treatment present a wide
variety of abnormalities, which, coupled with the fact that
its cytological threshold is separate from its toxicity
threshold, makes it a good basis for comparison with treat-
ments involving other organic substances.

It has long been known that colchicine has a disturbing
effect on the normal course of mitosis. Dixon (1905),
working on leucocytes, first noticed its action and inter-
preted the increase in the frequency of metaphases as being
due to the stimulating effect of the drug. Dustin (1934)
and Lite (1934) regarded colchicine as a very active agent

for increasing the number of mitoses in a tissue, thus also

-5-

regarding it as a stimulatin3 substance. However, in 1936
Ludford came to the conclusion that the increase in the
number of mitotic figures after colchicine treatment was due
to an accumulation of arrested.mdtoses rather than a stimu-
lation process. He attributed this effect to a failure of
the spindle to form and function in the normal manner (Levan
1958). Nobel and Ruttle (1958), who studied the effect of

colchicine on stamen hairs of Tradescantia, arrived at a

 

similar conclusion.

Levan, who, with his associates, has made an extensive
study of the effects of colchicine, has described its action
as consisting of three independent effects, namely: the
general poison effect; the cemitotic effect; and the in-

action of c-tumors (Levan and Gstergren 1943). The use of
the terms "c-mitosis” and "c-tumors" as first used by Levan
referred to the typical effects of colchicine upon the
mechanics of mitosis and implied the polyploid result in the
case of the first-named and to the formation of a tumor at
the tip of the onion root in the case of the latter. How-
ever, since that time many of the substances tested have
shown the typical effects without leading to polyploidy and
hence the term "c-mitosis" has come to refer to the two
outstanding effects of colchicine treatment which are spindle
disruption and a delayed Splitting of the kinetochores of the

over-contracted chromosomes causing x-shaped fi3ures scattered

at random throughout the cell.

-4-

Wada (1949) reported that there is an increase in time
of duration of c-mitosis over normal mitosis "due to the
greatly increased duration of the intactness or staying
period of the chromosomes. The increase of time necessary
for the formation of chromosomes and uncoiling, namely the
time required for the prophase and telephase, was not con-
siderable." 'his agrees with the findings of Levan (1943)
who reports the c-mitotic effect at metaphase in those sub-
stances tested.

Although most of the substances tested have been in-
effective as polyploidizing agents, there has been accumu-
lated a great deal of information about their abnormal
effects on somatic mitosis. These include scattered ar-
rangement of chromosomes at metaphase, "ball" or clumped
metaphases; diplo- and contracted chromosomes; "sticky”,
"lampbrush" and lagging chromosomes; "distributed", “ex-
ploded" or "reductional" groupings; "star" or "unipolar"
and multipolar spindles; "precocious reversion"; and micro-

uclei and pycnotic nuclei.

Various groups of workers have attempted to explain the
mechanics of ”c-mitosis" in terms of physico-chemical relations.
Levan and Cstergren (1945) demonstrated that there are many
substances which generally fall into the group of spindle
inhibitors. These substances include mono- and poly-cyclic
.ydrocarbons, napthalene acetic acid, nitrogen mustard,

insecticides, ethylene glycol, many colchicine derivatives,

-5-

and inorganic salt solutions. This discovery led them to
the principle that the activity of many c-mitotic agents is
conditioned not so much by their chemical preperties as es-
pecially by their physical ones, and this in turn caused
them to propound their theory of the narcotic nature of the
c-mitosis. They had noticed that the c-nitotic activity of
the substances increased at the same time as their water
solubility decreased. This fact, coupled with the wide
divergence in the chemical nature of the c-mitotic substances,
as well as the fact that many of the narcotics tested were
found highly effective, led them to the opinion that the
"cemitotic effect might be regarded as a kind of narcosis."
In order to explain the above named observations they formu-
lated the hypothesis, corresponding to the heyer-Overton
theory of narcosis, that "the decisive concentrations are
not the concentrations of the substances in the water phase
but in the lipoids of the cell." This assumption explains
the connection between the solubility and c-mitotic activity
which these authors had found to be characteristic of the
substances studied. Furthermore, they suggested that the
c-mitosis was simply to be considered as a narcosis of
certain enzymic functions of the cells. This hypothesis
agrees with the work of Dustin (1947) and his suggestion
that the various mitotic poisons act as enzyme-inhibitors.

q

Levan and Gstergren also considered the c-tumor eifect as

a narcosis of the growth control of the cells.

-6-

By 1944 Ostergren had arrived at a somewhat modified
theory. He laid stress upon the view that the c-mitosis
was to be considered simply as a narcotized cell division.
The spindle breakdown characteristic of the c-mitosis he
supposed to be caused by the fibrous comp nents of the
spindle being brought to assume a more or less corpuscular
shape by the protein-folding action of the c-mitotic agent,
and this in turn would lead to the disorganization of the
spindle.

The observations that some of these chemicals, in
addition to inhibiting he spindle, had other effects
started some new lines of investigation and thinking.
Berger and Witkus (1945) reported that under the influence
of colchicine Allium root tips produced an unorganized
spindle substance which took th form of an achromatic
sphere and about which ‘he diplo-chromosomes gathered at
c-metaphase. They attributed the many strange shapes of
the restitution nuclei as being due to the presence of the
achromatic sphere. The work of Kodani with sodium ribose
nucleate and Galinsky with phosphates suggested that perhaps
more than a single process was involved in all the “c-mitotic"
agents. Kodani (1948) noted, among the various effects of
sodium.nucleate solutions, an inhibition of chromosome re-
duplication and the formation of akinetic fragments.
Galinsky (1949) found that following his phosphate treat-

ments the deviations from normality were observed during

-7-

prophase in the delay of nuclear membra e "breakdown". he
pointed out that this effect had not been observed after the
use of the c-mitotic poisons. These substances had been re-
ported to permit mitosis to proceed normally until metaphase
(Levan 1958) when their specific effect on the spindle mecha-
nism.was manif 'este d. Accordingly, he concluded that, "the
unoriented prometaphase chromosomes must therefore involve
forces other than those associated with the oriented Spindle
mechanism." D'Amato (1949), after testing a series of
chemicals, observed. tha two different effects must be dis-
tingui shed: preprophase inhibition and an inhibition of
spin? e fo nation giving rise to c-mitosis. He was of the
opinion that "th spindle—inhibiting effect, manifested in
typical C-IlltOSlS poisons, may be of c'uite different sort
than that by which prepror hase poisons induce the c- -mitotic
effect." Ber3ner (19 50) found an arrangement of chromosomes
in the peripheral zone of a hollow sphere, as they were

arranged in late prop ebefore the nuclear membrane

l-’°

solated and interpreted this as ndicating that no spindle
was fo mead, and hat the chroraos01m s were not moved from
their prophase positions. This suggested that "the compound
was exerting its full effect before ritosis began or at
least no later than prophase."

It was first noted by Hushins (1948) that some sub-

1

being tested for c-m itos s

[-30

-ot only were not pro-

.3

stances

ducing polyploidy but were capsule of producing chromosome

-8-

groupings within the cell that might lead to somatic re-
duction. Such substances would be put in the category of
"unrecoverable“ and in a series of papers (Huskins 1947,

1948, Huskins and Cheng 1950, and Huskins and Chouinard 1949)
the occurrence and potential significance of these reductional
groupings was discussed. This type of effect has been re-
ported after treatments with sodium.nucleate (Huslzins 1948),
colchicine (Allen, wilscn and Powell 1950) and Acti-dione
(tilson 1E 50 and lthdO‘nv, un13ub1ished thesis 1951). Since
most of the work on plants with c-mitotic substances has

° 4"

been done witli the idea 01 producing polyploids, many
workers have probabli missed re‘uctional 3roupin3s and those
who did note such groupings (Levan 1959, Barber and Callan
1945, Witkus and Berger 1944, Nybom and Knutson 1947, D'Amato
1948 and Bergner 1950) did not realize its implications.

With the publication of the work of Hu$ {ins and his
associates there becare ap are nt tv .0 major points of view.
These, as expressed by Allen, Lilson and Powell (1950) were:
”(1) that the rechanism involved in the c-mitotic eerct is
probably the same re 3ardlsss of the chemical used, and in
any event, it is a phenomenon of little theoretical or prac ti-
cal importance, (Levan and Lotfy 1949) and (2) that the
mechanisms may b quite different and that there are, in any
event, important theoretical and practical implications (Eus-

kins 1948). The sepama tion of th e c-mitotic effects into

two groups was made by Allen, Wilson and Powell (1950).

-9-

They listed five points of difference between the effects

of the "mitotic poisons," e; which colchicine is the type

substance, and the "plysiological substances" of which the
sodium salt of nucleic acid is the type substance. These
were (1) arrangement of chrcmoscmcs, (2) types of groupings,
(5) chromosome d stribution within the groupings, (4)
frequency of reductional groupings, and (5) prophase to meta-
phase ratios. In 1951 Powell (unpublished thesis) showed
that the major point of difference between the effects of

the colchicine and sodium nucleate treatments in Pisum was
the time in the mitotic cycle that the effects were evident.

The nucleates showed effect- at late prophase while colchicine

C‘)

1

effects appeared at netapn -e. anthorne (1951, unpublished

9.?
C7

thesis) in an analysis of the cytological effects of Acti-
dione to be used as a quantitative basis for comparison with
other active chemicals, also found tiat the c-mitotic effects
represented a generalized reaction probably the result of
prephase or interphase effects which prevented "normal"

r3anization rather than destroying structures already
present.

-

It was srggested by Lilson, Hay

II

‘«

thcrne, and Tsou (1951),
reporting on the cytol 3ica1 effects of Lindane and Acti-dione,
that there are two, more or less independent, components of

the forces involved in the mitotic mechanism: "(1) a dipolar

cytoplasmic orientation possibly characteristic of the cell

at any sta3e; and (2) a nuclear component inherent in and

-10-

directed by the chromosome or the hinetochore thereof."

Then metaphase organization would be primarily the function
of the cytoplasmic component while anaphase separation would
be the function of the chromosome itself. Such an hypothesis
would explain the presence of "split but organized figures

as the esult of a weakness in cytoplasmic orientation;
multipolar anaphases would represent almost complete break-
down of cytoplasmic orientation without serious disruption
of the nuclear component; and completely dis rganizel meta-
phases and anaphases wculd indicate the elimination of both

‘

com ononts". As an ex mple of the latter, the unpublished

- 1

[—1.

pie may be c ed. In his preliminary studies
with short treatments of Allium with colchicine he has found
a reaction typical of the breakdown of both cytoplasmic and
nuclear components as evidenced.by (l) anaphases coming to

a stop and (2) metaphase chromosomes not reaching a plate
but beginning disorganized separation. After approximately
5 hours of recovery there is evidence that spindle action

has seen resumed with the chromosomes disorcanized, and the

initiation of cytokinesis gives rise to micronucleate cells.
It has been the purpose of this series of experiments
with 5 new or3anic substances; 0-1014, Systox, Thiolutin,
Eimocidin and P.A.-96; to compare their cytological effects
with those of cclchicine and other active c-mitotic sub-

stances to gain information that will lead to the elucidation

of the forces involved in the mitotic mechanism,

I}! 11' ai-r A“ ‘0 ‘T ‘r '.'IITYf\ o
L. LTLJ-LJ.I‘.1.ZL) AL.D l-'-A.J.L_-'V‘DI.J

The organic substan as tested in this series of e4-

‘

periments were obtained through tne courtesy of the few
Chemical Company of Iidland, Michigan and Ch s. Pfizer &
00., Inc. f Brcollyn, New York. System and C-1014 are
both insecticides produced by Dow Chemical Company. 0-1014
contains four pounds of Octaemethyl-pyropi ephoramide per
gallon, and is a water dispersible concentrate. The active
ingredient of Systox is a tri-alkylethiophosphate. The anti-
biotics, Thiolutin, Rinocidin and P.A.-96 were supplied by
the Pfizer Company. Although they have shown some anti-
bacterial properties, the main activity of Thiclutin and
Rimocidin has been indicated as antifungal in nature, and
they are being employed experimentally in the treatment of
dermatophytes. P.A.-9G, while being devoid of activity

against both bacterial and fun3al seecies, has shown a pro-

7
.5

nouneed effect against certain viruses.

All eXperiments were carried out on a single lot of

 

Allium cepa sets. According to routine already established
in this laboratory, the sets were rooted in vials of aerated
distilled water. When the roots were approximately one to
two inches lcng the root tips were examined and if they
showed a high rate of mitotic activity the sets were

-11-

-12-

transferred to vials of the solutions to be tested. In all
cases stock solutions vere made with Listille d water and
kept refrigerated until a series was run, at which time the
solution was brought to room tenperature before the root
tips were transferred. All e3cperiments were conducted at
room emperature and the onions were kept under normal con-
ditions ofe day and night. After treatment the roots were
washed with, and returned to vials of, distilled water and a
check for recovery was made at four, twenty-four and forty-
ght hours.

In all cases a preliminary trial was run w.ith selu ticns
ranging in concentration from 200 to 10,000 parts per million
with root tips collected at four, eijht and twenty—four hours
to determine the: est: romising concentration to be used in
an extended series m:ere root tips were collected at hourly
intervals until a L vel of toxicity was re ache d. Systox was
tested up to a concentration of 500,000 parts per million
and a series run at that concentration. The Pestox series
was run at a concentration of 10, 000 parts per million. The

hielutin, niqociain and P.A.-96 series were taken at con-
centrations of 1,000 parts per million.

Root tips were collected in a three to one absolute
alcohol-acetic acid mixture, fixed for l5 minutes at 60° C.,
hydrelized in one normal hydrochloric acid for 8 to 10 rzinutes,

Htion made.

'0
e
O
*3
{J
g

stained with Feulgen's stain and smear

After dehydration in a 95% ethyl alcohol to which a little

-13...

fi

fast green for counterstain had been added, the slides were
made permanent with diaphane.

At each cutting two root tips were taken and placed on
separate slides so as to obtain a duplicate series. Dr. G.
B. Wilson counted the slides from one series while the
writer counted th slides in the duplicate series as a
check upon the consistency of the data.

Ebr counting according to a standardized procedure,
each slide was placed on the calibrated mechanical stage,
the count was started at a constant, predetermined point
on the vertical axis and this reading recorded on the data
sheet. Consecutive high powered fields were counted from
edge to edge of the cover slip and these totals were re-
corded on the data sheet for each axis. The vertical axis
was increased by one unit each time and the count continued
until a total of approximately 100 division figures were ob-
tained from each slide. In the slides where the 100 figures
were totaled in the middle of the axis, the count was con-
tinued until the readines on that axis had been completed.
In those cases where, in the later hours of treatment, the
division figures were less numerous, it was frequently
necessary to go back over the sli‘e and tane readin
the one-half unit intervals.

It was recognized that, aside from tne subjective
element involved in the counting of the slides, two factors

might influence the counts: (1) the evenness of the

-14-

distribution of the haterial and (2) wzether or not the

*3
f.)

most actively dividing areas of the me i tem happen to have
fallen within the predetermined area of the ccrnt. hcwever,
since it can be assumed that all areas of the root tip
an equal chance of being ev nly distributed within the pre-
determined area of the count, it is believed that this
technique could be used to obtain an indication of trends

and should be interpreted accordingly.

 

Centrols
hany investigations have been undertaken in this
laboratory with control material. In accor*ance with the
established method in these experiments two root tips were
cut from every onion to be treated at the zero hour of
treatment. From the combined series of experiments 11 con-
trol slides were chosen at random.and counted to determine
the prophase: metaphase: post-metaphase ratio. In Table 1
it will be seen that this resulted in an average frequency
of 50.73'prophase figures, 22.4fi metaphase figures and 26.9%
post-metaphase figures. Plate I (figs. A, B, C) illustrate
normal figures of prophase, metaphase and anaphase divisions
found in control material.
Insecticides
0-1014
Root tips tested with a solution of 0-1014 at a con-
centration of 1000 parts per million showed no cytological
or toxicity effects after 24 hours of continuous treatment.
When the concentration of the solution was raised to 5,000
parts per million there was evidence of a very mild cyto-
logical effect after 24 hours of treatment. Prophase and
and metaphase finures were organized. Ketaphase and anaphase

-15..

-15-

figures showed a more than normal contraction of the chromo-
somes. The series run at a concentration of 10,000 parts

per million showed a mild cytological effect at prophase

after two hours treatment. The effect progressed as evidenced
by the hourly collections. At four hours the majority of
prophases were affected. As is typical with colchicine treat-
ment (Plate I, fig. L), metaphase chromosomes were greatly
contracted and were scattered throughout the cell in a dis—
organized manner (Plate I, fig. E). Post-metaphases were un-
affected. After six hours of treatment toxic effects became

dent witn ver

|J°

ev r "tight" prophases (Plateig fig. D). By 12

hours of treatneit there were very few divisions and these

'3

all showed high toxicity effects (Plate I, fir. F).

C

Recovery of the cytolOQical effects of C~1014 were
attempted after treatments of three, four, and five hours
with a concentration of 10,000 parts per million. There

sion stopped completely.

Po

were no recoverable effects; div
Hourly variation in the frequencies of the individual stages
of mitosis as compared to control slides was found to be
erratic (Table 2; Text fig. 1) and no major trend was ob-
served.
Systox

Systo‘~ was tested in solution with concentrations of
10,000; 20,000; 50,000; 100,000 and 500,000 parts per million.

In all cases after short treatments 01 one hour there was

aical effect. Kitosis

Q.

evidence of toxicity without cytolo

-17-

came to a complete standstill. Cells in interphase showed
toxicity effects with dark staining. hetephas and anaphase
figures were still organized but chromosomes were greatly

contracted and so clumpinr. hetaphases were

reverting to resting stages. A the highest concentration,

500,000 parts ner million, there were still many divi ion

\
IL

[I

O
..

figures in the raterial but all figures showed signs f

O

toxicity and reversion.
tibiotics

Thiolutin

 

’5

Root tips tested with solutions 0* r“intolutin at con-
centrations of 100 an 200 parts per million showed no cyto-
logical or toxicity effects after 24 hours of continuous
treatment. After four hours of treatment with the concen-
tration raised to 500 parts per million, the roots were still
normal but at 12 hours showed signs of toxicity without
cytological effect. With the concentration of Thiolutin at
1,000 parts per million and after three hours of treatment
there was evidence of a mild ytological effect. Kitotic
division figures were still organized but the chromosomes
were more contracted than normal. By four hours oi treat-
ment at this concentration chromosomes were greatly con-
tracted at metaphase, in some cases showing organization
(Plate I, fig. G) and in a few cases lying scattered through-
out the cell (Plate I, fig. E). At this tine ”asphase

figures were still organized but were seen only in polar

-13-

view (Plate I, fig. I). After eight hours of treatment

the first signs of toxicity were becoming apparent and by

twelve hours there were few divisions, with the material

at all stages of mitosis showing toxicity (Plate I, fig. J).
All attempts to recover cytolOgical effects after treat-

ment with concentrations of 500 and 1,000 parts per million

after three, four and five hours were unsuccessful.

Heurly variation in the frequencies of the individual stages

of mitosis as compared to control slides (Table 5, T-xt fig. 2)

showed a decline in the number of prophase anl post-meta-

igures with a complementary rise in the number of

PH

hase

*U

metaphases.

Rimecidin

 

In the s

(J

ries testing nimoc1din in solutions with con-
centrations of 100, 200 and 500 parts per million, after 24
hours of continuous tree ment, all material showed div sions
to be entirely normal. With a concentration of 1,000 parts
per million division continued normally until, after 12
hours treatment, toxicity was evident. There was no decline
in numbers of division but all stages of mitosis showed
toxicity, with the chromosomes shortened and beginning to
revert.
P.A.-96

An extended series was run with solutions of P.A.—96
at concentrations of 100, 500, and 1,000 parts per million.

Root tips were collected in each.series at two, three, four,

-19..

five, six, eight, twelve and thirty-six hours and after
five and one-half days of continuous treatment. In all
material examined, there was found normal mitotic division
(Plate I, fig. K). Hourly variation in the frequencies of
the individual stages of mitosis as compared to control
material shows a decline in the number of prophases with an
increase in the number of me apaases and post-metaphases
(Table 4, Tex fig. 3). A count of the number of divisions
per 1,000 cells (Table 5) showed no increase or decrease

from that of control material.

PmCEITAGES OF TOTAL DI VI SIC-I"?

I EDI VI DUAL

STAGES III

TABLE 1

FIGURES 0

CC KT EGL KAT IZIAL

F

 

 

 

Prophase Hetaphase Post-Meta.
Slide No. % to. % No. % Total
1 55 53 18 18 29 29 100
2 45 45 26 26 29 29 100
3 4O 4O 29 29 51 51 100
4 45 45 54 54 21 21 100
5 52 52 19 19 29 29 100
6 41 41 25 25 34 54 100
7 66 66 17 17 17 17 100
8 46 46 27 27 27 27 100
9 118 59 55 16.5 49 24,5 200
10 66 66 2O 2O 14 14 100
ll 45 45 ~15 15 4O 40 100
Total 1200
Average % 50.7 22.4 26.9
Standard Error‘: 2.815 : 1.855 : 2.25

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-30

 

 

PR OPH AS E

_ _ ' M ETAPHASE
"“ - POST METAPHASE

 

 

HOURS

* (‘1’ or:
- Lth-L- Lad. A'J l
v \ay~ ‘ .- I‘F"_ gm} (\f‘y s!" fi-Ffl‘vvfii’ — :‘f‘ 047-1 ”—‘FQ‘T f 9-er n—‘r‘
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PERCENTAGE

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PERCENTAGE

 

20

IO

 

-27-

PROPHASE

 

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T" “'POST METAPHASE

 

 

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~40 é ' t t 3—1
4 IO
HOURS
TEXT FIJJRE 5
IICUKLY X’nRIATI I? II? E’RJLCELIIZS CF IKEIVITLI. ETTASES

P “510-96

TTIL‘JI‘IOZ? c 1? 1 , 000

p ,a Tym ‘ 1':

. 15.-.... $

11'- 7"- TC;‘\?
43 LLLLL‘H

DISCUSSION

 

Ho

As it was the purpose of this nvestigation to compare
the cytological effects of the two insecticides and three
antibiotics with those of colchicine and other active 0-
mitotic substances, in the examination of results the
occurrence of five things was looked for:

(l) spindle abnormalities and c-mitosis,

(2) change in the relative numbers of prophase,

metaphase and post-metaphase figures,
(3) over-contraction of the chromoscmes,
(4) an increase in the percentage of reductional
groupings, and,
(5) necrosis.
Spindle Abnormalities and C-Mitosis

Ludford (1936) first suggested that the polyploidizing
action of colchicine Was caused by its specific effect upon
the spindle mechanism of the dividing cell. with the dis-
ruption of the spindle the chromosomes, at metaphase, were
left lying scattered throughout the cell. At anaphase th
chromatids separated, and then, with removal from the in-
fluence of colchicine, the spindle became reactivated and
the then polyploid cells were able to continue a normal
division. The term “c-mitosis", as first used by Levan
(1945), referred to the typical effects of colchicine upon

C‘Q

“flaw -

(‘0
(O

the mitotic mechanism and implied the polyploi‘ result.

Since that time, and as used in this instance, "c-mitosis"
has come to mean the two outstanding cytological effects

of colchicine treatment, i.e., spindle disruption and a
delayed splitting of the kinetochores of the over-contracted
chromosomes causing x-shaped figures scattered at random
throughout the cell. Fellowing the discovery of colchicine
as a polyploidizing agent much work was done in the follcwing

1

years with other organic suostances by various groups searching

for polyplo izing agents. The qualities to be looked for

H.
5" Q:

in such subs ances are: (l) a disrupting effect upon
spindle formation as evidenced by disorganized "scattered"
chromosomes, and (2) a cytological threshold separate from
the toxicity threshold with resunption of normal activity
:pon removal from the influence of the substance.

P.A.-96 had no effect upon the spindle mechanism. All
division figures appeared normal and organized. Rimocidin
and Systox showed neither evidence of Spindle disruption
or other c-mitosis effects. In both cases divisions showing
any effects of treatmen represented toxicity effects.
Thiolutin, at treatment of 1,000 parts per million for three
hours, began to show signs of c-mitotic effect with the over-
contraction of the chromosomes. After four hours of treat-
ment there were a few divisions at metaphase showing the

typical c-mitotic effect with disrupted Spindle mechanism

.1.

Ho

and scattered x—shaped chromosomes. however, in the major ty

-30-

of the meta phase divisions the figur es were or3an ized,

l._.'.

showing no s 3ns of spindle abnormality. rtrther treatment
produced toxicity effects. It can therefore be concluded
that Thiolutin is to::ic at any concentration that is capable
inducin3 mitotic abnormalities. At a concentration of
10,000 parts ee m llion C-1014 be3an to show signs of c-

.5

mitosis after three hours of treatment. After four hours

‘1

of treatment Instanapaases showed complete spindle disruption
and the chromosomes were over-contracted. This was considered
to be of particular importance since, potentially, such
fi3ures may lead to a polyploid condition. After six hours
treatment toxicity was evi nt. As all attempts to recover
from the c-mitotic effects were uns uccessxcl it must be con-
cluded that C-lOl4, like Thiolutin, is toxic at cone extrations
capable of inducin3 mitotic Chan3 es.
Change in the Relative Numbers of Prophase,
hetapha ass and P08 t-“eta'rase figures
Previous experiments have shown that one of the most
f

readily detectable effects 0- any substance on mitosis is a

more or less marked chan3e in the relative frequencies of
prophase, metaphase and post-met phase fi3ures. A detailed
analysis of these c :an3 es was made from material treated with
C-1014,Thiolutin and P.A.-96 (Tezct fi3s . l, 2, 3). As can
be seen from the graphs, he only consistent or significant

trends in the data was obtained with Th1 olutin. C-lOl4

shows only an erratic increase and decrease for all stages.

It may be noted that 'Wit P. A. - 96, contrary to the usual
trend, there would appear to he an increase in the n*mber

of post-1r.etaphase fi3: =;ures in combination with a rise in the
number of metaphases and with a decrease in prephases.
Thiolutin, however, showed a definite increase in the number
of metaphase figures while there was a decrease in the
relative number of prophase and post-metajhase fi3ures.

Such a crang e was folrn nd in the extensive study of Acti-
dione (IZawthorne, ur published tlme is) at the lowest, cyto-
lo3ically effective concentration used in continuous treat-
ment. With so many variables int olved in relation to the
frecuency chan3es; 6.3. such factors as the absolute fre-
quency of division figures, the rates with wl ich one sta
proceeds to the no xt and the de3ree of mitotic disruption;
it is difficult to draw any definite conclusions from the
data, especially in the absence of any other effects from

he treatment. however, it may be su3rested th at Thiolutin
had an effect upon prophase which is of a stalling ature
and which prevented onset of prophase and, in combination with
that, had a stallinge ffect upon metaphase W1 ichn prevented
that stage from pr03ress in3 to post-metaphase. This would
account for the rise in relative numbers of netaphase divi-
sion figures in combil lation "ith a decrease in the nur:bers
of prophase aid post-metaphase fi3ures, and rzi3 31t be cor-
related with the over-contraction of he chromosomes at meta-

phase as found with this concentration and tin of treatment.

-32..

Over-Contraction of Chromosomes
There was no evidence of over-contraction of chromo-
somes in material treated with P.A.-96, Rimocidin and
Systox. fiith Thiolutin, at a concentration of 1,000 parts
per million and after three hours of treatment, metaphases
began to show si3ns of over-contraction and after four hours

5

were "super-contracted". As was suggested above, this may
be correlated with the increase in relative nuxber of meta-
phases. C-1014, at a con centration of 5, 000 pa arts per

million, Showed si3ns of over-contra etion aiter a treatment

of 24 hours. When the concentration was increased to 10,000

parts per nzillion, the over-contraction of tr e c11r0mm1es
at metaphase appeared after four hours of treatment. As

over-contraction of the chroriosomes is usually correlated
with prolongation of the entire mitotic division, or with
prolongation of one sta3e of division, and since there was
no indication of a slowing down of propha se, metaphase or
post-metaphas e as evidenced in the hanges in the relative
numbers of these stages, it may be su33ested that C—1014 has
3e neral effect of prolon3ation of tze p100 es of mitosis.
Reducticnal Divisions

The occurrence and si3nificance of reductions l groupings
has been discussed by the 3roup at Wisconsin (flushins 1947,
1948, Lilson and Cher3 1949, flushins and Chang 1900, and

Itszins and Chouinard 1949).Li1son (1950) reported that a

variety of substances, unrelated to those previously tested

-55-

by the Wisconsin group, were capable of increasing the numbers
of such reductional divisions. Since the reductional group-
ings, as referred to by the above authors, have probably
been missed in many cases due to the fact that only the poly-
ploidizing activity of the substances was being tested, a
careful check was made on all material treated with C-lOl4,
Systox, Thiolutin, Rimocidin and P.A.-96. In no case was
there found an incidence of reductional groupings which
exceeded that found in untreated material. From.this it may
be concluded that these organic substances had no effect on
the occur ence of re‘uctional groupings at the degree of
concentration and length of treatment employed in these ex-
periments.
Necrosis

Necrosis, in this study, refers to the condition of
the nucleus when tie prophase chromosomes continue to pro-
ceed through their regular morphological changes while the
nuclear membrane seems to have been retarded in its activ-
ity; and then, having appeared to contract the membrane,
has drawn the contents of the nucleus into a tight ball.
Such an effect is evidence of toxicity and may arise from

1

a stalling effect of the substance on Livision or from re-
version of the chromosomes to intorphase. P.A.-96 showed
no signs of toxicity or necrosis. Systox and Rimocidin

showed only effects of necrosis at any level capable of

inducing changes from the normal. Necrosis was observed

-54-

in roots treated witi C-lOlé, with tight prophases, after
six hours of treatment with a concentration of 10,000 parts
per million, and after treatment of 12 hours all divisions
showed necrosis with evidence of tight prephases and meta-
phases the chromosomes of which were reverting to inter-
nhase. Similar effects of necrosis were seen in material

in

treated with Thiolutin after 8 hours of treatment

SIEECAI?

 

The cytological effects of two insecticides, C-1014 and
Eye oz, and of three antibiotics, Rimocidin, Thiolutin
and P. A.—96, were presented and discussed.

Studies were made of continuous exposures up to 12 hours
at concentrations raz:gi n; from 1,000 to 500,000 parts
per million for C-1014 and Svstox and from 200 to l, 000
parts per million for Himocidin, Thiolutin and P.A.-96.
The occurrence of five things was looked for: (l)
spindle abnormalities and c-mitosis, (2) change in the
relative numbers of prophase, metaphase and post-meta-
phase figures, (3) over-contraction of the chromosomes,
(4) an increase in the percentage of reductional
FPOLoln” o, and (5) necrosis.

The only cytological effects of Systox and :imocidin
were observed to be toxic.

P.A.—96 was found to have no evident cytolcgical effects.
T: e cytological effects of C-1014 and Thiolutin were
found to be an eve r-ccntraction of the chromos cm :es at
metaphase after some treatments. C-1014 showed signs

of unrecoverable c-mitosis.

D

.se in the

(D
o

A sugges ticn of correlation between an incr
frequency of metaphase figures and over-contraction of

chromosomes was dis cussed wit1 re ard to Thiolutin.

(l)

)

{‘3

(

(5)

(4)

(6)

(7)

LITJEL’LT ’RE CIT <

 

Allen, R. 3., G. B. Eileen and 3. Powell (1050)
"Corparative Effects of Colchicine and Sodium Eucleate
on Somatic Chromosomes of Allium and Tradescantia".
Jour. of lered. 413159-165.

 

T‘arber, K. K. and H. G. Callan (1943)

"The Effects of Cold and Colchicine on Hitosis in the
Hewt”.

P1700. R0 SOC. BOto 131:258-2710

Bercer, C. A., and 3. R. Witkus (1945)

"A Cy tolc:ica1 Study of C- Kitosis in the Polysoma atic
Plant cpinacia Olerace a, with Jompara tive Observations
on Allium C3pa".

Bull} Torrey Bot. 19:457-466.

 

 

Earp-311’ C o .510 , _“ R. In: b-.uS an
”The Cytological Effects of 3
Bull. Torrey Bot. Zl_:6.2 0- 623.

d 3. J. Sullivan (1944)
3nzene Vapor"

Bergner, A. D. (1950)

"“tudies on Jolchicine Derivatives III Effect on
Litotic Activity of House hecrnato :onia"

Cancer §;104-141.

Christoff, I. and I. A. Qhristoff (1942)

"heiosis in the Somatic Tissue Responsible for the
Reluction of Chromos cme Number in the Progeny of
Eierocium hopp-anrm Schult".

Geno §:56’42 o

 

D'“mato, F. (1948)
“h Sffect of Colohicine and Ethylene Glycol on
Sticky Chromosomes in Allium Cepa“.
Hereditas £4:81-105.

 

(9)

(10)

(ll)

(12)

(15)

(l4)

(l5)

D'Amato, 3. (1w 0)

"The Chromosome Breaking Activity of Ch micals as
Studied by the Allium C3na Te st".

Publ. Stazione 200. ai napoli. Vol. 22 S app.

ixon, W. E. (1905)
A hanusl of Pilarmacolo3V. London.

Dustin, P. (1947)
"Some New Aspects of 2
Nature 159:794-797.

tot ic Poisoning"

Galinsky, I. (1949)

"The Effect of Ce rtain Ph q31ates on Iitosis in
Allium 1100158".

JO‘Llro Of LCI’C CL. fig:229-2950

La horne, K. E. and G. 3. Iilson (1952)
"Cytological Effects of the Antibiotic Acti-dione"
In press.

Huskins, C. L. (1954)
"Anomalous Segregation of a Triploid Tomato"
Jour. of Herod. 25:281-264.

Huskins, C. L. (1948)

"Chromoscme Xultiplication and Refiuction in Somatic
Tissues".

Nature Ewe-£5.

ELISziinS, Co I... (104:8 )

"3e3r33ation and P36 action in Lomatic Tissues.
I. Initial Ooscrvations on Allium.Cepa".
Jour. of Hered. 52:511-325.

 

1181:1118, CO L. (1949)

LT-"The IIucleus in Development and Differentiation and
the prerirental Induction of 'Xeiosis'".

fiercditas Suppl. Vol.

-38-

(17) Huskins, C. L. and K. C. Cheng (1950)
"Segregation and Reduction in Somatic Tissues.
IV. Reductional Groupings Induced in Allium.Ce3§
by Low Temperatures".
qur. flared. §1;13-18.

 

(18) Huskins, C. L. and L. Chouinard (1950)
"Somatic Reduction: Liplcid and friplcid Roots and
a

a Liploid Shoot from Tcmfiploid Rhee".

(19) Kodani, :. (1948)
"fictium Ribose Nucleate and mitosis".
Jour. of Hered. 39:527-335.

(20) Levan, A.(1958)
"The Effect of Colchicine on Root Iitosis in Allium".
Hereditas 223471- 86.

(21) chan, A. (1939)
"Th Effects of Cclchicine on Root Iitosis in Allium".
Hereditae 25:87-96.

Levan, A. (1945)
"Cytclogical Reactions Induced by Incrganic Salt
Solutions".
Eature l§§:751-752.

rs
(0
N)

V

(23) Levan, A. and T. Lctfy (1242)
"Kapthalene Acetic Acid in the Allium rest".
Hereditas §§;357-374.

(24) Levan, A. and G. Cstergren (1945)
“The Eechanism of C-Kitotic Action. bservaticns
on the Kapthalene Series".
Eereditas 223379—4é5.

(25 Levine, I. (1245)
"Th Effect of Cclchicine and K-Rays on Onion Root 1ips."
Cancer Research 5:107-119.

(26)

(27)

-59-

vell (1949)
Node of Action of

Loveless, A. and S.
"New Evidence on the
Poisons'".

'fiitotic

nature 164:: 3’ 8-944.
Ludford, R. J. (1956)

”The Action of Toxic Suostances 1 son the Division of
Normal and: alignant Cells in Vivo and in Vitro".
Arcr iv. fur 1xp=riwencclle Zell for scnung 18:411-441.

Rebel, B. R. (1957)
"Cyt Clo: ical Observations on Colch
Biol. Bull. 75 551-552.

icine”.

Nebel, 3.11. and 1.2. L. Rattle (1958)
”Action of Colohicine on Iitoois"

Genetics 25:151-162.

1:3‘031’30 R0 811d 1‘. Lo Rdttle (10:8)
"Tne Cytolofical and Genetical Lignificance of

Colchicine"
Jour. of Kereo. 29:2-9.

Nybom, N. and E. Knutsson (1947)
"Investigations on C-Nitosis in Allium Ceoa. I.
Cytological dffect of Hexachlorocy clohexane"
Heredity 553220-254.

The

 

Cst:rgren, G. (1914)
“Colchicine Iitosis, Chromosome
and Brctein Cnain loloing".

Hereditas 50:429-457.

Contraction, Necrosis

Ostergren, G. (1944)
"An Efficient Chemical for tie Induction of Sticky
Chrome somes".
LBPGLJ'. uaS 159-: 512-316 .

and A. Levan (19é5)

"The conne ction Between C-Nitctic Activity a.d
Solvoility in you: Tcnocyclic Compounds"
Leroditas 29:495-498.

Cs tergron, 3.
4..

.18. L13 1?

(36)

(57)

(58)

(59)

(4O)

(41)

(42)

(43)

-40-

anc B. H. Nobel (1940)

Patton, R. L.

"Preliminary Observations on E-r“’olcg_csl and Jyto-
legical Effects of certain I3drocaroons in Plant

Tissues“.

Am. Jour. of Lot. 27:509-515.

Peteis, Josepl J. ( 945)

"C3tolo: ical 1ffects of Sulfanilamide on Allium Gena
Bot. Gaz. Vol. 27, I0. 5.

 

Powell, Shirley (1951)

"Com‘339rat ive 31f cts of Colchicine and Various Nucleic

Acid Salts upon somatic Iitosis".
Unpub. Thesis for Degree of 3.8. at ”ich. State Coll.
Wiiamura, T. (1959)

h

"Cytological Studies 01 Pclyploidy Induced by Colchicine".
Cytologia 9:455-494.

Ste ineQ ger, E. and A. Levan (1949)
”The C-I. Iitotic Qualities ofColc‘11icine, Trimethyl
Colchicinic Acid and Two In enant :rene Derivatives"

Vaarama, A. (1947)
”Emperimental studies on the Influence of
Insecticide Upon Plant Iitosis"
Iereditas 55:191-219.

DODOT.

Wade, 9. (19 49)
"Enrther Studies on the Effect of 30101 iicine upon
the Mitosis of the Stamen Iair in Tra iescsntia”.
CytologiaIl§:88-95.

 

Wilson, 9. s. (1950)
”Cytological «ffects of So: as Antibiotics".
Jour. Herod. 41:225- -251.

fiilson, G. 3. and C. C. Bowen (1951)
"Cytological Effects of Some Icre Anti
Jour. of Hered. 42: 251 -25 .

biotios”

I
\

4

4)

5)

I‘Jil Son, '2'.
"Segrse gaticn and

II. THC
Trillium
W
Jour. 01

Jilson, G.

:— .1.
"spontanc

Jour. of

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.thcrle and Te flay Tsou lJ
and ndiced Variations in Iitosis".

-41-

9 :eng (1949)

chu ction in Lomatic Tissues

Iomolo:ous Chromosomes in

-"’1E;90

A CI’ITO"..1:§13C—I.I$?TS

 

The writer wishes to extend her profound gratitude

to Dr. G. B. Wilson, whose interest and encouragement,

O V

helpful discussion, advice and criticism nave made th

C!
U

M

work possible. Kis critical reading of the manuscript
and count of one of the two series of slides is deeply
appreciated.

The writer also wishes to express her thanks to
Ir. Ihilip G. Coleman for his photomicrography and sug-

gestions for final preparation of graphs.

-42-

 

PLATE I

Iormal prophase from control material.

ornal r23 Mailase from control raterial.
Zormal anaphas o fr-m contr rel material.
"Tight" propha so after six hours treatment "ith C-1 014
at a cor centra tion of 10,000 parts per million.
Over-contracted, scattered chromosomes at metaphase
after four hcurs treatment with C-lOlé at a concentration

10,000 parts per nillio-.

x'city leects from treat23nt witn C-lOl4 at a con-
centration of 13,000 parts ior million after 12 hours
Organized, over-c ntract3 C chro " mes at metaphase
after four hours troat23nt with Thiolutin at a con-
centrat ion of 1,000 parts per million.

Scattered, over-contracted chromosomes at metaphase

after four hours troat~on with Thiolutin at a con-

centration 1,000 parts per nillion.

Polar View of ana thase a;‘ ter four horrs treatnent with
hiolutin at a concentr tion of 1,000 parts per million.

ionicity eifi3cts after eight hours of treat:eu with

Tliolutin a a cancentr ation of 1,000 tarts pe million.

Letaphase and najhase from mate rial trea tad 12 Lours

with P.A.-96 at a concentiation of 1,000 parts per

million.

Typical c-::i tosi s from material treat-3i 15 minutes

with colc21ic inc at a concentration of 1,000 parts per

millio ani r3covero d for 5 hours.

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