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WCEHSAN SM'E‘E UR‘EVERSETY

Gabrieie Mnhhng

1957

MITOTIC RESPONSES IN EISUH SATIVUH

TO THREE HERBICIDAL COMPOUNDS

By
Gabriele Mt'ihling

AN ABSTRACT

Submitted to the College of Science and Arts of Michigan
State University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

1&st OF SCIENCE
Department of Botany and Plant Pathology

1957 ~fi“ tug

Approved by.I§E§E%£¢ié:§§¢¢z;

 

Gabriele Mdhling

The 12.2.“; test was used to determine the cytological effects of
three phenolic compounds, tris and bis phosphite forms of 2.1+ dichloro—
phenoxyethanol and 2,1; dichlorophenoxyethanol. laboratory formulation
numbers of these herbicidal compounds are 3Y9, 7Y5 and Jon respectively.

Root tips of young seedlings of m sativum were treated for
3—1» hours at concentrations ranging from 60-90 ppm and 200 ppm for 3Y9,
90—135 ppm for 715 and 180-270 for Jon. Treatment solutions contained
the compounds plus a balanced nutrient solution. Boot tips were col-
lected at predetermined times. Slides were prepared by the Images-
squash technique from which cytological data were obtained. Some seed-
lings were recovered and observed for morphological effects.

Scattered metaphases and anaphases were the key aberrant configu-
rations produced by the three compounds. A few other abnormalities
such as clumps and multinucleate cells were also recovered. These
effects were then compared with those produced by the c-mitotic agent.
colchicine.

Conparing the effects of colchicine and the three phenolic com-
pounds, identical cyt010gical aberrations namely scatters and clumps
are produced. A similar time-dose relationship of effect is also
shown, i.e. a straight line based on log dose/log time. Production of
clumps appear comparable in the two types of substances. Multinucleate
cells were recovered in material treated with the phenols but no fur-
ther analysis was made. In no case was polyploidy found in material
treated with the phenols. A difference also exists in the effect
which these compounds have on mitosis in general. The mitotic index

remains relatively constant throughout colchicine treatment. In the

Gabriele Mdhling 2

case of the phenols, it falls suddenly after extended treatment. The
differences however are probably not significant in view of the toxi—
city factor, which is always encountered with these phenols.

In conclusion, 3Y9, 7Y5 and.JOh can be tentatively considered as

c-mitotic agents.

REFERENCE

Hadder, J.C. 1957. Cytological measurement of c-mitotic and prophase
poison actions. PhD Thesis, Michigan State University of Agri—
culture and Applied Science, East Lansing, Michigan

NINTH! RESPONSES IN PISUM SATIVUM

T0 m HERBICIDAL COM’OUNDS

By

Gabriele Mahling

A THESIS
Submitted to the College of Science and Arts of Michim
State University of Agriculture and Applied Science
in partial fulfillment of the requirements

for the degree of

MAS'ER OF SCIENCE

Department of Botany and Plant Pathology

195?

/.:>"/2>

q‘)

FRONTISPIECE

A group of scattered metaphases illustrating those produced by
the 3 phenols, 3Y9, 7Y5 and Jon.

AC mowmncmnmurs

The author wishes to express her deepest gratitude to
Dr. G.B. Wilson fer his abundant assistance in the directing and
carrying out of this study and for the words of encouragement which
were always there when neededi

Also sincere appreciation for numerous helpful ideas obtained
from other members of the cytology group and botany department.

To Mrs. Natalie Knobloch fer her technical assistance,
Dr. 3.3. Grigsby for kindly supplying the compounds investigated.and
Mr. P.G. Coleman for his photographic services, many thanks are given.

In.sddition deepest appreciation is expressed to the National

Institute of Health for their financial aid in this project.

INTRODUCTION
nmms AND METmDS
1.1mm EVEN
OB SERVAT IONS

DISC USS ION
SUMMARY
CONOLUSIONS
mus

TEXT FIGURES
PLATES
BIBLIOGRAPHY

TABLE 01' CON'IENTS

16
21

21

27
31
33

TABLES

I.

II.
III.

IV.

VI.

VII.

LIST OF TABLES

Doses Expressed in Parts Per Million and in Milli-
equivalents of 2,h—dichlorophenoxyethanol per milliliter.
Percentage of Aberrations Using 319.

Clamp-Scatter Indices for 319 Treatment with 200 PPM.
Number of Abnormlities per 100 Post-prophese Divisions
after 3Y9 Treatment at 200 PPM.

Mitotic Indices.

Time in Minutes of Twenty Percent Effect

Highest Number of Abnormalities Obtained per 100 Post

Prophase Divisions.

PACE

22

23
21+

21+

25

26

26

LIST OF EXT FIGURES

TEXT FIGURES PAGE
1. Percentage of Aberrant Configurations Obtained upon

Treatment with 3Y9. 27
11. Percentage of Aberrant Configurations Obtained upon

Treatment with 7‘15. 28
111. Percentage of Aberrant Configurations Obtained upon

Treatment with 301+. 29
IV. Log dose/Log time Relationships of 3Y9, 735 and Jolt

Based on 20% and 30% Effect Intercepts.

LIST Ol‘ PLATES

PLATES PAGE
PROMISPIICE Group of Scattered Metaphase Plates.
1. A Series 'of Nomi and Aberrant Division

figures. 31
II. Seedlings Illustrating Abnorml Morphology. 32

INTRODUCTION

One of the more imortant groups of chemicals known to effect
autosis are the substituted phenols, many of which.are used.as the
basis for'pesticides of various kinds. Preliminary cytological inves-
tigations of three phenolic substances, having laboratory formulation
numbers of 3Y9, 7Y5 and.JOb, indicated that their effects were quali-
tatively comparable to those of colchicine. A review of the literature
on phenolic compounds and their relatives also indicated this. The
indication of a colchicine type reaction was the impetus which started
the experimentation to be discussed herein.

The three substances, mentioned above, though not available COIF
mercially, are all presumptive herbicides. 3Y9 and 715 are trio and
bis 2,h dichlorophenoxyethyl phosphites respectively and.J04 is 2,“ di-
chlorophenoxyethanol (to be referred to hereafter as 2,1» DOPE) itself.
Of the three, 3Y9 has the highest herbicidal value on a weight basis.
Experiments were also conducted to determine whether the degree or
rate of cytological effect varied according to the molecular structure.

Since many of these substituted.phenols differ only with respect
to number*and position of a common substituted atom or radical, it may
be thus possible to determine or define the active site or'active coup
figuration of such compounds in the near future. .A program is being

conducted with additional phenols for evidence of this possibility.

MATERIALS AND METHODS

The meristemtic tissues employed in these studies were the
actively growing root tips from m m var. Alaska, a relatively
homogeneous genetic strain which is also disease free. The peas which
were kindly donated by the Perry-Morse Seed Company were not treated
with any chemical by the growers prior to their use.

The experimental technique used was essentially that develOped by
Bowen (1953). Peas were first soaked in distilled water over night or
until fully imbibed. They were then rolled in paper toweling and the
rolls placed vertically in beakers approximately one third filled with
distilled water. Previous investigators showed that the toweling used
for germination was inert. i.e. caused no deviations from normal mito-
ses. Peas were allowed to germinate in the rolls for approximately 36
hours at room temperature, at which time seedlings having roots 2% to
3% cm. long were selected for treatment. The seedlings were put on
paraffin coated wire mesh and suspended over 250 ml. bealners.

The phenolic compounds used were kindly supplied by
Dr. 3.3. Grigsby. Because these substances are not water soluble they
were incorporated in Carbowax so that they could be put into solution.
A Carbowax blank was run at the beginning of these studies on pea
seedlings and no deviations from normal were found in the number and
kind of division figures. The structural formlae available for these

compounds are:

CL
( aw) 64:;th W
' ' 3
3Yq 7r:-
a<i§ww~
.104

Stock solutions of 1,000 ppm and 10,000 ppm of the chemicals were
used from which the desired concentrations were made. It should be
pointed out that the accuracy of the concentrations was only within
several parts per million. Because of the often semi-liquid or par-
tially solidified state of the compounds it was inpossible to be more
accurate. Stock solutions were kept refrigerated from time of mixing
to actual use. Fresh stocks were used each time.

Treatment solutions contained the desired concentrations of the
chemical in a balanced salt solution. Triple distilled water was used
in all treatment and control solutions. The treatment chambers,

250 ml beahers, were placed in a 22°C controlled water bath and allowed
to equilibrate before seedlings were suspended in them. pH was also
taken at this time and at the end of each run. A pH of 5-6 was consi-
dered satisfactory, since previous work has shown that a pH variation
within this range has no effect. A control was always run along with
each experiment. Samples from control and treatment were taken at pre-
determined times. A zero hour control was taken at the beginning of
every experiment. Other collections were taken at 15 minute intervals

for the first hour, half hour intervals for the second and third hour

-u-

and hourly thereafter. Three root tips per beaker were collected at
each sampling. This procedure was followed throughout the experiments
except for a slight mdification of steps when high doses were run.
In the case of the 500 ppm or more treatment concentrations, 21m
roots were suspended for 15 minutes in solutions, thoroughly rinsed in
distilled water, and allowed to recover in a balanced salt solution.
Samples were then taken as described above. Five root tips per beaker
were collected at each interval instead of three. After completion of
sampling the mining seedlings were in all cases recovered and
observed for morphological effects. Seedlings were rolled in paper
toweling, placed in distilled water, and allowed to grow four to seven
days at which time any deviations from the moral morphology were
recorded.

fixation was in a 3 to 1 mixture of absolute ethyl alcohol and
glacial acetic acid. After infiltration for 10-15 minutes by evacua-
tion the root tips were placed in a refrigerator and stored over night
before staining. Tips were then hydrolysed in 1H hydrochloric acid at
60°C for 15-18 minutes, then stained by the l‘eulgen reaction. Squash
preparations were nude in a drop of fast green, acting as a counter
stain, and dehydrated in a 9:1 mixture of tertiary butyl alcohol and
absolute ethyl alcohol. This was found to be advantageous over 95%
ettwl alcohol since less shrinkage of cells resulted. The slides were
nude parliament in Diaphane.

Cytological Examination
Slides were emined with a 451: high dry objective and 20X oculars.

A random sampling of the meristematic tissue was made for all types of

-5...

analyses by viewing alternate strips of the material on the slide. One
hundred cells in a particular division stage or stages were counted per
slide when percentage of abnornals was being determined. All slides
for each treatment and treatment time were analyzed. Mitotic indices
were obtained by counting the number of divisions per 1500 cells. At
least two slides per sample were scored. Data obtained in this mnner
form the basis for all quantitative and qualitative analyses in these

studies.

LITERATURE REVIEW

There is some question concerning the correctness of including
some of the compounds listed in the literature in the substituted
phenol group. Nevertheless it was felt that they were pertinent to
this study and therefore were included.

Perhaps the most critical consideration of the effects of some
phenolic compounds was that given by Hindmarsh (1951) in her studies on
nitrophenols. She reports that at low concentrations (3 ppm), meta—
phase and anaphase chromsomes of All-Lug appeared short and thick but
otherwise norml. Only a few scattered chromosome configurations were
found upon extended treatment. Higher concentrations (30 ppm), however,
ave metaphase and anaphase figures with short, thick chromosomes which
were frequently scattered or clumped. Stickyanaphase chromosomes with
resulting bridges were also evident. At still higher concentrations
(300 ppm), nuclei became pycnotic after two hours treatment. In no
case was polyploidy found. All seedlings recovered from a 21+ hour
treatment in the 3 ppm solution. When treated with 30 ppm concentra-
tion for 21+ hours and 300 ppm for one hour, no seedlings recovered.
Boots became soft and flaccid.

Configurations resembling 'c-metaphases' were also reported by
Lena and TJio (19148) and Steinegger and Levan (19148) in AM 9.029.
when treated with various phenols. Some of these were, hydroquinone,
pyrogallol, p-amino phenol, hydroxyhydroquinone, p-phenylenediamine,

phsnanthrens derivatives, etc. Higher doses ave a strong toxic effect

-7-

or pycnosis. Bridges and fragments also occurred due to stickiness.
Parmentier and Dustin (19%) seemed to agree with these results in
their work on the effect of hydroquinone in mouse intestine mitoses.
Irregular distribution and clumping of chromsomes plus pycnosis were
observed.

In addition to these effects, polyploidy was also recovered by
other workers in material treated with such ring compounds as scena-
phthene, naphthalene, ot-naphtlalene-acetic acid, phenyl mercuric
hydroxide and nitrate, halogenated benzenes of all sorts, etc. (Levan,
19140: Carey and McDonough, 1943: Ostergren, 191m; Berger and Witkus,
19148; hcfarlane and Erlanson, 1951+; Bradley and Crane, 1955). Some
workers (Ostergren, 19m; Oroker, 1953) also reported swellings on root
tips of recovered seedlings merficially resembling 'c-tumors“ after
treatment with phenolic compounds.

In short, most workers using these substances reported scattered
and clumped configurations in their material and perhaps polyploidy in
some cases. At high doses pycnosis often became evident. Swellings on

root tips seemed to accompany some of these effects.

OBSERVATIONS

The key aberrant configuration produced by 3Y9, 7Y5 and Jon in
root tips of’giggg,g§tivum is what has been termed.a 'scatter' (Frontie-
piece). There still remains a question as to the point in the division
cycle when this aberration is formed upon treatment. Perhaps it arises
during the transition between late prophase and prometaphase or during
the movement from prometaphase and metaphase. In order to distinguish
in this discussion between these "scatters” and the scattered.anaphases
(Plate I, Figs. 8,9) which may result when kinetochore cleavage of
these aberrant chromosomes take place, the former shall from now on be
referred to as scattered metaphases. Scattered metaphase chromosomes
generally have very definite characteristics. In.addition to being
scattered.about the cell, these chromosomes are usually highly con?
tracted and resemble the c—pairs produced by colchicine as described by
Levan (1938) (Plate I,rig. 6,7). This is the only significant abnorb
mality found in.zi§gg_root tips when treated with lower concentrations
of these substances. Thur different concentrations were used.with each
of these three compounds from which the data on ”scatters” were derived.
These four doses of each of the compounds range from the threshold con-
centration to the maximum concentration which can be used, i.e. up to
the point where the treatment becomes too lethal in too short a period
of time. Table I lists the doses used for this part of the experiment
in.parts per million.and in milliequivalents of 2,h DOPE per milliliter.

The latter type of expression of the doses was given so that the differ-

 

-9...

ant concentrations could be compared on the basis of the common unit of
structure in the compound namely, 2,14 DOPE.

Since 3Y9 was chosen arbitrarily as a type substance for the reac-
tion, a mare detailed analysis of its observed effects will be given.
The first indications of effect with 3Y9 were noticed after 15 minutes
of treatment in the highest of the four doses, 90 ppm. By 30 minutes,
"scatters" were found in all doses. This reaction increased with time
up to approximately 90 minutes of treatment, after which the frequency
of aberrations decreased while still subjected to the chemical (Table II).
This puzzling observation will be elaborated on a little later in this
discussion. Although the maximum effect increased also with dose, at
no concentration was 100% effect over reached. Analysis of the scat-
tering reaction was made from data obtained between 30 minutes and 90
minutes of treatment with 3Y9. The percentage of abnormalities (scat-
tered metaphases and anaphases) was plotted for each interval of time
at every dose. The mean of the abnormlities for each interval of time
was plotted also. Vith every concentration a simid curve relationship
evolved as the reaction procsded (Text Fig. I). From these curves,
times of 20% and 30% effect were estimated and these intercepts analyzed
on a log/log (dose-time) basis. Each case gave a straight line rela-
tionship with all lines being parallel (Text Fig. IV). The differences
between the slopes of the lines were tested statistically . None
were significant.

In addition to “scatters", multi-nucleate cells were found some-
times resulting from these ”scatters" which had entered resting stage

in this condition. No further study was made of these.

Besides 'scatters", “clumps" (Plate 1, Fig. l1,,5) appeared simulta-
neously at higher concentrations. 'CIumps" seem to result from spindle
destruction at prometaphase. 3Y9 was again enployed to detect and ana-
lyze this aberration. The concentration used was 200 ppm. Data were
collected from 15 minutes to 120 minutes of treatment, since this
appeared to be the critical period. The clup-scatter indices given in
Table III were derived in the following manner. A value of zero was
arbitrarily assigned to normal division figures, one to scatters and two
to clumps. The total values of each were amused and divided by the num-
ber of cells counted, usually 100 in this analysis. Table IV lists sep-
arately the average number of clumps and scatters found at the desig—
nated times and also a scatter-clump ratio average.

To begin with, scatters did not give rise to clumps since the lat-
ter did not appear at any time at the lower doses. From Table III one
observes that the index of total aberrations increases to a point and
then decreases while still in treatment. This same mode of action was
mentioned earlier when the scattering effect alone was discussed. From
Table IV a downward trend in the scatter-slurp ratio seems to be indi-
cated from the onset of the reaction through at least the first hour.

Even at the higher dose and using the combined frequency of scat-
ters and clumps, 100$ effect was never attained. 100$ effect would be
a value of two in Table III. The highest value reached was 1.38. The
reason for this seems to be general toxicity. Before these reactions
can procede to their naximum point, the cells become pycnotic from poi-
soning by the phenolic compounds. This undoubtedly is part of the rea-

son for some of the unexpected irregularities in the data as a whole.

-11..

In addition to this, the rate of absorption and reaction of these com-
pounds is so rapid that all cells can't “keep up" and thus account for
some of the variation.

11933 seedlings were given extended 3Y9 treatment but at no time
we polyploidy ever recovered. This again nay be because of the toxi-
c1 ty which always is encountered when treating with these compounds.
Before polyploidy could be attained, the cells were poisoned.

Mitotic indices showed no definite trend during the period of time
When the scattering and clump reaction was analyzed. Eventually a point
We reached at which the mitotic index dropped suddenly (Table V).
Cells became pycnotic and everything generally appeared poisoned.

As was mentioned at several places in the observations, the effect
Of 3Y9 seemed to increase up to a point and then, while still in the

treatment solution, decrease as was evidenced by the rise and fall of
the frequency of aberrations. This rather strange mode of action
Prompted a series of experiments from which it was hoped an explanation
would be forth coming. 80 ppm and 100 ppm solutions of 3Y9 were used.
m seedlings were treated at these concentrations for one hour after
which they were removed and a new untreated batch of seedlings were put
into these same treatment chambers for one hour and then removed. In
both cases samples were taken in the usual nanner and slides were ana-
lyzed. The analysis from the first batch of treated seedlings agreed
Vith the previous experiment but when the second batch was scored,
a-1most no aberrations were found. This would indicate that the seed-
1lugs apparently take up so large a portion of the compound in a short

1lime that the concentration in the treatment chamber falls below the

-12..

effective threshold of the substance and seedlings appear to ”recover.”
That is the frequency of aberrations decreases. However the recovery
is too rapid to be explained by this lowering of the external concen-
tration alone. Perhaps the following mechanism is involved also. The
initial large and quick uptake of the toxic substance by the-seedling
may disrupt or partially destroy the absorption mechanism of the root.
As the active sites of the compound are used or tied up in the cells,
the effective dose is lowered below the reaction threshold within the
cell. Because of the disturbance in the absorption mechanism this def-
1cit can not be compensated for by additional uptake. l'rom other exper-
iments 60 ppm was found to be the reaction threshold concentration.
Since from 20 to 21+ EL!!! seedlings were used per beaker each seedling
took up approximately 2 ppm of the compound in the initial absorption.

In order to rule out the possibility of breakdown of the 3Y9 upon
standing, test solutions were allowed to stand for an hour before seed-
lings were suspended in them. No differences, quantitatively or quali-
tatively were found in comparison with solutions which were used imme-
diately.

However the possibility that the plants enzymtically destroy the
compon and thus account for the apparent recovery is not ruled out by
any of the above tests. This can not be determined by the cytological
experiments employed in this study.

Some seedlings were always allowed to recover for four to seven
days after treatment and then observed for morphological aberrations.
The following morphological cinnges appeared (Plate II):

1. Lower doses produced a swollen region at end of primary

-13..

root, superficially resembling o-tumors of colchicine.

2. The cortical region was slit open with an excess of secon-
dary roots.

3. Higher doses killed primary roots or produced a shrunken
region on the root where the chemical was absorbed.

lb. The entire seedling was generally stunted.

With 3Y9 the above morphological effects were found down to 15 ppm.
Cytological effects first appeared at 60 ppm. Cytological examination
of secondary roots was made. No aberrations were found.

Similar experiments were run using 7Y5 and J0“. Qualitative cyto-
10gical and morphological results were identical to those outlined
above for 3Y9. Mode of action was identical also. A sigmoid curve was
obtained as the reaction proceded in both 7Y5 and JOI+ (Text Fig. II and
III). Intercepts from extimated 20% and 30% effect gave straight line
relationships on a log dose/log time basis (Text Fig. IV). These lines
were parallel to those of 3Y9. Differences in slopes between all these
lines were tested statistically and none found to be significant. The
toxicity problem was also encountered throughout the work with these
two phenols.

One can conclude that qualitatively the activity of the compounds
is associated with the non-phosphite portion of the molecule, since .101;
Vhich does not contain phosphite produces effects similar to the other
two phosphite containing compounds.

Quantitative variations however were found. Table VII gives the
ulenximum abnormalities reached with the three compounds at each dose.

some fluctuation exists for which no real explanation can be given

 

.. ll; ..
other than perhaps a toxicity effect. The greatest variation was found
in the length of time the compounds took to reach a certain level of

effect. Table IV gives the times at which 20$ effect was obtained with

the three compounds at each dose. Of the three compounds, 3Y9 was

found to reach 20% effect in the shortest period of time with J04 taking

the longest and 7Y5 falling slightly below the length of time of Job].
The data from Tables I and IV show that a concentration of .0028 milli—
equivalents of 2,1; DOPE] m1. as 3Y9 produces 20% effect in approximately

the same time as .0059 milliequivalents of 2,14 DCPB/ ml. as 7Y5 and

- 0130 milliequivalents of J04. Therefore when expressed in terms of

milliequivalents of 2,4 DCPE/ ml., 3Y9 is about twice as effective as

'7 Y5 and about four and one half times as effective as J01». In terms of

molar concentration, 3Y9 is about four times more effective than 7Y5 and

about thirteen and one half times more effective than 301+.
0f the three compounds, 3Y9 has been found to be the most effective

herbicide. The above observations are certainly in keeping with the

Observed effectiveness of 3Y9 as an herbicide.
However the possibility that the phosphite may affect the rate of

the reaction of these compounds can not be ruled out without further

experimentation. l‘rom Text Figure IV it appears at first glance that

this radical is not important since the non-phosphite compound J04 does

not appear widely separated from the two phosphite containing compounds

735 and 3Y9. Looking at the problem from another angle one would expect

the graph of 7Y5 to at least be approximately coincidental with that of

the 319 if the phosphite affected the rate of reaction. This of course

“as not the case according to Text Figure IV. Because of the possible

-15..

difference in pl! among these three compounds, this factor can not be
disregarded. One would assume the tris (R3?) and the ethanol (ROH)
compounds to be relatively unassociated. However the his form (R2P0H)
probably exhibits at least some dissociation. If the rate of effect of
these compounds was plotted on the basis of the concentration of urns-
aociated compound, the graphs of 7Y5 and 3Y9 may be coincidental. Only
after such data are available would the effect of phosphite on the

reaction be known.

-16..

DISCUSSION

Superficial examination of the effects on mitosis in.§i§gglroot
tips from these substituted phenols indicates that they may cause a
c-mitotic action. Before these substances can be designated as c-mito-
tic agents, a detailed examination of their effects must be made and
then compared, point for point with the mode of action of colchicine.
By use of the 'Pisum" test the following characteristics have been
established for the reaction of colchicine (Hadder, 195?):
l. Produces cytologically distinguishable aberrations-macab—
ters and/ or scatters and clumps.
2. These cytological aberrations show a definite time-dose
relationship.
3. Time—dose relationship of the degree of effect is a straight
line on a log/log basis.
h. Does not inhibit mitosis.
5. Polyploidy, multinucleate cells, or both are recovered.
Any compound which, when tested in a similar manner, produces
these results can be regarded as a c—mitotic agent. The effects pro—
d-‘uced by 3Y9, 715 and Jon have been discussed in a previous section and
for ease of comparison shall be briefly outlined here:
1. Scatters and clumps are produced.
2. Degree of effect as measured by scatters shows a definite

time-dose relationship. This could not be determined for

clunps .

-17-

3. Tine—dose relationship is a straight line on a log/log basis.

h. Mitotic index fluctuates for a period of time and then drops
suddenly.

5. At no time is polyploidy recovered.

6. Multinucleate cells sometimes appeared.

Qualitatively, colchicine and the three compounds used in this
study, produce identical effects, i.e. scatters and clumps are found
with all. The time-dose relationship of scatters was also obtained in
all cases and could be plotted as a straight line on a log/log basis.
Although clunps were recovered in mterial treated with the phenols, a
relationship could not be established as was possible with colchicine.
Before tests could be run for a sufficient time to determine the mode of
action of clumping, the cells were poisoned. The data show however,
that the scatter-clump index increases and decreases in a reasonable
Orderly mnner, i.e. without any definite breaks. That the clump action
does not differ from the scatter action is thus indicated. It therefore
appears to be similar in this respect to colchicine, except that this
reaction can not be carried to completion.

Mitosis was inhibited by the phenols which was not‘ the case with
colchicine. An explanation of this inhibition is however necessary
before any conclusion, one way or the other, can be drawn concerning
this apparent difference in action. Mitotic indices of the phenolic
treated cells showed some fluctuations up and down for a time after
which the number of divisions dropped suddenly. This can undoubtedly
be explained mainly by the poisoning action of the compounds which

became very apparent after one and a half hours with most effective

..18..

doses. The variation in results with respect to polyploidy and multi-
nucleate cells can also be expected for the same reason. In general the
data here presented do not show any major differences in action between
the three compounds and colchicine.

There are a number of other problems related to this investigation
which my be worth considering at this time. The question arises as to
What stage of the mitotic cycle is affected by these substituted phe-
nols. No abnormlities were found in early and late prophases in
treated material. However the following stages became aberrant upon
treatment as was evidenced by the clumps and scatters which appeared.
This would mean then that our reaction occurs immediately following late
Prophase. What these substances do, is of course still another question.
Since prOphase movement and morphology were norml, one could probably
Say that the nuclear membrane is not affected. when it is disrupted in
some way, preplnse movement seems to be abnorml (Wilson and Hyypio,
1955). Perhaps these chemicals disrupt enzyme systems by binding sub-
ctrate or inhibiting enzyme action which is in some way related to
Chromosome movemnt at this particular time of division. This of course
18 where the biochemist should take over. If the question of what these
compounds do to disrupt normal movement and morphology were answered,
lllilchprogress would be make in more fully understanding the mechanics of
mitosis.

Ever since cytologists have studied mitotic aberrations caused by
drugs, chemicals, etc. the question has always arisen as to what part of
the structural molecule is responsible for a particular action. Inves-

tigtors have often wished that they could accurately predict the action

-19..

of a substance by its structural composition and configuration. Com-

pounds are classified into the various reaction categories based on

their cytological and genetical effects. If this could be done accord-

ing to whether they possess a particular molecular group or configure-e
tion, the problem of classification would be solved. Although this
Vould appear like a very simple criterion to use, much more is involved

than one might expect at first glance as for example, in the substances

used in this study. All have one structure in common, a substituted

benzene ring. Many other compounds listed in a previous section, which

have this group in common, also have a similar action on mitosis, i.e.

cause scattering of the chromosomes. However some with a benzene ring

have no action of any kind on the mitotic cycle. Why? This may best be

explained by listing some of the may differences which are possible in

a. substituted benzene ring.
The type and complexity of the substituted group, e.g. halo-

1.
gene, amino group, nitrates, sulfates, organic acids, alde—
hydes, ketones, etc.

2. Position of the substituted group-—-ortho, meta, para.

3. Amount of substitution. Partially or completely substi—

tuted: if partially, what positions are open or free.

14. Spatial position of the substituted group—i somerism.

These factors would be concerned with not Just the type of chemi—

can, group or groups present but also the position and therefore acces-

sibility of the groups for chemical reactions. Screening tests have

813own that the later factor is of utmost importance. This is of course

where the complexity of the problem comes in because of the many iso—

-20..

meric possibilities.
Considering the structure of the phenolic compounds employed in

this study and those used in other work done by the cytology group at
this institution one could speculate that although it is important what
and where the various groups are attached to the ring, the negative
point of view, i.e. what positions are open or free, my be more vital
for a proper understanding of the relationship between the stmctural
configuration of a molecule and the cytological effect it produces.
her]: in physiology done by Leaper and Bishop (1951) at least indi—
cates that such a hypothesis is feasible. With substituted phenols,
they found that the compounds were most effective if they had present
two unsubstituted positions in the benzene ring opposite one another.
with a cytological'measurement of the type of effect described in
this paper, one can obtain by a process of elimination valuable clues

as to the structure and constitution of the compounds producing this

effect.

-21..

SUMRY All!) CONCLUSION

In sumry, colchicine, tris and bis forms of 2.1+ dichlorophenoxy—
ethyl phosphite and 2.1+ dichlorOphonoxyethanol have the following char-
acteristics in common: -

l. Identical cytological aberrations, namely scatters and
clumps.

2. Similar time-dose relationship of effect is shown, namely
straight line relationship based on log dose/log time.

However they differ with respect to:

1.. Effect on mitosis in general. Mitotic index falls after

extended treatment with the phenols. In colchicine it is

not affected.

2. Production of clumping, polyploidy, and miltiimcleate cells.
It is doubtful that these differences are significant in view of
the toxicity problem involved with these phenols.
From these experiments and pertinent data from work done by other
investigators I therefore conclude that the three phenolic compounds,

3Y9. W5 and Jolt can be called c-mitotic agents.

TLBLE I

Doses expressed in parts per million and

in millisquivalents of 2,h—dichlorophenoxyethanol per

 

milliliter
319 715 :fiigfl
Milli— Mill 1- Mill 1--
equivalents PPM equivalents PPM equivalents PPM
.0028 60 .0039 90 .0087 180
.0032 70 .00u6 105 .0101 210
.0037 80 .0052 120 .0115 240
.00h2 90 .0059 135 .0130 270
Molecular Equivalent
weight weight
3Y9 649 216
7Y5 #60 230

30h 207 207

Percentage of Aberrations‘Using 3Y9

TIME

in minutes

15
30
1+5
60
90
120

180

- 23 -

TKBLB II

I

CONCENTRATION ;9_rrn

 

60 70 80 90

7 36
1a 15 28 39
25 29 3a 51
34 #7 56 77
36 31 25 62
23 ‘49

17

.. 21;...

TABEE III

Clump-Scatter Indices

for 3Y9 Treatment with 200 ppm

 

TIME
.lflalfllflflfiflil. ‘AYEEAEELJE!!§£__
15 .02
30 .96
#5 1.38
60 1.27
120 1.22

TABLE IV

Number of Abnormalities per 100 Post

Prophase Divisions after 3Y9 Treatment at 200 ppm

 

TIME AVERAGE .LVERLGE

in minutes or CLUMPS OI SCATTEBS SCATTEH/OLUMP
15 15.0 12.2 .93

30 bfl.h 2U.5 .86

as u9.s 38.6 .71

60 50.0 27.u .61

120 39.0 37.“ .95

- 25 -

MEIEV

MITOTIC INDICES

Number of Divisions per 1000 Cells

 

TIMI

in minutes CONCENTRATION in.PPM

319 0 60 70 80 90
0 50

15 “a
45 06 03 58 40
60 52 56 he #1 02
9o 58 hi 35 42
120 63 1h
:15, 0 90 105 120 135
60 53 39 an 61 68
120 5a— 47 48 5a 62
2% 1+1 25 35 #2
300 68 19 20 38 01
‘ggg 0 180 210 240 270
0 58

60 76 30 he 58 us
120 61 34 I41 1&7 32
180 53 29 37 40 21

 

 

 

 

 

-26..

TABLE VI

Time in Minutes of Twenty Percent Effect

 

3Y9 7Y5 3'01» V
CONCENTRATION CONCENTRATION CONCENTRATION TIME
in PPM TIME in PPM TIME in PPM

60 38 90 81 180 96

70 33 105 63 210 71

80 2b 120 #8 2&0 57

90 18 135 42 270 1+2

TABLE VII

Highest Number of Abnormalities

Obtained Per 100 Post Prophase Divisions

 

.__ _g 319 715 ,juNL_
C ONCENTBATI 0N ABNOR- CONCENTRATION ABNOB- CONCENTRATION ARBOR.-
in PPM ms in mm ms in PPM MALS
60 36 90 22 180 29
70 u? 105 25 210 35
80 56 120 5a 240 £18

90 62 135 88 270 65

-27...

mmugde a“ 339

 

 

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man sum 8 D 1:.
a
assets.“

cessation ma oat/satnmouqv :0 ”qu

TOW!

-28...

Se and m9 s
new 2mm can 0
3:. in mg I
mi. and cm a

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L

oma . owe

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oo

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in

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-29..

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:2. 2mm SN I

don xmm omH D

can

IO

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b

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ba

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N

6 I
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seswqdoag qsog oat/ssnnmauqv JO .10qu

(5
to.

 

5m

DOW!

mmTION of Text Figure IV

These lines are plotted on a log dose/ log time relationship.
Points are 20% and 30% intercepts taken from the graphs in Text
Figures I, II and III.

Differences between the slepes were tested statistically and

none found significant.

DOSE in milliequivalents of 2,l+ DCPE/ 1111.

N

 

-30..

Text Fig-ire I?

a - 30% J01»
b - 20% Jon
c - 305% 715
d— 20% 715
e~3fl3w
f- 20% 319

 

f

.3 .11 j .5 .5 .7 {8,9 10 Q

TIM of treatment in tenths of an hEur

LOG Doss/1.00 TIME

DESCRIPTION OF PLATES
PLATE I

Figs. 1-3 Norml mitosis in Pisum m. Fig. 1, prometaphase:

 

Pig. 2, metaphase: fig. 3, anaptnse.

Figs. 14,5 "Olmsps' produced by 3?? (200 ppm) treatment in m
roots.

Figs. 6,7 Scattered metaphases illustrating those practiced by the
3 phenols, 3Y9, 7Y5 and Jon in Piggy; roots.

Figs. 8,9 Scattered anaphases illustrating those produced by the

3 phenols, 3Y9, 7Y5 and .101; in Pisum roots.

PLATE II

Swollen root tips, excess secondary roots and shrinkage of
treated root region illustrating those produced by the 3 phenols,

3Y9, 7Y5 and .104. Control seedlings are the first pair on left.

Each division of the scale represents ten microns.

'1 9 , ~ ‘9) -
. h .. . -
. W at! 3
e ‘- q s ‘ o 4\,‘ |,{
‘ , .. “j, 9
o . p
x .- 0 ‘3'» ’
\ ‘~ ‘ ,‘a. .
Q” “5 ‘ ‘ -’ ' - A"
U I 3'?" % I
’ #:1‘1‘ ‘3' I
a. .
e ‘. .7. a ‘ . y . 3’s . L
I.' I'm I '
i so;
‘ . ,~\ 'I - ._
. $ ,. . fl"
1 he 2 v~ “3

 

   

' n:
/ j‘
/
.s ‘ "l‘.
I

 

 

PLAT! I

 

-33...

BIBlIOGBAPHI

\

Berger. OJ. and 3.8. Witlcus. 19’48. Cytological effects of alphanaph—
thaleneacetic acid. Jour. Hered. 32: 117—120.

Bowen, C.C. 1953. A comparative study of the effects of several anti-
mitotics. PhD Thesis, Michigan State College of Agriculture and
Applied Science, East Lansing, Michigan

Bradley, M.V. and J.S. Crane. 1955. The effect of 2,h,5-trichloro-
phenomcetic acid on cell and nuclear size and endOpolyploidy in
parenchyma of apricot fruits. Am. Jour. Bot. 52: 273-281.

Carey. M.A. and E.S. McDonough. 19113. On the production of polyploidy
in Allium with paradichlorobenzene. Jour. Hered. 111: 238-2140.

Croker, B.H. 1953- Effects of 2,4—dichlor0phenoxyacetic acid and
2,h,5—trichlor0phen0xyacetic acid on mitosis in A1112 m.
Bot. Gaz. _1_1_1;: 27b-283.

Hadder, J .C. 1957. Cytological measurement of c-mitetic and prophase
poison actions. PhD Thesis, Michigan State University of Agricul—
ture and Applied Science, East Lansing, Michigan.

Hindmarsh, M.H. 1951. A critical consideration of c-mitosis with ref-
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Zé: 5—6.

Kostoff, D. 1938. Colchicine and acenaphthene as polyploidizing agents.
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Leaper, J.M.l'. and J.H. BishOp. 1951. Relation of halogen position to
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acids. Bot. Caz. _l_l_2_: 250-258.

Lev-an, A. 1938. The effect of colchicine on root mitoses in Allium.
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Loves, A. 19140. The effect of acenaphthene and colchicine on mitoses
of mm and W. Hereditas g6: 262-276.

Bevan, A. and C. Ostergren. 1914.3. The mechanism of c—mitotic action.
Observations on the'naphthalene series. Hereditas a: 37941113.

Levan, A. and J .H. Tjio. 19148. Induction of chromosome frayentation
by phenols. Hereditas 11;: 1153-1184.

-311-

Macfarlane, E.W.D. 1951+. Some phenyl mercurials as polyploidogenic
and radiomimetic agents for plants, compared with colchicine with
remarks on antagonism. Rev. de Cytol. et de Biol. Veg. 15:
139-146.

Ostergren, G. 19%. Colchicine mitosis, chromosome centration, nar-
cosis and protein chain folding. Hereditas 39,: 1129-1467.

Parmentier, R. and J.P. Dustin, Jr. l9h8. Early effects of hydro-
quinone on mitosis. Nature _1_§_l_: 527-528.

Steinegger, E. and A. Levan. 19148. The o-mitotic qualities of colchi-
cine, trimethyl colchicine acid and two phsnanthrens derivatives.
Hereditas 35L: l93~203.

Wilson, G.B. and P. Hyypio. 1955. Some factors concerned in the
mechanism of mitosis. Cytologia 2.0: 177-181-1.

3‘5"! . "v.
:‘ai. :71 BEE Griz-x,

ibfite’iflu‘é

Demco-293

 

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