ANTEMWQNC EFFEQTS 0F SIMPLE SiFBSTITUTED
PHENOLS AND AROMATiC 7
QRGANEC PHOSPHQRUS COM?GUHDS

Thesis far the; Dsgme of P31. D.
MECHEGAN STATE UNIVERSWY
Arthur Alan Nefhery
1965

LIBRAR Y ' '3
Miclligt‘. L State
University

 
     

THESXS

This is to certify that the

thesis entitled

ANTIMITOTIC EFFECTS OF SIMPLE SUBSTITUTED
PHENOLS AND AROMATIC ORGANIC PHOSPHORUS
COMPOUNDS

presented by

Arthur Alan Nethery

has been accepted towards fulfillment
.of the requirements for

i
Ph. D degree in (Plant Cytologfll *
Botany and Plant Pathology !
I

\

“fie/a. w

Major professor

 

Date May 11: 196;

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ABSTRACT
ANTIMITUTIC EFFECTS OF SIEPLE
SUBSTITUTOD PHENOLS AND AROMATIC
ORGANIC PHOSPHORUS COMPOUNDS

by Arthur Alan Nethery

An attempt was made to determine the cytologically
recoénizeable effects of a series of substituted phenols
and of a group of aromatic organic phosphates derived from
the Simple phenols, and by comparison of the effects, to
determine whether a relationship exists between the chem-
ical structures and biological activity.

Treatment of the root meristems of the garden pea

(Pisum sativum, var. Alaska) was accomplished by adding

 

the chemicals to an aqueous nutrient medium used to culture
the pea roots under standardized conditions. By microscop-
ical examination of the root meristems, analyses were made
of the cytological changes induced by the chemical treat-
ments. The relative toxic levels of the chemicals were
obtained from toxicity tests on DrosOphila melanogastgr.
Three types of antimitotic effect were produced
by all members of these two series of compounds--pre-pro-
phasic inhibition, late prOphase blockage and spindle dis-
ruption--all of which occur at sub-toxic dose levels. The
general pattern of pre-pronhasic inhibition produced a de-
pression in the mitotic index first occurring at from one
and one-half to three hours after the initiation of treat-
ment. The mitotic index remained depressed for two to eight

hours, depending on the chemical used and the dose level.

Arthur Alan Nethery
Subsequent recovery was very rapid and resulted in a sharp
peak of mitotic activity significantly above the control
level, before the index returned to the normal level.

The prophase block caused an increase in the relative
proportion of late prophase configurations after treatment.
Spindle disruption was of the type produced by the alkaloid
colchicine.

The nature, number and position of substituents affects
the relative capabilities of the compounds to produce the
antimitotic effects. Nitro groups were found to be most
effective and methyl groups the least. Bromo and chloro
substituents had an intermediate effect on the activity of
the molecule. Three substituents caused a greater effect
than two, and the ggghg positions were the most influential
in causing cytological changes.

The organic phosphorus compounds were found to be non-
effective when the phosphorus atom was bonded to a sulfur
atom, but were effective when the phosphorus was bonded to

an oxygen atom.

I}

 

ANTIMITOTIC EFFSCTS OF SIMPLE
SUBSTITUTED PHENOLS AND AROMATIC
ORGANIC PHOSPHORUS COMPOUNDS

By

Arthur Alan Nethery

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1965

ACKNOMLEDGNENTS

The author expresses his deepest gratitude to Dr. G. B.
Wilson for his guidance and encouragement throughout this
program of study.

To the members Of the cytology group at Michigan State
University, I offer my thanks for advice and willing assis-
tance at all times during this investigation.

To my wife, I offer my most sincere appreciation for
patience and cheerfulness when the pressures of study were
great, and for typing this manuscript.

I express my appreciation to the Agricultural Experi-
ment Station, the National Science Foundation and the
National Institutes Of Health for their financial aid in

support of this research.

Ho
Ho

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . .

LITERATURE REVIEW

A.

C3 '33 L11 U 0 CD
0

Classification of Antimitotic Effects

Spindle Disruption. . . . . .
Prophase Blockage . . . . . .
MitOtic Inhibition. e e o e o

Cytological Test Systems. . .

Biological Activity and Structure

Biological Activity and Structure
Organic Phosphorus Compounds. . .

EXPERIMENTAL PROCEDURES

A. Cytological Experimental System

B. System for Determining Toxicity
OBSERVATIONS

A. C-mitotic Activity. . . . . . .

B. Prophase Stalling . . . . . . .

C. Mitotic Inhibition. . . . . . .

D. Toxicity Data . . . . . . . . .

DISCUSSION . g g g o o e e o e o o o e

S UMMA RY .

BIBLIOGRAPHY e e e o e o e e o e e o 0

APPENDIX

iii

of Phenols.

of Aromatic

PAGE

mfiflwmp

13

16
23

25
28
31
37
LC
59

66

LIST OF FIGURES

FIGURE

1.

2.

3.

h.

5.

. 9.

IO.

Colchicine indices of colchicine, Ruelene and
2,h,5-TCP plotted versus time . . . . . . . . . .

The percent of polyploid cells recovered after
treatment with 155 mg/liter of Ruelene (II) ver-
sus tine. O O O O O O O O O O O O O O O O O O O 0

Ratio Of early to late prophases resulting from
treatment with 60 mg/liter of 2,h,5-TCP, compared
with the control. . . . . . . . . . . . . . . . .

Mitotic indices as percent of control of the re-
covery le S of the effect curves from treatment
with 5 mg liter and 7 mg/liter of 2,L-DNP . . . .

Mitotic indices as percent of control of the re-
covery legs of the effect curves from treatment
with 50 mg/liter 60 mg/liter, 70 mg/liter '80
mg/liter, 90 mg/liter and 100 mg/liter of §,h-

Dc.oeooooooeeeoeeeeoeeeee

Mitotic indices as percent of control of the re-
covery legs of the effect curves from treatment
with 40 mg/liter, 50 mg/liter, 60 mg/liter, 70
mg/liter and 80 mg/liter of 2,h,5-TCP . . . . . .

Effective doses of 2,h-DMP plotted as probit per-
cent kill Of Drosgphila melanogaster versus log

dose 0 O O O O O 0 O O O O O O '0 O O O O O O O C O

Diagrammatic interpretation of the points of mi-
totic disruption, compared to the Pisum mitotic
eyele O 0 O O O O O O O O O O O O O O O O O O O Q

Mitotic index in percent of control from treat-
ment with AG mg/liter of 2,L,5-TCP versus time. .

Probit percent effect plotted versus log dose of
Ruelene treatments of 125, 1&0, 155, 170, 185 and
200 mg/liter. . . . . . . . . . . . . . . . . . .

iv

PAGE

26

29

30

3h

35

36

38

#2

#5

A9

LIST OF APPENDIX TABLES

TABLE
1. Abbreviations for Substituted Phenols . . . . . .
2. Structures of Simple Substituted Phenols
TQSthoooeoooeeoeeeeeeooooco
3. Chemical NOmenclature of Organic Phosphorus
compoun‘SOOOOOOOOOOOOOOOOOOOO
L. Structures of Organic Phosphorus Compounds Tested
5. Structures of Other Non-effective Organic Phos-
phorus Compomlds 1‘33th 0 e e e e e e e e e e e e
6. Colchicine Indices of Substituted Phenols and
Organic Phosphorus Compounds. . . . . . . . . . .
7. E/L Ratios of Substituted Phenols and Organic
Phosphorus Compounds. . . . . . . . . . . . . . .
8. Average Control Mitotic Indices . . . . . . . . .
9. Mitotic Indices of all Compounds Tested as Per-
cent of Control Values. . . . . . . . . . . . . .
10. Minimal Effective Doses and Maximal Sub-toxic
Doses of all Chemicals Tested Causing Mitotic
Inhibition....................
11. Orders of Effectiveness for Mitotic Inhibition
and Toxicity Based on Minimal Effective Doses
and Maximal Sub-toxic Doses . . . . . . . . . . .
12. LD50 Values for Chemicals Tested by the Dro-

sthila System in Order of Effectiveness. . . . .

PAGE

67

68

69
7O

72

7h
76

77

8O

81

82

INTRODUCTION

This research program was initiated as an attempt to
determine the differential cytological effects of a series
of substituted phenols and of a series of aromatic organic
phosphates, in order to establish whether a relationship
exists between their chemical structure and biological activ-
ity. This type of approach has been fairly successful as
applied to the structure-activity relationships of several
groups of plant growth regulators, namely the phenoxy-alkyl
acids (Linser, 1956 and Aberg, 1956) and the naphthyloxy-
alkyl acids (Linser, 1956; Luckwill and Woodcock, 1956: van
Overbeek, 1956), and in the area of the bactericidal and
insecticidal activity of phenols (Kagy, l9hl; Metcalf, 1955;
Tattersfield, 1925). The value of the correlation Of the
structures of a series of related compounds to their rela-
tive biological activities may be several-fold. First, such
a system can be of predictive value in the synthesis and
manufacture of commercial pesticides, and in the routine
analysis Of these compounds, where reasonable expectations
may be gained as to their primary and general usefulness,
appropriate formulations, and possible deleterious side ef-
fects on plants and animals. Secondly, the elucidation of
the chemical groups requisite for the biological action to
occur provides an indication of the probable chemical and
physical factors responsible for the effect, and may be
helpful as a first step in the study of the mode of action

of these compounds.

2

The study of antimitotic agents is important both from
the standpoint of determining the action of specific causal
factors and of inferring from such disruptions the "normal"
activities of the mitotic cycle. Although a great deal of
work has been done on the antimitotic effects of various
physical and chemical agents (Biesele, 1958), it was felt
that there was a need for a systematic comparative study of
the effects of a series of closely related compounds. The
experimental system for this comparison must necessarily be
rigidly standardized insofar as possible, in order to recog-
nize subtle differences in the effects of compounds of sim-
ilar activities.

Since several phenols were known to have a recognize-
able effect on mitosis at physiological concentrations and
because of their commercial importance as disinfectants and
pesticides, this group of compounds was selected as a model
for developing a method for the comparative assay of struc-
ture-activity relationships. The phenol ring also has the
capacity for ring substitution, by which means a large num-
ber of relatively stable compounds of only slightly differ-
ent structures may be produced.

In order to describe antimitotic effects, we must first
classify the various possible aberrancies arising from the
disruption of the mitotic cycle. According to Wilson (1965),
only two classes of events may be readily recognized as changes
from the normal course of mitosis (other than gross chromo-

some damage). These are: 1. inhibition of the onset of

3
mitosis and 2. production Of deviant figures in mitosis.
In the second category, essentially two types of disruption
occur, namely failure of the transition of late prophase to
pro-metaphase and failure of the spindle to form or to func-
tion.

These three criteria of mitotic interruption are more
or less separable as distinct effects on the division cycle,
and are suggestive of the occurrence of several reactions
or modes of action. An analysis of the comparative effects
of a group of closely related phenolic compounds was made,
based on these criteria. The ends were: 1. to develop an
analytical method for the comparison of chemical structure
with biological activity, 2. to describe a general type of
antimitotic reaction for phenolic compounds and 3. to cor-
relate the biological activity of the compounds with their
chemical structures and physicochemical properties by point-
ing up specific differences between the effects of related

compounds.

 

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LITERATURE REVIEW
A. Classification of AntimitOtic Effects

The general field Of antimitotic chemicals has been
reviewed by Biesele (1958), who discussed several methods
of classification of antimitotic action based on the suscep-
tible stages of the mitotic cycle or on observable damage
induced, and on the physiological changes or biochemical
reactions affected. Bauch classified mitotic poisons into
1. spindle poisons, 2. cell division poisons and 3. chromo-
some poisons. Levan discussed chemical effects under 1. 1e-
thal and toxic reactions, 2. reversible physiological reac-
tions and 3. mutagenic reactions. D'Amato grouped mitotic
poisons into 1. inhibitors of cytokinesis, 2. spindle inhib-
itors and 3. preprophasic inhibitors (Biesele, 1958). Wilson
(1960) classified chemical effects on the cell into four
types--mutagenesis, fragmentation, carcinogenic activity
and antimitotic activity--and briefly described the general
nature of antimitotic action. Other systems include group-
ings for chromosome breakage, "perfect" or "partial" radio-
mimesis, prolongation of metaphase and faulty chromosome
separation (Biesele, 1958).

The classification proposed here, for the sake Of clar-
ity, places chemical agents producing cellular effects under
five headings - 1. irreversible lethal and toxic reactions,
2. mutagenesis, 3. fragmentation, h. carcinogenic activity
and 5. antimitotic activity. The last category is further

subdivided into a) spindle disruption, b) prophase blockage,

h

5
c) pre-prophasic mitotic inhibition and d) failure of cyto-
kinesis.
B. Spindle Disruption

The field of experimental cytology dealing with the
chemical and physical factors which influence the mitotic
cycle began in earnest with the work of Nebel and Ruttle
(1938) and Levan (1938) on the effect of the alkaloid col-
chicine on mitosis. Nebel and Ruttle reported that colchi-
cine was an important tool for producing polyploidy in plants,
which led to an abundance of applied research, but not to the
elucidation of the basic cellular effects or of the mode of
action. Levan showed that colchicine suppressed spindle
formation, and consequently resulted in the failure Of chrom-
atid separation. The term "c-mitosis" was applied to this
distinctive action. Later the scope of the field was broad-
ened by the reports of many other compounds giving "c-mitot-
ic" effects (Levan and Sandwall, 19h3; Levan and Ostergren,
19h3; Ostergren, l9hh; D'Amato, l9h8; Levan and Tjio, l9h8).
These chemicals were classified as c-mitotic primarily because
Of the production of scattered configurations, and thus may
not all fall under the description of c-mitosis as set forth
below.

The independent work of Auerbach (19h3) and Oehlkers
(l9h3) proving that mutations can be induced both in plants
and in animals by chemical agents gave an additional impetus
to the study of chemicals exhibiting various cytological

effects.

6

Although the many compounds classified as c-mitotic
agents are, for the most part, completely unrelated both
structurally and in chemically reactive groups, it must be
supposed that, if the cytological effect truly is the same,
the mode of action should be common to all. However, since
a serious error could be made in attempting to infer a com-
mon mechanism from the observation Of the end result, the
aberrant configurations, the c-mitotic reaction will be
defined here as adhering to the following criteria outlined
by Hadder and Wilson (1958): 1. no inhibition Of the onset
of mitosis, 2. production Of the following deviant config-
. urations in order: a) "scatters" (c-metaphase) associated
with partial effect, b) "clumps" (stalled pro-metaphase)
associated with full reaction, 3. for a given dose under
standard conditions there should be a steady increase in the
degree of effect with time until either full effect or an
equilibrium is reached, h. polyploid or multinucleate cells
or both should be recoverable.

C. Prophase Blockage

The prophase poison reaction was first described by
D'Amato (19L8 and 19h9), and later by Hawthorne and Wilson
(1952) and Hadder and Wilson (1958), the latter two papers
describing the effects Of the anti-fungal antibiotic Acti-
dione in this regard. Since the activity Of this chemical
has been thoroughly described, it may be used as the type
reaction for the prophase stalling effect. It should be

added here that Acti-dione also affects the pro-mitotic

7
competence of the cell to a great extent.
D. Mitotic Inhibition

Although mitotic inhibition is sometimes thought of in
terms of a physiological effect rather than a cytological
one (Biesele, 1958), it should be considered in conjunction
with prophase stalling and spindle disruption, particularly
since it Often occurs simultaneously with prophase blockage
(Wilson, 1965).

E. Cytological Test Systems

Many variations Of the Allium test, as described by
Levan (1938) and D'Amato (19h9), have been used widely in
testing the effects of chemicals at the cytological level.
While this is to some extent a standardized system, there
are several drawbacks to this test, namely the variability
in mitotic index over a period of time in the root tip, the
lack of uniformity due to the differences in lengths and
ages between individual roots, and the difficulty in main-
taining uniformity among several stocks of onion bulbs.

The Ei§2m_test, which has been described by Bowen and
Wilson (195h) and re-examined in some detail by Wilson (1965)
provides a greater uniformity of material, due to the pos-
sibility of selection of individual roots for testing and
to the intrinsically more stable mitotic levels over a per-
iod of time. This particular system has also been highly
standardized with respect to such variables as temperature,
humidity, pH, and total ion concentration of the nutrient

medium.

8

F. Biological Activity and Structure of Phenols

According to Sexton (1963), any biological activity
which is recognized as being due to an exogenous chemical
may be considered either "the end result of a series of
interlinked chemical reactions or the observable manifes-
tation of an interference with a delicately balanced system
of interdependent chemical and physical processes." He
also states that chemical compounds do not necessarily pro-
duce biological activity by inhibiting the function of spe-
cific essential metabolites, but also bring about such ef-
fects by mechanisms which may be determined by physicochem-
ical factors. Therefore, the chemical structure may be
correlated with the physiological effect, to the extent
that the physicochemical properties relate to the structure.

Various substituted phenols have been studied with a
view to their effect on mitosis or chromosome structure.
Levan and Tjio (19h8) reported a c-mitotic effect for the
mononitrophenols, and also claimed radiomimetic effects.
The antimitotic activity of various other phenols has also
been reported (Loveless and Revell, l9h9; Hindmarsh, 1951;
Muhling 32 gl., 1960; Clowes, 1951).

Very little is known about the mode of action of the
phenols as mitotic inhibitors. A number of theories relat-
ing the physical or chemical molecular characteristics to
the biological activity of phenols have been proposed.

According to Ferguson (1939), Richet stated that chem-

ical toxicity is roughly inversely proportional to the sol-

 

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9
ubility of the compound. He also reported that Moore claim-
ed that the toxicity of insect fumigants increased with the
boiling point and consequently is in an inverse relation-
ship with the vapor pressure. The Meyer-Overton lipoid
theory of narcosis (Meyer and Hemmi, 1935) assumed that
isonarcotic effects are produced by extremely diverse chem-
ical structures when their molar concentrations in the cell
lipoids are identical. Levan and Ostergren (1943), from a
study of numerous chemicals inducing polyploidy in plants,
found that the introduction of ~OH, -NH2, -COZH, and -803H
radicals into the reactive molecules extinguished the c-mi-
totic effect. They also noted that -N02 and haloids some-
times had this extinguishing effect. Since most of these
are typical hydrophilic groups which as a rule increase
water solubility (and thereby decrease lipoid solubility),
this correlation was taken to suggest that the partition of
substances between water and lipoids plays an important
part in the mechanism of c-mitotic action. Ferguson (1939)
believed that a parallelism between physiological action
and the oil/water distribution ratio is really a case of
Richet's rule, and that the same may be said of the paral-
lelism between physiological action and surface tension
lowering. From the work of Fuhner, Ferguson (1939) draws
the conclusion that it is apparent that there is no intrin-
sic connection between the two, and where a correlation)
exists, it is due to the fact that highly surface-active

substances are also usually very slightly soluble.

THE

 

 

 

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Comparative studies of structure-activity relationships
for bactericidal activity of phenols have been carried out
(Wolf and Westveer, 1952; Suter, 19hl). Wolf and Westveer
determined the phenol coefficients of 16 of the 19 possible
chlorophenols and showed that they increase in germicidal
activity in the mono-, di-, and trichloro- series, but de-
crease in the tetra- and penta- series. According to Tat-
tersfield (1925), the toxic value of nitrocresols as contact
insecticides was greatest when the nitro group was EEEQ to
the hydroxyl group, and when the ring was alkylated in the
ggthg position as in h, 6-dinitro-grcresol (DNOC). With
respect to the mononitrophenols, he found para:>meta:>gg§hg.
He also found that the introduction of a third nitro group
to 2, h-dinitrophenol to form picric acid reduced toxicity
to Aphis rumicis.

The physiological activity of phenols has been studied
by Blackman gt_a1,(l955 a and b) by the induction of chlor-
osis in Lemna minor and the reduction of radial growth of
Trichoderma viride. They concluded that when the results
are corrected for the degree of dissociation of the molecule,
as an alkyl chain is lengthened or the number of chlorine
atoms or alkyl groups substituted in the ring is increased,
then the biological activity is progressively augmented.
They also found that the position of the substituents was
important. Blackman gt 3;. (1955a) emphasized the impor-
tance of correcting comparative concentrations of different

chemicals for the difference in dissociation of the mole-

ll
cules. They found (1955b) that all phenols tested showed
a general linear relationship between pK value and the log-
arithm of activity, except those which are substituted in
both ggghg (2 and 6) positions, at least one of which sub-
stituents is a chlorine atom. All of these compounds failed
to conform because the activity was considerably higher in
relation to the pH values. Considering the hydroxyl group
of prime importance in activating the biological response,
they interpreted these results in terms of the enhanced
ionization of the phenol by adjacent chlorine atoms and of
the tendency to repel the compound from possible negatively
charged bonding sites. According to Metcalf (1955), Dierick
found that a 0.12% aqueous solution of h, 6-dinitroggrcresol
(DNOC) applied to Ephestia eggs produced 100% mortality at
pH 2 and none at pH 5. The importance of considering the
amount of dissociation of the molecule is obvious, since
DNOC is undissociated at pH 2 and completely dissociated
at pH 7. It should also be noted that the amount of dis-
sociation affects the oil/water phase distribution of the
chemica1--the greater the dissociation, the greater the
affinity for the aqueous phase.

2, h-dinitrophenol and 2, h-dichlorophenol are known
to be uncouplers of oxidative phosphorylation, and several
other phenols have been reported to have this activity at
varying degrees of efficiency. Chance and Hollunger (1960)
found uncoupling activity with 2, h-dibromophenol; Gaur

and Beevers (1959) list the mono-substituted nitro-, chloro-

l2

and bromo- phenols as exhibiting uncoupling of oxidative
phosphorylation.

In his study of the herbicidal action of compounds
with phenyl nuclei, Aberg (1956) shows a correlation between
the lowering of the "intrinsic auxin activity" and the in-
crease in the van der Waals radius of the £333 substituent.
He found that the effect of 235g chlorination is to increase
the dissociation constant, leading to the notion that 233g
substitution could increase the affinity of a substance for
receptor sites.

According to Pauling (1960), phenols form stronger
hydrogen bonds than aliphatic alcohols because of the in-
crease in electonegativity of the oxygen atom resulting

from resonance with structures such as:

_. «(—
o :::£qg+

He reports that hydrogen bond formation is of importance

in affecting the melting point, boiling point, dielectric
constant, solubility of organic liquids in water and other
solvents, and proton magnetic resonance. In phenol and
substituted phenols the C-0 bond has some double bond char-

acter, so that cis and trans forms are possible:

OH. HO
‘Cl CI

cis trans

13
Thus, intramolecular bonding results in the presence of
more of the cis form, at least in the gaseous phase, since
it is more stabilized. In the liquid and crystal phases,
intermolecular bonding may tend to keep both forms in some-
what equal abundance.

Sundt (1961) states that the influence of substituents
on the physical properties of phenols is due partly to their
nature and partly to the position of the substitution. The
chief effects are: a) inductive effect, b) steric hindrance,
c) hyperconjugation and d) mesomeric effect.

A comparison of structure-activity relationships of
phenols made a the level of antimitotic activity was reported
by Muhling gt‘al. (1960). Their work indicated the impor-
tance of the ggghg and EQEQ positions, in addition to de-
scribing to some extent the cytological effects of 2, A-
dinitrophenol and 2, h-dichlorophenol. Clowes (1951) re-
ported the inhibition of cell division in sea urchin eggs
with nitro- and halo-substituted phenols. Inhibition oc-
curred with the dinitrophenols and all dihalogenated phenols
except those substituted in the ggthg (2 and 6) positions.
The mgga and 2323 mononitrophenols were less active, and
the ggthg-nitrophenol and all of the monohalophenols were
inactive.

G. Biological Activity and Structure of Aromatic Organic
Phosphorus Compounds
As far as the aromatic organic phosphates are concerned,

the only report of their antimitotic activity was made by

1h
Nethery ggnal. (1965) on the studies of the acaricide and
anthelmintic ruelene. In considering the structure-activity
relationships of these complex molecules one must take into
consideration variations in groups X, Y, and Z, and both
the position and the particular chemical group involved in

the R substitution. Z

o—a/X
\\
Y
R
In discussing the insecticidally active phosphorus esters,
Metcalf (1955) makes the generalization that methyl esters
(at groups X and Y) are less toxic than the ethyl esters,
and that activity falls again with increased chain length,
although there are numerous exceptions. He also states
that the stability of the dialkyl phosphorylated enzyme
complex involved in acetylcholinesterase inhibition is
approximately the same for various inhibitors containing
the same dialkyl groups and varies in the order dimethyl
phosphorylation<1diethyl phosphorylation<:diisopropyl
phosphorylation. Group 2 usually is either 0, S or Se.
The first two are the most common and are of commercial
value as pesticides. The S substitutions are much less
toxic, which is partially explainable by the fact that, at
least in some cases, the thionophosphates must be converted
enzymatically by the organism to the phosphate in order
.for the toxic action to occur (Metcalf, 1955). Such an

<enzyme system may also operate in some plants which are

l
c

1961).

EXPBHIHENTAL PROCEDURSS
A. Cytological Experimental System
The experimental procedure used to determine the anti-
mitotic effect of the phenolic compounds was a standardized
system for the treatment of the root meristem of the garden

pea, Pisum satiyum, var. Alaska. The dried peas were for

 

the most part supplied by the Ferry Morse Company and were
guaranteed to be free of chemical treatment. The final
experiments were conducted on peas of the same variety sup-
plied by the Vaughan Seed Company; the results obtained were
entirely equivalent to the earlier runs.

The dried peas were rinsed three times in de-ionized
distilled water, then soaked for six hours in de-ionized
distilled water at 22.50C. 1:0.500. The soaked peas were
rolled in absorbent paper toweling moistened with de-ionized
distilled water. The rolls were wrapped with waxed paper,
leaving about one inch uncovered at the lower end of the
roll to facilitate water absorption, and placed in approx-
imately one inch of water in 600 ml beakers. These rolls
were left to germinate for 36-h2 hours in an incubator at
22.5°C. i;0.5°C., with the relative humidity increased by
means of open pans of water on the lower shelf of the incu-
bator. After germination, the paper toweling was unrolled,
and the pea seedlings were selected for a root length of
15-2 cm. These peas were suSpended by means of wax-coated
wire mesh with spacings of about 9 mm (3 to the inch) placed

over 100 mm x 50 mm crystallizing dishes containing approx-

16

l7
imately 390 ml of modified Hoagland nutrient solution made
as follows:

12.5 ml of each of the five stock solutions was

used per 937.5 ml of de-ionized distilled water

to make each liter of nutrient solution.

Stock A - 3.80 g Calcium nitrate Ca(N03)2. LH20

in 500 ml H20

Stock B - 5.16 g Ammonium nitrate NHAN03 in 500

ml H20

Stock C - 7.20 g Magnesium sulfate Mg SOh’ 7H20

in 500 ml H20

Stock D — 5.34 g Potassium monobasic phosphate

KHzPoh in 500 ml H20

Stock E - 0.29 g Potassium dibasic phosphate

KZHPOh in 500 m1 H20
The pH of this solution was found to be 5.6-5.7.

Air filtered through a water trap and through a char-
coal-glass wool filter was bubbled through the nutrient
solution to provide oxygen and to supply a stirring action
for homogeneous oxygen distribution.

The treatment dishes were maintained at 22.500. i 0.500.
by means of a controlled temperature water bath in an air-
conditioned room with the relative humidity held at h0-50%.
The peas were allowed to acclimatize to the culture condi-
tions in the treatment dishes for four hours, at which time
the wire grids carrying the peas were transferred to other

crystallizing dishes containing the chemical being tested

18

in solution or suspended in the nutrient solution. The
chemicals used for treatment were dissolved in the nutrient
solution at the desired concentrations, if sufficiently
soluble. Most of the phenols required heating up to 70°C.,
while being stirred on a magnetic stirrer-heater. At this
temperature there should be no appreciable breakdown of the
phenolic compounds studied. The abbreviations, symbols,
and chemical structures of all compounds tested are given
in Tables 1 throuph 5. Several of the less water-soluble
compounds were suspended in the nutrient medium by the use
of surface-active agents. The most commonly used was Tween
90, supplied by Nutritional Biochemicals Corporation, which
was used at concentrations below 0.5 g/liter of nutrient
medium. At this concentration, no cytologically detectable
effect due to this compound occurred. The technique used
was to add the weighed amount of chemical to the measured
Tween 80, and then to pour on nutrient solution previously
heated to 70°C. Stirring for up to one-half hour, while
holding the temperature at 70°C. sufficed to produce appar-
ently homogeneous suspensions with no sedimentation in most
cases.

Samples were taken at various regular time intervals
after the treatment for cytological analysis. A zero hour
control (before treatment) of each treatment dish was taken.
and a continuous non-treated control dish was run for the
duration of each eXperiment. Samples were taken by remov~

ing at random five pea seedlings for each composite sample

19
at a given time, breaking off approximately the first cen-
timeter of the distal end of the root and immediately kill-
ing and fixing the specimens in a shell vial containing
approximately five ml of fixative consisting of 6 parts
methanol, 3 parts chloroform, and 2 parts propionic acid.
The vials containing the fixed pea root tips were

evacuated for 10-15 minutes. The material was prepared

for being made into slides by fixation for at least twelve
hours under refrigeration or 20 minutes in a 60°C. oven.
The fixative was decanted and 60°C. 1.0 N HCl poured on.
The vials were left in the 60°C. oven for 18-20 minutes,

to allow mild hydrolysis of the deoxyribonucleic acid, lib-
erating aldehyde groups which react with leucobasic fuchsin
(Schiff reagent) to give a purplish color. The acid was
poured off and Schiff reagent was added. The meristematic
tissue develOped a deep purple color in 15-30 minutes.

Slide preparation was carried out by removing the dark-

ly stained meristematic region (approximately 1 mm), and
macerating it with a flat-end glass rod in a drop of 0.5%
fast green stain in LS% acetic acid. A cover glass was
placed over the material with 1/8 inch extending beyond
the edge of the slide. The slide was heated gently over

an alcohol flame and squashed firmly between several layers
of paper toweling. The preparation was then dehydrated for
at least six hours in tg§§.-butyl alcohol, diluted to 90%
with absolute alcohol.

After dehydration, the cover slip was removed, and the

20
slide was made pennanent by adding a drop of'diaphane mount-
ing medium.

Four of the five slides prepared for each sampling time
were usually scored by microscopical examination, and the
fifth saved as a spare in case of breakage or inferior slide
quality.

In order to retrieve meaningful information from ob-
servation of these slides, it was first necessary to set
up guidelines for types of analyses reouired, as based on
preliminary examination of the material. The type of data
obtained depends on the analyses used, which in turn are
dependent on the range of observable effects that could
possibly occur.

From the preliminary work, it was seen that three tvpes
of effect could be distinguished: l. mitotic inhibition,

2. prophase stalling and 3. c-mitotic action. Analyses
were therefore arranged to distinguish between the presence
or absence of each of these effects, singly or in combin-
ation.

Mitotic inhibition results from any action which pre-
vents the cell from entering mitosis. There are many points
(in time) where inhibition may occur, and presumably as
many different reaction mechanisms involved, depending on
the specific causal factor. In many cases mitotic inhib-
ition is linked with the inhibition of the prophase-to-pro-
metaphase transition, but this is not always so. Presum-

ably this correlation holds only for inhibition at some

21
Specific site or for some specific induced chemical change
and not for others. Inhibition of mitosis is recognizeable
by the lower proportion or complete lack of dividing figures
at some time subsequent to the initiation of treatment.
An analysis known as the mitotic index consistently proved
to indicate the presence or absence of mitotic inhibition.
The mitotic index is defined as the number of dividing cells
per one thousand total cells counted. The analysis is per-
formed by the counting of cells in random microscope fields,
covering as much of the total slide area as possible, until
1000 cells are counted.

Chemicals which induce prophase stalling have been
found always to have an associated effect on mitotic inhib-
ition, although, as noted above, the reverse is not neces-
sarily true. This suggests the probability that only one
mode of action exists for inhibition of late prophase, and
that this same reaction is one of several means by which
cells may be prevented from entering mitosis. Prophase
stalling has the result of preventing the normal transform-
ation of a nucleus from the late prophase to the pro—met-
aphase stage. There is an associated failure of the nuclear
boundary to break down and failure of the chromosomes to
become oriented toward the center of the cell, as in a
normal pro-metaphase. In fact, all subsequent orientational
movements (including alignment on the metaphase plate, sep-
aration of the chromatids and polar movement and grouping

of the separated chromatids) are typically absent, although

22
the mqrphological changes of the chromosomes continue at
about the same rate as the normal to the interphase condi-
tion. Such affected nuclei therefore have the appearance
of late prophase configurations with considerable over-con-
traction of the chromosomes relative to a normal late pro-
phase, and eventually the chromosomes show a telomorphic
appearance. A computation of the number of dividing cells
in each mitotic stage was found to be useful in determining
prophase stalling. A comparison of the ratio of early pro-
phase stages to late prophases (E/L ratio) at a given time
showed a relative accumulation of late prophases where sig-
nificant stalling occurred. Admittedly, this reduction in
the ratio was due both to the piling up of cells which were
prevented from leaving late prophase and to the lower pro-
portion of cells in early prophase due to the inhibition
of mitosis. However, a significant drop in the ratio could
be taken as proof of a late prophase block. This analysis
was carried out by counting 200 dividing cells, and clas-
sifying each according to its mitotic stage. Again, the
fields were selected at random over the major area of the
slide.

C-mitotic action is defined in terms of the effect of
colchicine on cell division. Colchicine is a spindle poison,
causing partial or complete disruption of the spindle mech-
anism, and resulting in the formation of c-mitotic aberra-
tions, namely "scattered" and "clumped" chromosome config-

urations. Due to the failure of both karyokinesis and

23
cytokinesis, a certain number of polyploid cells may be
found in the second division cycle following the treatment.
The c-mitotic effect may be analyzed in several ways. In
some cases, a count of the abnormal post-prophase stages
was made. This group of abnormal configurations was made
up largely of "scatters" and "clumps". Although qualitative-
ly showing the presence of a c-mitotic effect, these data
gave no indication of the quantitative efficiency of such
an effect relative to colchicine or to the effect of other
phenols. For this reason, some scoring was also done by
counting the number of "scatters" and "clumps" and compar-

ing these data by means of the colchicine index:

no. of "scatters" +-2(no. of "clumps")

 

Colchicine index :
total post-prophase cells

Chromosome damage was assayed primarily by scoring
anaphase configurations for chromosome fragmentation and
for the formation of chromosome bridges.
B. System for Determining Toxicity

The toxic dose levels of the various chemicals were
obtained on Drosophila melanogaster. Chemicals in acetone
solution were added to nutrient agar medium, which was
poured into disposable plastic petri dishes. Four dishes
each containing 25 m1 of nutrient were poured for every
dose level tested. Thirty flies, Oregon R strain, were
placed in each dish, providing four replications of thirty

flies each for every dose. After 2h hours at 22.5°C. 1:

2h
0.500., the dead flies were counted. By plotting the per-
cent kill from several doses on a probability scale versus
log dose, the LDSO concentrations could be interpolated for
each chemical. The various chemicals used in this test
system also are listed and the chemical structures are

given in Tables 1 through 5.

OBbERVATIONS
A. C-mitotic Activity

Some c-mitotic activity, as evidenced by the presence
of "scatters" and "clumps" was found with virtually all of
the simple substituted phenolic compounds tested. The ef-
fect is compared by means of the colchicine index. The col-
chicine indices of all compounds tested during the first
four hours of treatment are found in Table 6.

From studies on the effects of colchicine (Hadder and
Wilson, 1958), it was found that for maximum effectiveness,
the initial peak of c-mitotic activity should occur within
two hours after treatment. One of the clearest demonstra-
tions of the c-mitotic activity of a phenol is shown by the
effects of 2,h,5-TCP. Used at a dose level of no mg/liter
of treatment solution, the 2,u,5-TCP-treated cells reached
a colchicine index peak of 0.07 at 2 hours, as compared to
1.75, the effect produced by 200 mg/liter of colchicine
(Figure 1). It was noted that dose levels of 2,h,5-TCP up
to 80 mg/liter resulted in no further increase in c-mitotic
effect, as measured by the colchicine index (Table 6).
Doses greater than 80 mg/liter proved toxic.

2,h,6-TBP at 200 mg/liter also reached a peak slightly
(greater than 1.00 at 2 hours; 2,3,6-TCP at 100 mg/liter

IDeaked at 0.87 at 1% hours; 2,h,6~TCP at A0 mg/liter reached
0.50 at 1% hours; and 200 mg/liter of 2,3,5-TMP at 1 hour
reached a colchicine index of 0.69.

The di-substituted phenols had a somewhat less well-

25

Figure l. Colchicine indices of colchicine, Ruelene and

2,h,5-TCF plotted versus time.

INDEX

COLCHICINE

DO-

26

 

 

'TIAAEI

Colchicine

Ruelene

2,L,5-TCP

 

IN HOURS

27

defined effect on the spindle. One of the most effective
was 3,h-DCP. 60 mg/liter caused a peak of 0.h6 at 1 hour;
90 mg/liter resulted in an index of 0.61 at 1 hour. 2,3-DCP
at 100 mg/liter produced an index of 0.97 at 1 hour; 50 mg/
liter of 2,6-DCP reached o.a3 at 2 hours; 3,5-DCP at 50 mg/
liter peaked at 0.91 in 1 hour; 2,h-DBP caused an index of
0.hl at 2 hours with a dose of 50 mg/liter; and 2,h-DMP at
200 mg/liter resulted in a peak of 0.h7 at 2 hours.

The highest sublethal dose (200 mg/liter) of the or-
ganic phosphate, Ruelene (II), is compared to the effect
of 200 mg/liter of colchicine in Figure 1. Again, the
maximum effect reached is considerably below that induced
by colchicine, since the bulk of the deviant figures may
be classified as "scatters" (partial effect) rather than
"clumps" (complete effect). The maximum index obtainable
with Ruelene is about 1.00, as compared to a theoretical
maximum of 2.00, if all deviant figures were "clumps".
The other actively antimitotic organic phosphate compounds
(IV and VI) did not show a particularly striking tendency
toward spindle disruption as measured by the colchicine
index, but did produce great enough numbers of "scattered"
and "clumped" configurations to indicate that at least some
such action does occur (Table 6).

One of the diagnostic features of c-mitosis is the
appearance of polyploid cells in several division cycles
subsequent to treatment. Slides were scored at these times

for polyploid cells, but only in a few cases were any sig-

28
nificant amounts found. Figure 2 shows the percent poly-
ploidy recovered after treatment with 155 mg/liter of Rue-
lene (II). Low levels of polyploidy were also found with
2,h-DNP, 2,h,6-TBP and 2,h,5-TCP.
B. Prophase Stalling

The extent of prophase stalling was gauged primarily
by the drop in the E/L ratio. This change was noted in
almost every treatment to a greater or lesser extent. As
can be seen from Table 7, this drop may be recognized within
the first hour of treatment, and usually reaches its lowest
point at from one to two hours after the initiation of the
treatment.

Figure 3 shows the change with time in the E/L ratio
obtained by treatment with 60 mg/liter of 2,h,5-TCP as com-
pared with the control. All of the substituted phenols
showed a very definite drop in the ratio, all falling below
1.00 at some time within the one to two hour period, and
at some effective concentration. It was also noted (Table 7)
that increasing the dose level did not materially increase
the prophase inhibition. Probably the lack of a direct
proportional relationship of the effect to dose is due in
part to the fact that only a fairly complete inhibition
is recognizeable, because of the tremendous variability of
E/L ratios in untreated material.

The di-substituted phenols appeared to be at least as
effective as the tri-substituted phenols in producing a

drop in the E/L ratio; all chemicals of this type tested

Figure 2. The percent of polyploid cells recovered after

treatment with 155 mg/liter of Huelene (II) versus time.

29

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31

showed a very definite effect of this nature.

0f the antimitotically effective organic phosphorus
compounds, para-oxoanI) showed the best evidence of pro-
phase stalling, reaching a low value of 0.53 two hours af-
ter treatment with a dose of approximately 100 mg/liter.
The other chemicals of this type tested showed a drop, but
the values did not fall significantly below 1.00, and are
therefore only indicative of the presence of such an effect.

As a result of the release of large segments of root
meristem cells from the depression of the transition rate
from late prophase to pro-metaphase, the subsequent recov-
ery from the treatment of these segments may lead to an ex-
treme reversal in the ratio, with peak figures of L.30 or
5.00 in some cases (Table 7).
C. Mitotic Inhibition

In order to compare the changes in the mitotic cycle
arising from inhibition at various points of the cycle,
which resulted in longer average cycle times, a control
mitotic index was scored for each chemical tested. The
control mitotic indices averaged over the duration of the
sampling period are tabulated in Table 8. The overall mean
mitotic index was 60, with the individual averages ranging
from 45 to 75, a deviation ofitlS index points, or2t25%.
The mitotic indices from each of the treatments have been
computed as the percent of the control mitotic index for
that eXperiment, and are found in Table 9. Any deviation

from the control value of greater magnitude thanit25% will

32
be considered significant.

All treatments which showed any cytologically apparent
effect inhibited the onset of mitosis to some extent. The
drop in mitotic index did not occur immediately; in some
cases, the higher concentrations particularly seemed to pro-
duce even a slight stimulation at one-half to one hour after
treatment. The 2,h,5-TCP provides a good example of stim-
ulation at higher dose levels. From Table 9, it may be
seen that, at one-half hour following treatment, levels of
llh% and 120% of the control value were caused by dose lev-
els of 70 mg/liter and 80 mg/liter, respectively. With
2,h,6-TCP, indices of 11R% and 132% of the control level
occurred at one-half and one hour subsequent to treatment
with 25 mg/liter, and 128% of the control at one-half and
one hour after treatment with no mg/liter. This apparent
stimulatory effect was also noted with the di-substituted
phenols (125% at one-half hour with so mg/liter 2,t-DBP)
and with the organic phosphorus compounds (119% at one—
half hour with 100 mg/liter of para-oxon (VI)).

After the initial stimulation or maintenance at cone
trol level, the drop became apparent at from one and one-
half to three hours, depending at least in part on the dose
level. The lowest point was reached at three and one-half
to four hours. An eventual rise, usually quite steep, to
supra-control levels was found in most cases during the re-
covery period.

It may be seen from Table 9 that all of the simple

33
substituted phenols at four hours following treatment had
a mitotic index below 75% of the control value at almost
all detectably effective dose levels. The higher dose lev-
els of the organic phosphates also resulted in indices below
75% of the control at four hours, except for para-oxon (VI)
which reached a low of R0%. The amount of the drop was in
most cases not very dependent on the dose level, possibly
since the drop must be of fair magnitude to be recognizeable.
However, the rate of recovery of the tissue may be in part
dependent on the dose. Figures h, 5 and 6 show respectively
the recovery legs of the effect curves of 2,h-DNP, 3,h-DCP
and 2,h,5-TCP. Once recovery began, the build-up of divid-
ing cells appeared to ensue rapidly. Therefore, a straight
line between the last sub-control point and the first supra-
control point should be a good approximation of the true
shape of the curve. Although it may be seen that, in gen-
eral, higher dose levels tended to retard recovery, and that
a relationship approaching linearity was found with an arith-
metic dose progression, no rigorous proof of linearity can
be offered, due to the approximated shape of the recovery
curves.

The mitotic index also serves as a means of comparing
the minimal effective molar dose levels of different com-
pounds in order to classify them according to their rela-
tive effectiveness. The minimal effective and maximal sub-
toxic dose levels for all compounds tested which were found

to be antimitotically effective are listed in Table 10.

Figure a. Mitotic indices as percent of control of the re-
covery legs of the effect curves from treatment with 5 mg/

liter and 7 mg/liter of 2,h-DNP.

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Figure 5. Mitotic indices as percent of control of the re-
covery legs of the effect curves from treatment with 50 mg/
liter, 60 mg/liter, 70 mg/liter, 80 mg/liter, 90 mg/liter
and 100 mg/liter of 3,4-DCP.

35

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Figure 6. Nitotic indices as percent of control of the re-

covery legs of the effect curves from treatment with LO mg/

liter; 50 mg/liter; 60 mg/liter, 70 mg/liter and 90 mp/liter
of 2;h;5-TCP.

36

 

 

 

 

 

 

 

 

 

 

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Concentrations are given both in mg/liter and in molarity.
The various compounds may be classified in several ways in
order to compare their relative effectiveness. First, they
have been listed in order of effectiveness, from the most
effective to the least, by placing them in order from the
lowest minimal effective dose to the highest. The relative
toxic values may also be compared by listing the compounds
according to their maximal sub-toxic dose levels. Both of
these orders of effectiveness have been recorded in Table
11.

It can be seen readily that the nitro substituents are
the most influential in causing the phenols to disturb the
mitotic cycle, and that the methyl substituents are the
least effective. Both the bromo and chloro groups fall
within these two extremes, and are more difficult to sepa-
rate as to order of effectiveness. Several interpretations
of these data will be offered later in order to distinguish
between the biological activity of these two chemical group-
ings.

D. Toxicity Data

The comparative LD5O values in Table 12 were derived
by plotting the actual experimental doses used on a log
scale versus the probit percent kill of DrOSOphil§_melano-
Easter. An intercept was dropped from the 50% line to de-
termine the LD5O value. A characteristic plot is shown in
Figure 7, from which the LDSO value of 30 mg/liter was de-
termined for 2,4-DMP.

Figure 7. Effective doses of 2,4-DMP plotted as probit per-

cent kill of Drosophila melanogaster versus log dose.

 

38

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90....

80—

 

20-—

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Probit Percent X111

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Dose in mg/liter (10‘ scale)
Figure 7.

39

In general, the same order of effectiveness was found
for the compounds in regard to toxicity to Drosgphila melan-
ogaster as with the pea system. The 2,h-DNP was by far the
most toxic, resulting in 50% mortality with a concentration
of only 2.0 mg/liter or 1.1 x 10'5M. AS was the case with
mitotic inhibition of the pea root meristem cells, the
methylphenols required the highest concentrations to produce
50% mortality. The bromo- and chlorophenols had effects
ranging between the two extremes. Generally, the tri-sub-
stituted phenols were more toxic than the di-substituted
ones, the only exception being 2,h,6-TCP, which was less
toxic than 2,h-DCP, 2,3-DCP, 3,4-DCP and 3,5-DCP, but more
toxic than 2,6-DCP. In regard to the minimal effective
dose and the maximal sub-toxic dose in the pea system,
2,h,6-TCP was both more effective in producing mitotic in-
hibition and toxicity than any of the di-substituted phen-

ols.

DISCUdSIUN

Although the three antimitotic effects just described
appear to be distinctly separable, at least in many cases,
they may occur also in combination. This, of course, does
not necessarily preclude the possibility of a distinct mode
of action being required to initiate each effect. Therefore,
it should be pointed out that no contradiction exists when
mitotic inhibition occurs simultaneously with a c-mitotic
reaction. According to our definition, a true c-mitotic
reaction does not cause inhibition of mitosis, and indeed
this does not occur with colchicine treatment. Alternative-
ly, the mitotic inhibition found with treatment by many
phenols may be interpreted as a distinct effect acting si-
multaneously with the c-mitotic reaction. In fact, the
inhibition may or may not be observable, depending on the
chemical used and the dose level.

We have shown that the general effect of substituted
phenols as a group on mitosis is three-fold. Firstly, an
inhibition of mitosis occurs, secondly the transition of
late prophase to pro-metaphase is inhibited and thirdly
the spindle mechanism is disrupted. Each of these should
be considered a separate reaction or the recognizeable ef-
fect of a separate reaction for several reasons. First,
these mechanisms may not all be associated with the use
of certain chemicals. For instance, colchicine is probably
the most effective chemical for producing spindle damage

below toxic levels, and for allowing the recovery of poly-

ho

Al

ploid cells during subsequent division cycles. However,
no recognizeable mitotic inhibition or prophase stalling
occur. A second reason for considering the effects as sep—
arate is the Specific point during the mitotic cycle at
which each effect must be produced. That is to say that
cells in the presence of the chemical will continue their
normal mitotic changes up to the specific susceptible point.

Figure 8 shows diagrammatically the susceptible points
of the mitotic cycle in the pea root meristem. Van't Hof
£3 a1. (1960) reported an average mitotic cycle duration
of twelve hours for the pea root meristem by colchicine
tagging. This figure has since been confirmed in the same
laboratory by a somewhat different method by Bekken (1965).
More recently, Van't Hof (1965) reported a fourteen hour
cycle with colchicine tagging, and, from tritiated thymidine
treatments has calculated mitosis and "62" to be 3.5 hours,
the "S" (DNA synthetic) period to be 5.8 hours and "Cl" to
be h.7 hours, by difference. However, he calculated cycle
time from the beginning of colchicine treatment, rather than
from the colchicine index peak which usually occurs about
two hours later. The polyploid cells which one sees during
the second mitotic period correspond to those which are
affected by the colchicine and are apparent two hours after
treatment. This timing, then, should correlate with the
other reports of a twelve hour cycle, and the "Cl" period,

which is computed by difference, would then be about 2.7

Figure 8. Diagrammatic interpretation of the points of mi-

totic disruption, compared to the Pisum mitotic cycle.

 

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hours in length.

In order to correlate the chemical effects with var-
ious portions of the mitotic cycle, mitosis will be con-
sidered first. Van't Hof (1965) allotted 3.5 hours to "G2"
and mitosis, 1.8 of this to mitosis itself. However, mitosis
may require as long as 2.0 to 2.5 hours. The length of
mitosis, as measured by microscopical examination of the
nuclear configurations, may vary considerably, depending
on the investigator's subjective judgement as to the be-
ginning of early prophase stages and the end of the telo-
phase stage. Since the post-prophase stages occupy less
than half of the total mitotic time, spindle disruption
occurs within the last hour or slightly earlier. That
c-mitosis is induced during the actual presence of the spin-
dle is shown by the almost immediate production of c-mitotic
configurations by chemicals with very rapid ingress and
egress, such as the carbamates (Wilson, 1965). Prophase
stalling occurs just slightly prior to this point, since
the accumulation of late prophases can be seen within one-
half hour subseouent to treatment. The point of mitotic
inhibition is somewhat more difficult to localize, and it
probably occurs over an extended portion of the cycle.

Since no immediate response is noted, there is certainly

no inhibition of cells very near the onset of mitosis.

For this reason, a period known as "B" has been indicated,
which probably corresponds to some segment of the "62" per-

iod. Once into the "B" period, cells are not prevented

Lb

from continuing mitosis, unless they encounter a later
blockage point. However, cells in the "A" period are inhib-
ited. Since no reduction in the number of dividing cells
is seen until after two hours after treatment, the center
of period "A" must occur at least two hours prior to the
center of the mitotic period. Therefore, the "A" period
has been extended back into the "8" period. Although the
inhibition may be due in part to reactions occurring in
"02", the susceptibility probably also is present in the
late "S" phase.

Each of these three types of activity is manifested
in a definite pattern over a period of time. Figure 9
shows the mitotic index pattern produced by treatment with
to mg/liter of 2,h,5-TCP. The curves of the other phenols
closely follow this type of pattern. As noted previously,
certain chemical treatments seem to produce a slight stim-
ulation during the first one or two hours after treatment.
The low point is usually near four hours, as in this case.
Although the depression is seen to be from two to eight
hours in duration, depending on the chemical and dose used,
the inhibition is not necessarily still in force at the time
at which the depression is recognized by the lack of divid-
ing cells. In a moving cyclic system such as the mitotic
cycle, an inhibition which occurs several hours prior to
the presence of recognizeable configurations is not noticed
until several hours after it occurs. Likewise, the release

from inhibition is not seen until several hours later.

Figure 9. Mitotic index in percent of control from treat-

ment with LO mg/liter of 2,h,5-TCP versus time.

 

#5

 

 

 

 

160 r-
120 -
80 #—
llo (—

1
)L/

mu: onoam IO-IWOO JO WOO-19d

12

10

Hours

Figure 9.

L6

The recovery portion of the curve reaches a point con-
siderably above the control level. The time required for
the index to cross the 100% line on the recovery leg is de-
pendent on the dose level. This may be due to the need of
a "threshold" concentration in order for the reaction to
ensue.

The rates of movement of the phenols into and out of
the cell are not known, but it is evident from the immed-
iacy of some of the antimitotic effects that their entry
is fairly rapid. This is not surprising, since all of these
compounds are very lipid-soluble, and should certainly pass
through the cell.membranes with ease. However, the exit
of the chemicals at the end of treatment (and the consequent
reduction in their concentration at the active site) does
not occur with as much ease. The phenols have good bind-
ing qualities which permit inter-molecular hydrogen-bonding
and hydrogen-bonding between the phenols and other sites of
appropriate attraction. Since most of the phenols also
dissociate with fair ease at the proper pH values, ionic
bonds also could occur. LipOphilic attractions may also
serve to maintain high levels of these compounds in the tis-
.sues even after removal from the treatment solution. Thus,
the cell may require a longer period of time to reduce the
higher concentrations of phenols to levels below the effec-
tive "threshold" level.

The plot of the E/L ratio versus time resulting from

treatment with a 60 mg/liter dose of 2,h,5-TCP in Figure 3

A7
is typical of the effects of prophase stalling induced by
the phenols. A very immediate drop is noted, indicating
an immediate blockage of the transformation of late pro-
phase nuclei to the pro-metaphase stage. This results in
an increase in late prophasasas compared to early prophases.
By the time of the lowest point, usually 2-2% hours after
treatment, the mitotic inhibition may also begin to affect
the ratio, in that most early prophases have moved on to
late prophase, and no more cells are permitted to enter '
early prophase. However, the ratio begins to increase from
this point, indicating that cells are being allowed to move
on to later stages from late prophase. Since the mitotic
inhibition is still in effect, very few cells are permitted
to enter the prophase range, but of these few cells, the
ratio of early to late prophases returns to more normal
levels, usually by four hours. Although supra-normal levels
may occur with recovery, the great variation in normal con-
trol root meristems (from about 1.50 to almost 3.50) makes
it difficult to distinguish a significant increase.

The pattern of c-mitotic action of the phenols, where
it occurs at fairly high levels, is similar to the specific
curves shown in Figure 1. Again, the rising portion of the
curve is much more rapid than the drop-off, which tends to
form a shoulder. The colchicine index may be used as a
guide for the prediction of the amount of polyploid cells
in subsequent division cycles. Since the maximum indices

obtainable with the phenols does not exceed 1.00, one would

#8
expect very few polyploid cells to be recovered. This was
found to be true, and only small, but significant, numbers
of polyploid cells were found with several treatments. The
c-mitotic effect was used to determine the dose-dependence
of this reaction as induced by the organic phosphorus com-
pound, Ruelene (II). The effect was scored as the percent
of abnormal post-prophase cells, consisting entirely of
"scattered" and "clumped" configurations, without weighting
the "clumps" as two and the "scatters" as one, as is done
with the colchicine index. The curves were plotted for six
doses of Ruelene (II) ranging arithmetically from 125 mg/
liter to 200 mg/liter. The area under the curve of each of
these concentrations was calculated in arbitrary units for
the period of maximum effect, 1/2 to 2-1/2 hours after
treatment. These arbitrary units were computed as the per-
cent of total area possible between these limits and then
plotted as the percent effect on a probability scale versus
the log dose (Figure 10). A straight line was obtained,'
confirming the sigmoid character of the dose-effect rela-
tionship.

The fact that mitotic inhibition is fairly sensitive
to dose makes possible the comparison of the relative bio-
logical activity of the various phenols, from which certain
general principles regarding the nature and position of
the substituent groups may be drawn.

It should be pointed out that inhibition of mitosis

and toxicity may be the end results of two very different

Figure 10. Probit percent effect plotted versus log dose
of Ruelene treatments of 125, 1L0, 155, 170, 185 and 200
mg/liter.

 

—.¢-_ *.* -——-—. —-—-——-—- -—

#9

 

Am.._<om >._._..=m<momn: hummum 8

I00

IN PPM (LOG SCALE)

DOSE

50

reactions or mechanisms, so that no correlation need neces-
sarily exist between the minimal effective doses and the
maximal sub-toxic doses. Rather, the order of the compounds
classified by the toxic levels should differ from the anti-
mitotically effective order depending on the breadth of the
dose range of each compound below the dose necessary to
produce the toxic reaction.

Many factors are undoubtedly involved in the biolog-
ical activity and effective dose levels of compounds at
the cellular level. Any one compound may undergo various
separate reactions within a cell, and may be involved in
several metabolic pathways. The simultaneous action of a
compound on numerous enzyme systems is a common phenomenon.
Effects may even be caused without chemical reaction, by
the alteration of the physical environment at enzymatic or
structural sites. From the diversity of structure in the
many compounds found to exhibit the c-mitotic reaction and
other mitotic aberrations, there arises the possibility that
at least part of the biological reactivity of these molecules
may be due to physical characteristics such as energy po-
tentials, particularly regarding electronegativity, rather
than, or, in addition to, generalized biochemical reactions.
Factors influencing the biological effects of compounds
such as the phenols include such considerations as the rate
of entry and exit of the chemical, which are dependent on
such physical properties as the lipid solubility of the

compound, its charge, and on its propensity to hydrogen-

51
bond or otherwise form attachments of less than covalent
bond strength; the specificity of the chemical for the ac-
tive site; and the ability of the compound to concentrate
at the site of action.

Different physical or chemical factors may be influ-
ential for each reaction which a compound undergoes. Con-
versely, the same factors may influence the action of one
compound differently from that of another. Thus, it is
not surprising that different relative activities are found
for the same compounds when the criteria of biological ac-
tivity are mitotic inhibition, pea root toxicity or toxicity
to Drosqphila melanogaster, although a certain general order
is common to them all.

Comparisons have been made on the assumption that an
equilibrium exists between the lipid and water phases, and
that the chemical potentials of the compounds are the same
in each phase, so that the chemical potentials may be ob-
tained from measurements in the circumambient phase. It
should be cautioned that, although any measure of compar-
ative toxicity must involve comparison between the amounts
required of different chemicals to produce the same effect,
the measured toxic concentration of a given compound is not
necessarily an indication of the concentration at the site
of action.

Without consideration of the position or number of
substituent groups, it can be stated that the nitro group

is the most effective in inducing mitotic inhibition, and

52

that the methyl group is the least effective. The bromo
and chloro groups are midway between these activities, and
are rather close in comparable effectiveness. However,
2,h-DBP is both more effective antimitotically and more
toxic (in both test systems) than are any of the dichloro-
phenols. For this reason, it is likely that the bromo sub-
stitution results in more biological activity than the
chloro substitution. The failure of 2,4,6-TBP to be more
active than the trichlorophenols (although close to them
in value) may be due in part to stereochemical interference
at the active sites due to the larger size of the bromine
atom (van der Waals radius of 1.95 2) as compared to the
chlorine atom (van der Waals radius of 1.80 R). In 2,4,6-
TBP, two bromine atoms flank the hydroxyl group of the
phenol.

In general, also, the tri-substituted phenols are
more active than the di-substituted ones. This might be
expected if one considered the substituents to influence
the reaction of the compound as a whole; thus, if one group
created a given amount of activity, two groups could pro-
duce a greater activity, and so forth. There is the ex-
ception of 2,4,6-TBP being less effective than 2,h-DBP in
the pea test system. Again, this may be accounted for by
steric hindrance.

The position of the substituent groups is undoubtedly
of some importance also. From the order of mitotic inhi-

bition, it is seen that amongst the chlorinated phenols,

53

the four most effective ones are all substituted in both
.95339 (2 and 6) positions. The next lower one in activity,
2,h,5-TCP, has only one ggthg position filled, but would
be expected to manifest considerable activity because of
the triple substitution. 2,h-DCP is the next lower chloro-
phenol and has one ggghg position substituted. The 3,5-DCP
and 3,4-DCP are indistinguishable from 2,L-DCP in activity,
although they lack the ggghg substitution entirely. In
the same manner, 2,h,6-TMP with two filled 25239 positions
inhibits mitosis at considerably lower doses than 2,3,5-TMP,
which has only one ggghg substituent. It would appear also
that the combination of only one ggthg group plus possible
steric hindrance renders the 2,3,5-TMP less active than
2,h-DMP, which in turn is less active than 2,4,6-TMP.

2,h-DNP is well known as an uncoupling agent. 2,L-
DCP also has some uncoupling capacity, but considerably
less than that of 2,h-DNP. The order of magnitude, and the
reports of uncoupling by 2,h-DBP and several mono-substi-
tuted phenols have led to the possibility that the antimi-
totic activity of phenols is related to their uncoupling
capacities. However, there is no proof that this is the
primary action which results in the cytologically apparent
disruptions.

The question of how the various substituent groups
mediate their effects on the activity of the molecule is
a problem falling into the realm of physical chemistry.

Several possibilities of their physical effect on the mol-

5h
ecule, however, stand out because of the good correlation
with the biological data.

The more electron-withdrawing substituents are found
to result in higher activities, when incorporated into
the phenolic compound. Fieser and Fieser (1957) list as
electron-attracting N02, C1 and Br, with dipole moments
of 3.97, 1.56 and 1.53 respectively. CH3 is classified
as electron-releasing, with a dipole moment of 0.Ll. The
electron withdrawing substituents tend to deactivate the
ring to electrophilic attack, while the electron-releasing
substituents, by displacing electrons into the ring and
increasing electron density at the available carbon centers,
activate them for electrophilic attack. Thus, several
factors may be involved. If we consider the theory of
Blackman gt g1. (1955b) of negatively bonded active cell-
ular sites, it can be seen that the rings deactivated by
electron-attracting substituents to attack by positive ions
are still free to bind to negative sites. Further, the
phenols activated by electron-releasing substituents to
electrophilic attack may become much more susceptible to
hydrolysis and metabolic degradation or detoxification.

The capacity of the phenols to accept or give up elec-
trons could also be involved in a possible mechanism of
interference with hydrogen bonding (for instance, in the
tertiary structure of proteins) essential for the normal
transition of a nucleus from one stage to another.

The theoretical consideration of the problems involved

55
in comparing the relative activities of a series of compounds
such as the phenols are complicated further by the practical
aspects of providing mechanical contact of the test chem-
icals with the experimental materials. For the most part,
these chemicals are soluble in water up to the concentra-
tions used in these experiments, and present no serious
hindrance to uptake. However, 2,h,5-TCP, 2,h,6-TCP and
the organic phosphates IV and VI, were not soluble in
aqueous solutions at the concentrations desired for testing.
These chemicals were suspended in the treatment solutions
by means of the surface-active agent Tween-80, which was
found to have no cytologically detectable affect when used
alone. It is not known what interactions occurred between
the chemicals and the surfactant, nor how strong were the
forces of attraction (and thus the ease of dis-association),
nor the effect of this complex on the uptake by and local-
ization within the cell. However, when used in conjunction
with other chemicals, which also could be dissolved in water
alone, the results could not be distinguished.

No attempt was made to correlate the relative effect-
iveness of the organic phosphorus compounds, because of the
influence not only of the nature, number and position of
substituents on the ring, but also the influence of the
chemical groups attached to the phosphorus atom.

The most important finding in regard to the organic
phosphates was the total inactivity of all compounds with

a sulfur atom bonded to the phosphorus, regardless of the

56
ring substituents or the esteratic groups attached to the
phosphorus atom. Insofar as toxicity to insects is concerned,
the P== 3 form is also inactive, and must be enzymatically
converted to P:: 0, which is considered the active form
(Metcalf, 1955). If the P== S form is also inactive in
causing lethality in plants, then enzyme systems similar
to those in the insects must be present in certain plants,
since Zytron (X) is used as a selective herbicide for crab-
grass (Crafts, 1961). Pisum sativum apparently is not cap-
able of such an enzymatic conversion at a rate sufficient
to supply effective levels of the active form.

The P:: 0 forms of these organic phosphates are very
active phosphorylating agents, and it is by this means that
they mediate their toxic action on insects. The following
is an interpretation of the phosphorylation mechanism sug-

gested by Metcalf (1955):

 

O. (I) ['77“\ 0,. I (_)
GH + C -R ——+ m—Io P-O
o o R R/ ,5

SH F
-I—O ¢ (3) [LO (2) I; ,I_ + //’” \

I' +IH20
HO- -P—’(0R)2 (3R)2 (0W2) R "
0 represents an electron transferring group and H an acidic
portion of'some susceptible enzyme. By the phosphorylation

of the enzyme, it is deactivated. At the same time, the

57
corresponding simple substituted phenol is released. Ap-
plying this system to the data obtained from treatment of
the pea root meristem, it could be suggested that the anti-
mitotic action of the organic phosphorus compounds is due
to the liberation of the phenol. Further, step 3 is rate
determining, and if sufficiently slow, will allow the build-
up of inactivated enzyme, which could result in other side
effects or toxicity. Comparative toxicity data given by
Metcalf (1955) of diethyl aryl phosphates on topical ap-
plication to figsgg domestics agree well with our results.

With compounds of the type

 

the LD5O values in X/g for substitutions in the para posi-
tion were: nitro-~O.5, chloro-~150,,Eg§§.-butyl-—)>500
and methyl--)>500. With Apis mellifera the LD50 values
were: nitro--O.6, chloro—->100, _t_e_r_§.-butyl-->100 and
methyl--200. Thus, the order of effectiveness of the sub-
stituent groups in the phenolic organic phosphates appears
to follow the same trend as in the simple phenols, i.e.
N02>Cl> CH3.

From this reasoning, then, it follows that any effects
of the groups on the phosphorus atom are not directly in-
volved in the reaction causing mitotic aberrations, but
influence the solubility, the stability to hydrolysis or

other physical properties which prevent uptake, movement

58
or phOSphorylating capacity of these compounds.

The analysis of chemically induced mitotic deviations
in the standardized pea root meristem test system was found
to be as useful in determining the relative biological ac-
tivity of a series of related compounds as are the more
usual insect toxicity tests, growth regulator systems and
bactericidal Systems. Biological activity of one type,
such as antimitotic activity, is not, however, strictly
comparable to activity of other types, such as toxicity or
metabolic disturbances of other types. Although parallels
in activities and relative effectiveness may be drawn from
data obtained on separate test systems, the divergent met-
abolic schemes and protective devices of different organ-
isms prevent them from reacting in the same way to the same
stimulus. Because of this, it is to be hoped that data
from other workers in agreement with ours will tend to
strengthen our conclusions, while that in opposition can

be attributed to inherent biochemical and physical dif-

ferences.

S UM:~ .1 ARY

All compounds tested of the series of substituted phen-
ols and the series of organic phosphorus compounds with
phenyl nuclei were found to have three distinct effects on
the mitotic cycle. These are: pre-prophasic inhibition,
prophase blockage and spindle disruption.

The substituent groups on the phenol ring have an in-
fluence on the antimitotic activity of the compounds. Those
compounds with nitro substitutions were most effective and
those with methyl substituents were least effective. The
bromo- and chlorophenols were intermediate in effectiveness.

Compounds with three substituent groups generally
showed an antimitotic action at lower doses than those with
only two groups. The ggthg positions on the ring appeared
to be especially important for activity.

Presence of the P22 8 group in the organic phosphorus
compounds eliminated their antimitotic effect. Those with

2:0 were all effective.

The structure-activity relationships may be explained
in terms of the physical and chemical characteristics of
the molecules insofar as these are determined by the struc-

tures e

59

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65

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APPENDIX

66

67

Table 1. Abbreviations for Substituted Phenols

2,h-DCP 2,h—dichlompheno1

2. 3-DCP 2, 3-di ch10 rophenol

2. 6-DCP 2. 6-dichlo rophenol
3.14430? 3.14-dichlor0phenol

3. 5-130? 3 , 5-6.1 chlorophenol

2, h-mlP 2.u-un1trophono1
2.14433? 2.1+-d1bronophsnol
2.144119 2.14-d1methy1phono1

2. a. 6-m 2.1;, 6—trinetlwlphenol
2. 3. S-THP 2. 3. 5-trimethy1phenol
2, h. 6—TBP 2. 1+, 6- tribromophenol
2.h. $410? 2,h.6-trichlor0phenol
2,3.6—‘1‘0? 2,3.6-trichlorophenol

2.h.5—TCP 2,1}.5-tr1chlor0phsnol

68

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Table 8. Averag- Control Mitotio Indicoo.

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Table 10. Minimal Effective Done and "axial Sub-toxic Doses of all
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M term! IQ'uM, finial-MI 10-h“:
234-136? < 50 < 3.07 70 b.29
2.3-1»? 100 6.13 200 12.27
2,6—DCP < 25 < 1.53 200 12.27
3,24.” < 50 <. 3.07 100 6.13
3.5-1»? < 50 < 3.07 100 6.13
2.1mm; 5 0.27 7 0.38
2.14433? < 25 . < 0.99 100 3.97
2.1531? 100 8.19 > 300 >21». 56
2.0.64}? 100 7.31» > 300 >22.03
2.3.5—M 200 10.69 > 300 >22.o3
2.0.643? < 50 < 1.51 200 6.05
2.0.640? 10 0.51 > “0 > 2.03
2.3.6-m? < 25 < 1.27 > 100 > 5.06
2,l+,5-'rcr < 1+0 < 2.03 80 n.05
II 125 n.29 200 6.86
N 75 2.56 125 0.2?
V1 <100 < 3.63 > 100 > 3.63

81

Table 11. Orders of Effectiveness for Mitotic Inhibition and Toxicity
Based on Minimal Effective Doses and Mimi Sub-toxic Doses.

 

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2m 1221;121:2314 mm nos- (x 10-51.)
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2.0.6.011)? 0.51 2.h.6—1'C.P > 2.03
2.1mm? < 0.99 VI >3.63
2.3.640? < 1.27 2.u-DB? 3.97
2.0.643? < 1.51 2.0.540? 0.05
2.640? < 1.53 n 0.27
2.u.5-T0? <2.03 23:40? 0.29
N 2.56 2.3.640? >5.06
2.0-00? <3.o7 2.96m 6.05
3.5-1»? < 3.07 3.5-1»? 6.13
3.040? <3.07 3.9-1»? 6.13
VI < 3.63 11 6.86
11 n.29 2,640? 12.27
2.3—nc? 6.13 2.340? 12.27
23.5.99 7.31. 2.0.641? > 22.03
2.1mm? 8.19 2.3.5419 > 22.03
2.3.5.111? 1h,69 2.144)? >2b.56

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Table 12. L1) 0 Values for Chemicals Tested by the 21‘0'021’1118 System in
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2.3.640? 13.0 0.66
2.11.6.1!” 22.5 0.68
2,h,5-'1‘CP 114.0 0.71
2.14-1.73? 22.0 0.87
2.14430? 15.8 0.97
2,3—DCP 19.5 1.20
3.5-DCP 20.0 1.23
3.15-DCP 23.0 1.111
' 2,h,6-1‘0P 28.5 1.44
2.640? 31.0 1.90
2.11.6410 28.0 2.06
2.3.5-1‘14!’ 30.5 2.211

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