Tissue Culture Experiments on the Biological Action of Methyl bis (s-Chlorethyl) Amine and Its Hydrolysis Products

HONOR B. FELL* AND C. B. ALLSOPPt

(From the Strangeways INTRODUCTION Research Laboratory, Cambridge, England)

The $-chlorethylamines
mustard gas and

differ chemically
(the

from

vestigation concerns the action of methyl bi$(fi chlorethyl)amine on fibroblasts cultivated in vitro.

its analogues

$-chlorethyl

It was made during 19492 and 1943 on behalf of the

Chemical
Ministry

suiphides), whose effects on tissue culture were described in previous communications (5, 8), only in the replacement of a sulphur atom by an amino radical. In many respects, the biological effects of the two classes of compound are similar, especially in their growth-inhibitory action on tissues which are in a state of active proliferation (6), and for this reason the nitrogenmustards in particular have already found clinical application, e.g. in the treatment of Hodgkin's disease. The present in

Defence
of Supply,

Research

Department

of the
described

and was originally

in two confidential reports (4). We are indebted to the Chief Scientist of the Ministry of Supply for permission to publish the results. The experiments were made with the free base,

(compound I). In aqueous solution, this under

goes a complex series of reactions. The first stage,

achieved rapidly, involves cyclisation to the qua ternary ethylenimonium chloride, (compound II):

Cl.CH,.CH2\
Cl.CH2.CH/

CHI\
CH/

+ ,CH3
Cl; \CHI.CHI.C1

@N.CH@I N' @
(I) (II)

A slower second stage yields either the mono- or di- hydroxy compounds, (III), (IV) or (V): /CH2@CH2OH
CH,.N'

7CH2.CH,.OH
\

\CH2 CH2 .Cl@CH3NCH2

\@H2

+/

Cl

(III)
and

(IV) /CHI.CHI.OH
CH3.N'

\CH2.CEI2.OH
methyl diethanolamine, (V) ; or the piperazinium dimer:

CH3\+/CHICl .CH, . CH/

(Hornby et al, 7).

CHI\/CHI
92C1

\dH,

CH,/

\cH,Cn,Cl

(VI)

aFoulerton Research Fellow, Society. Royal

t Now ReadernPhysicstGuy's i a Hospital Medical School, London.

9238

FELL AND ALi@1soPPChemotherapy

in Tissue

Culture its application,

9239
and in the

The aqueous solutions of methyl bis(@-chlor ethyl)amine develop an acute toxicity (as test ed on mice), associated with a rapid convulsive and lethal action, and remain toxic indefinitely (3). In neutral solution, the maximum toxicity is
reached after about two days. It has been attrib

tinuously

for some time after

although

a slow coagulation

then began as mdi

cated by the absence of Brownian movement side the cell and a certain rigidity of its surface,

protoplasmnever became opaque and refractile as in cultures treated with mustard gas. When the
excess liquid was drained away, that infiltrating the cells disappeared and their structure could be

uted to the half-hydrolysis product, (III), or to the cyclised form, (IV), into which it is converted
On standing. The dimer is not toxic. The effects on fibroblasts of the crude aqueous solutions of the
base and of the individual hydrolysis products

examined in detail. They appeared greatly at tenuated, their outlines were indistinct, and their nuclei extremely pycnotic, again in strong con
trast to the cells of cultures mustard gas (Fig. 1). treated with liquid

present

in them

are described

in this paper.

MATERIAL
The tissue was

AND METHODS
from the choroid and

obtained

II. The effect of the chiorethylamine vapour on cells

in vitro.

sclerotic of 1 1 to 192 day chick embryos and was grown by the ordinary hanging-drop method on 1@

inch square coverslips;

mixture of equal parts

the culture medium was a

of plasma, to which the ac

1. Saturated vapour. The coverslip of each prepa

ration was raised, a single drop of the liquid chlorethylamine was placed in the hollow of the slide below but not in contact with the tissue, and the coverslip was then re-sealed in position with

tive agents were added as described later, and of embryo extract made with Pannett and Compton's
saline. The cultures were used for experiment after one or two passages in normal medium; for his tological study they were fixed in S per cent acetic

paraffin wax and the culture was incubated.

small volume (92to 3 cc.) enclosed

In the

in this arrange

alcohol for 3 to .5 minutes,

haelnatoxylin, and mounted

stained with Ehrlich's

whole in Canada

ment, equilibrium

between liquid and vapour was

balsam.
EXPERIMENTAL

rapidly established, and the cultures could be as sumed to be in contact with the saturated vapour.

When observed on the warm stage of the micro scope, the cells were seen rapidly to withdraw their
cytoplasmic processes; they often became de

I. The effect of liquid chiorethylamine on cells in

vitro. The coverslips carrying the hanging-drop prepa

tached from the glass and threw out from the sur face balloon-like protrusions which continually
broke off, the cells disintegrating without coagula

rations were removed from the hollow ground slides and the cultures were placed in contact with drops of the liquid base on flat slides, the cover slips being supported on two strips of paraffin wax painted across the glass. The preparations were

tion

(Fig.

92). Saturated

mustard

gas vapour

causes little distortion because, like the liquid, it quickly coagulates or fixes cells (5). the 92. Vapour at lower pressures. For studying the

then re-sealed with wax and either observed on a microscope stage maintained at body temperature
or returned to the incubator for various before being fixed and stained. periods

cytological

effect of lower concentrations

of the

Immediately

after the cells were in contact with

the liquid, the fat globuies in the cytoplasm began to swell rapidly, and in a few minutes the cells became heavily laden with refractile droplets pre cisely as in cultures treated with liquid mustard gas (5). Sometimes the infiltration was so complete

vapour of chlorethylamine, was incorporated in the wax opened into the air space of as was described previously the liquid could be introduced with paraffin wax. The

a narrow inlet tube seal so that one end the culture chamber, (5). A small drop of into the tip of the diffused slowly

inlet pipe, the outer end of which was then sealed

vapour

down the tube, and the vapour pressure in the air

space increased only gradually.

that the globules fused with each other and the

cells appeared as silvery, homogeneous bodies. Apart from the infiltration of the protoplasm

The effect of the chlorethylamine

vapour

was

with droplets of the chlorethylamine,

the cytologi

cal effects of this substance were very different from those of liquid mustard gas. Whereas the latter immediately coagulated the protoplasm, so fixing the cell with little distortion of either nucleus or cytoplasm, the liquid chlorethylamine caused both nucleus and cytoplasm to shrink con

indistinguishable from that of mustard gas vapour under similar conditions. The cells gradually with drew their long processes, while balloons of ac tively streaming cytoplasm were continually pro truded and withdrawn at the surface. The shape of the cell and its nucleus became more and more distorted and fantastic, and small vacuoles often appeared in the interior; eventually the cells broke down.

@1r@@-'

.
4

4
s'@.
@!s':

@.

@f.
2
P

*1

@
All the cultures were fixed in acetic alcohol, stained with
Ehrlich's haematoxylin and mounted whole. Magnification: X875. (Photographed by Mr. V. C. Norfield).
FIG. 1.Four-day stained. Note culture of fibroblasts incubated in con outline of

4
rated chlorethylamine vapour for 30 mins. before being fixed and stained. The cells are greatly deformed and beginning to
disintegrate.
FIG. 3.Culture of fibroblasts grown for 2 days in me

tact with liquid chlorethylamine

the pycnotic culture nuclei

for 30 mins., then fixed and

and the indefinite in contact

dium containing 200 -j/cc. of methyl diethanolamine. Note

the shrunken nuclei and enormously vacuolated cytoplasm.
FIG. 4.Normal (control) culture of fibroblasts from the

the greatly attenuated

FIG. 2.Similar

cytoplasm.
incubated with satu

same experiment

as the preparation

shown in Fig. 3.

FELL AND ALi@oPpChenwtherapy

in Tissue

Culture

9241

III. The effecton cells in vitro of low concnfi'ations the end of the first day, when S of the 6 cultures
of methyl bi@($-chlorethyl)amine dissolved in had no outgrowth at all, and the sixth very little.

the culture medium, (Table 1).

For these experiments, cultures were grown for two days in normal medium. They were then
grouped known After into a number concentration a further of equivalent sets of six and

A sudden, surprising improvement occurred dur

ing the second 924hours; the behaviour of the cul tures suggested that the toxicity of the medium
was diminishing, so that if a culture was not killed

each set was transferred being returned to normal

two days'

to medium containing medium

incubation

of chlorethylamine,

one set the cultures

as a control.

were fixed and stained. The descriptions in Table 1 refer to their condition at the end of this second incubation period.

at the beginning of the experiment, it was subse quently able to revive and grow. It seemed that the culture medium itself might be exerting a detoxicating action, and this possibility was in vestigated in experiment 92.

Experiment
chlorethylamine

@. solution of 0.01 cc. of the A

in 10 cc. plasma was incubated

TABLE 1 SUMMARY OFEXPERIMENTSSHOWING(A) THE EFFECTONGROWTHOF DIFFERENTCON

CENTRATIONS OF METHYI@ bis(fl-CHLORETHYL)AMINE IN THE CULTURE MEDIUM,

(B) THE DETOXICATING ACTION OF PLASMA ON THE CHLORETHYLAMINE.

Concentration of chlorethylamine in medium (-,/cc)

Conditions

of preparation
solution

No. of
cultures

Growth after
48 hours

No. of cap.

of chlorethylamine

0.0 0.5 5.0

50.0

Control
Added just before use

6 6 6
6

6:+++ 6:+++
@1:

0.0 So .0 50 . 0
100 . 0 100 . 0

Control Added just before use Incubated

Added Incubated Incubated just

6 6 6
6

24 hours in plasma
before 24 hours 24 hours use in plasma in plasma

I 2: + + I 1: 6: ++ I 1: +
I 4:
+ 6: 6: 6:

6:

+++

200 .0
200.0

Added just before use

6 6 6
6

++ ++

0.0
200 .0

Control
Added just before use

6:+++

18 18

f1:+
@17:

@ +++

200 . 0

Incubated

good growth; ++

f12: +++ 6: fair; + = poor; = very poor; no growth. 24 hours in plasma

Experiment 1. The experimental culture media contained respectively 0.5, 5.0 and 5O.0@yper cc. of chiorethylamine. They were prepared by vigor

for 924 hours

at body

temperature.

Culture

media

ously stirring 0.01 cc. of the liquid with 10 cc. of fowl plasma at room temperature until it had dis solved. Portions of the solution were further di
luted with appropriate volumes of plasma and im

were prepared from it, as described above, to con tam respectively 50, 100 and 9200'yper cc. of the base in the clot, and a set of cultures was trans
ferred to each of these media. Three further sets

were transferred

to media containing

the same

mediately chilled at ()OC. The final clots to which the cultures were transferred were composed of equal volumes of embryo extract and of these
plasma dilutions.

concentrations of chlorethylamine, which had been prepared without the initial incubation. It was found (Table 1) that whereas a concentration

Table 1 shows that by the end of 92days' incuba tion, a concentration of 5O'y per cc. of the chlor ethylamine had produced a slight but definite inhibiting effect on growth. This retardation of
growth, however, was much more pronounced at

of 9200-y per cc. prepared immediat4y before use was lethal to the cells, the same concentration prepared from the chlorethylamine solution in plasma

which had been incubated for @24ours, had little h

effect on them. This result was confirmed in Experiment 3, which was a repetition of experiment 92at the high

92492
est concentration of the agent. Of 18

Cancer Research
cultures

transferred

to freshly-prepared this exception

medium

containing

92007 per cc. of chlorethylamine,

slight growth;

only 1 showed

may have been due to

a slightly uneven distribution of the agent in the clot. On the other hand, 18 cultures in the same concentration of chlorethylamine prepared from a solution which was initially incubated for 924hours, grew almost or quite as well as did the controls.
TABLE 2
EXPERIMENT SHOWING THE EFFECT ON THE DETOXI CATION OF METHYL BIS(fi-CHLORETHYL)AMJNE BY PLASMA, OF VARYING THE PERIOD OF INCUBATION
Concentration of

92.The effect on detoxication of increasing the con centration of the chiorethylamine in the parent solu lion. Table 3. Experiment 5. To 10 cc. of plasma in each of 4 tubes were added respectively 0.01 cc., 0.05 cc., 0.92 cc., and 0.5 cc. of liquid base. The tubes were thoroughly shaken and were then in cubated for 924hours. Each solution was diluted
with fresh plasma immediately before use to give a concentration of 92007 per cc. in the final culture

medium, and each was tested on a set of 6 cultures; a control set was kept in normal medium. The detoxicating action of the plasma, as can be seen
from the table, decreased with increasing concen

tration
Incubation period of No. of chiorethylamine cultures
in plasma used

of chlorethylamine.

chlorethylamine
No.
Exp.

of

in medium
(y/cc)

Growth after 48 hours

0.0
200.0

Control 2 hours 6 , 12

6 6 6

6:

+++

6:

6:
(1: +-@@2:+

6 6

ts:
24 6:++

V. The effecla on cells in vitro of aged aqueous solutions of methyl bis(fi-chlorethyl)amine. 1. Comparison of the influence on the toxicity of the chlorethylamine of incubation in (a) saline, (b) plasma. Table 4. In experiment 6, 3 sets of cul tures were grown in media containing 50, 100 and

92007per cc. respectively of chlorethylamine which

had been previously incubated at 37 . for 924 C
hours in Pannett and Compton's saline; S other sets were grown in media containing the same con centrations of base which had been similarly in

It may therefore be concluded that : (1) the lethal

concentration of untreated ethyl bis($-chlor m ethyl)amine for fibroblasts in vitro is between 100 and 9200'y per cc. ; (92) incubation in plasma di
minishes the toxicity of this substance.

The sublethal concentrations of chlorethyla mine, though not preventing cell growth, were not without effect, and the cultures grown in them dis played many abnormal mitotic figures. Clumping and fragmentation of chromosomes, multipolar
figures and multinucleate cells derived from ab

cubated in plasma. The parent, incubated solu tions each contained 0.0925 cc. of chlorethylamine in 5 cc. of the solvent ; after incubation the plasma solutions were diluted with fresh plasma and the saline solutions with embryo extract in such a way that all the culture clots ultimately contained the same proportion of embryo extract.
TABLE 3
EXPERIMENT SHOWING THE EFFECT ON THE DETOxICA TION OF METHYL bis(fl-CHLORETHYL)AMINE OF RAIS ING THE CONCENTRATION OF BAsE IN THE PARENT SOLUTION. INCUBATION PERIOD: 24 HOURS. Concentration of chlorethylamine Volume of chlorNo. of No. of in medium ethylamine in 10 cc. cultures cap. (rny/cc) of parent solution used

normal divisions were fairly common, though less abundant than in cultures grown in sublethal con centrations of mustard gas (5, 8).

IV. Observations on the detoxicating action of

plasma on methyl bi@(@-chlorethyl)amine.

1. Variation of the degree of detoxica.tion with the

time of incubation in plasma, Table 2, Experiment 4. 0.01 cc. of liquid chlorethylamine was added to 5 cc. of plasma in each of 4 glass stoppered tubes,
which
370 C.

Growth after 48 hours

0.0
200.0
,.

Control
0.01cc.
0.05,

6
6
6

6: +++ f5: +++ 6: ++ f2:

f2: +

@1:nfected i

were
for 92,

vigorously
6, 192 and 924

shaken
hours

and

incubated
Each

at

respectively.

0.20,
0.50

6
6

@4:

Of the 18 culturestransplanted to medium con taming the saline-incubated chlorethylamine, only cubation. A set of 6 cultures was grown in each 92,both in the lowest concentration, i.e. 50@y er cc., p medium, and a control set in normal medium. The showed any growth. In contrast, all those grown in results are summarized in Table 92.Under the con the media with plasma-incubated chlorethylamine ditions of the experiment, there was no detoxica grew except one the 9200y per cc. concentra in tion until after 192 hours' incubation in plasma; tion. The detoxicating action of the plasma in this detoxication was not advanced until after 924 experiment appeared to be rather less than in
centration when there has been no previous in hours' incubation. earlier ones.

solution was diluted with fresh plasma shortly be fore use so as to give a concentration in the culture clot of 9200-yper cc. of free base, i.e. the lethal con

FELL AND ALLS0PPChemotherapy

in Tissue

Culture

9243

92.Increase in toxicity of the chlorethylamine

incubation in saline. Experiment

after

7. Table 4. Sets of

cultures

were grown in media containing

50 and

1007 per cc. of the agent, which had been prepared

from (a) fresh chlorethylamine added immediately before use, (b) chlorethylamine incubated in Pan nett and Compton's saline as in experiment 6, and (c) chlorethylamine similarly incubated in plasma. The results set out in Table 4 show that at a con centration of SOy per cc. the untreated base did not prevent, though it greatly restricted, the growth of the cultures, whereas the chlorethylam me which had been incubated in saline for 924
hours completely inhibited it, and the agent in

of Pannett and Compton's saline and incubated for 924hours : an equal volume of saline was then added and the solution incubated for a further 924 hours; (f) 0.1 cc. of chlorethylamine was shaken with 92.5cc. of saline and incubated for 924hours; an equal volume of serum was then added and the solution incubated for a further 924hours. All the solutions were diluted with fresh plasma imme
diately before use in such a way as to produce in

the final culture media containing

solutions

(b) to

(f) a concentrationequivalentto 92007 cc. of per

the free base, i.e. the concentration found to be lethal to cultures in the earlier experiments. A set of 6 cultures was transferred to each medium. The results are set out in Table 5. Serum and

cubated

in plasma was again largely detoxicated.

TABLE 4
SUMMARY OF EXPERIMENTS SHOWING (A) THAT THE DETOXICATING ACTION OF PLASMA
ON METHYL bis($-CHLORETHYL)AMINE IS NOT DIRECTLY INCREASES RELATED TO HYDROLYSIS AND (B) THAT HYDROLYSIS Concentration of chlorethylamine in medium (7/cc.) 0.0 THE ToxIcITY OF THE BASE.

No. of cap. 6

Treatment of chlorethylamine before being added to the culture medium

No. of cultures used 6

Growth after 48 hours 6:+++

Control
24 hours'

50.0 50.0
100.0 100.0 200.0 200.0 7 0.0 ,50.0 50.0 50.0 100.0 100.0 100.0

, , , ,

, , , ,

incubation

, , , ,
,

in saline
, plasma

f2:
6 6 6 6 6 6

,saline ,plasma ,saline ,plasma

6

14: 6@++ 6: 6: + 6:

f5: +
ii: 6:+++

Control Dissolved in saline shortly before use

24 hours' incubation in saline

6
6 6

12:+
@4:
6: 6:+-I-

Dissolved in saline shortly before use 24 hours' incubation in saline

, , ,plasma , , , ,plasma
6
6 6

6:
6: 6:-@--@-

3. Detoxicating a@ticn of serum on hydrolysed chiorethylamine. Table 5. For these experiments,

serum was used instead of plasma because at the required concentrations the agedaline solu s tions of the chlorethylamine did not prevent the

plasma (solutions b and c) were equally effective in detoxicating the unhydrolysed chiorethylamine. Chlorethylamine hydrolysed in saline after 48
hours (solution e), at a concentration of 9200'y per

plasma from clotting during incubation. This sub stitution was controlled as described below, and did not influence the results. Experiment 8: The following solutions were prepared : (a) con trol : normal serum was incubated for 924hours at 38.5 ; (b) 0.01 cc. of chlorethylamine was shaken C with 5 cc. of serum and incubated for 924hours; (c) 0.01 cc. of chlorethylamine was shaken with
5 cc. of plasma and incubated for 924 hours ; (d)

cc., killed all the cultures. In contrast, when it was initially incubated in saline for 924hours and then treated for 924hours with fresh plasma (solution f), it only partially inhibited growth. This observation was confirmed in experiment 9.
Solutions (i), (@)and (k), Table 5, were repetitions

of solutions (d), (e) and (f) of experiment 8; but the two control solutions (g) and (h) were differ ent. Solution (g) contained the same proportion of
saline as did solution (j), and to solution (h) the same quantity of previously incubated serum was

control : 0.1 cc. of chlorethylamine

92.5 cc. of serum and incubated

was shaken with

for 924 hours; an

other 92.5cc. of serum was then added and the solu tion incubated for a further 924hours ; (e) control: 0. 1 cc. of chlorethylamine was shaken with 92.5cc.

added as was present in solution (i). The experi ments indicate clearly that the hydrolysis prod ucts of methyl bis(j3-chlorethyl)amine can be partially detoxicated by the action of serum.

@
9244

Cancer

Research

VI. The toxicity of the substances produced during

hydrolysis of methyl bis(13-chlorethyl)amine.

1. Methyl diethanolamine (compound V). The toxicity of the di-hydroxy-derivative of the chlor ethylamine was compared with that of the par ent chloro-compound in experiment 10. Fifteen mg. of the compound was dissolved in 1 cc. of plasma, and diluted with fresh plasma so as to give concen
trations in the culture clot of 9200, 1000 and 920007

1000 and 920007 per cc. as compared with 92007 per cc. of the chlorethylamine, i.e. it is less toxic. Un der the conditions of the experiment, it was not detoxicated by incubation in plasma. In all the concentrations used, methyl diethan olamine had a fairly drastic effect on the struc ture of the cells (cf. Figs. 3 and 4). The nuclei were rather shrunken and the cytoplasm of the fibro blasts in all the treated cultures became honey

TABLE S
SUMMARY OF EXPERIMENTS SHOWING THAT HYDROLYSED METHYL bis(@-CHLoRETHYL)AMINE IN SERUM CAN BE PARTLY DETOXICATED Concentration of chlorethylamine No. of
cap.
in medium

BY INCUBATION

afterSolution

(7/cc.)

hours(a) Normal ++(b) (c) serum ,

added to the culture mediumcultures incubated 24 hours66:

No. ofGrowth used48

0.0 200.0
+++,

Chlorethylamine ,
,

incubated
,

24 hours in serum
24 , , plasma6 66: 6:

+++

,(d)
9 0.0 0.0 200.0

48
48

,
,

, serum
, saline

(e)

6:

(f)

24 (a) (b) 24 incubated

, ,

, saline 1@ , serumf

6
6{@

6
6:

(g) Normal saline6 +++(h) Normal serum +++(i)

incubated 24 hours66:

(j) (k)

Chlorethylamine

, 48 , (a) 24
(b)24

48 hours in serum

, ,
,

, saline , saline@
,serumf6

6
66:

6:
6@

+++

per cc., solutions

tions were used

(b), (c) and (e), table 6; the solu

shortly
as

after
solutions

preparation.
(c) and

Solu
(e) re

tions (d) and (f), which contained

centrations of agent

the same con

combed with vacuoles ; many of the cells grown at a concentration of 10007 per cc. were reduced to a mere shell of protoplasm enclosing an enormous
central vacuole. These vacuoles contained a clear

TABLE 6
EXPERIMENT SHOWING (A) THE EFFECT ON GROWTH OF DIFFERENT CONCENTRATIONS OF METHYL DIETHAN OLAMINE AND (n) THAT METHYL DIETHANOLAMINE IS

Nor DETOXICATED
Concentration

BY INCUBATION IN PLASMA.

of diethanol
amine in Treatment of

No. of
cultures Growth after

No.of exp.

medium (7/cc)

dietlianolamine

used 6 6:

48 hours +++

fluid in which particles in active Brownian move ment were seen. The formation of cytoplasmic aqueous vacuoles . in fibroblasts treated with various nitrogen compounds has been described by other observers (9, 10). 92. Methyl @9-hydroxy-eth@jl-fl-chlorelhylamine; compound III. Experiments were made to deter mine the lethal concentration of the mono-hydroxy derivative of the chiorethylamine. On account of
its very hygroscopic nature, it proved impossible

10

(a)

0.0 Control

(b) 200.0
(c)1000.0

Added just before use

Incubated24 hoursin plasma

6
6 6 6 6

6: ++
6: + 6: + 6: 6:

(d) 1000.0

(e)2000.0 Added just before use Incubated 24 hours in (f) 2000.0

plasma spectively, were prepared from an initial solution

of 9292.1 mg. in 1 cc. plasma which had been in cubated for 924hours at 37 . before being diluted C with fresh plasma. One set of cultures was grown in each medium and a control set in normal me dium. It will be seen from Table 6 that the lethal con centration of methyl diethanolamine lies between

to prepare culture media in which exactly weighed quantities of the small order required were dis solved; for this and other technical reasons, direct comparisons on sister cultures, of the toxicity of this substance with that of the parent compound, were found to be unreliable and they will not there fore be described in detail. Sets of cultures were, however, transferred to media containing concen trations of the hydroxy-derivative of the order of
7, 13, 927, 50, 100, 9200 and 4007 per cc. After 48

hours,

the cultures

in concentrations

of up to 9277

per cc. showed no growth and those at 137 per cc. were subnormal. The mono-hydroxy-derivative, therefore, is at least as toxic to cells in vitro as is

FELL AND ALLSOPPChemotherapy

in Tissue

Culture

9245

ate cells. As noted elsewhere (5), the abnormalities resemble those caused by carcinogenic compounds and by x-rays. Similar mitotic abnormalities have Examination of the cultures grown in a concen been observed by Bodenstein (92)in the ectoderm tration of 137 per cc. after 924 hours showed no of amphibian embryos treated with chiorethyl significant outgrowth, and that of the explants amine, and Auerbach and Robson (1) have shown in 77 per cc. was very poor. Both these sets under that this agent has a strong mutagenic action on went a marked recovery during the second 924 Drosophila. hours' incubation, suggesting that this substance also is detoxicated by the plasma. SUMMARY 3. NN'-di-$-chlorethyl-NN'-dimethyl piperazin 1. Liquid methyl bis($-chlorethyl)amine, when ium chloride (the piperazinium dimer, compound applied directly to tissue cultures, infiltrates the VI). Addition of relatively high concentrations of cells via the fat droplets as does liquid mustard the dimer to the culture medium had no effect on gas; but, unlike the latter substance, it distorts the cultures grown in it. It appeared to be nontoxic. cells, which become attentuated and their nuclei the parent chlorethylamine
the latter

and

it is probably

more toxic than

material.

pycnotic;
DISCUSSION

it

does

not

immediately

coagulate

The results described above show that there is a close parallel between the relative toxicities of methyl bis($-chlorethyl)amine and the products of its interaction with water, for cells in vitro and when injected into mice (3). Thus the chlorethyl amine and its mono-hydroxy-derivative are very toxic both to tissue cultures and to mice, whereas the di-hydroxy-derivative is much less injurious and the dimer is non-toxic in relatively high con centrations. The toxicity of the parent substance and of its solutions in saline to fibroblasts in vitro may therefore be attributed to the presence in the culture medium of the mono-hydroxy derivative. Both the chlorethylamine and the mono-hydroxy compound lose some of their toxicity when kept in contact with plasma at body-temperature; this suggests that the chloro-base reacts with protein, probably after partial hydrolysis since detoxica tion is not marked until after 924hours' incubation. Evidence will be adduced elsewhere that such a protein-chloroethylamine compound of low toxic ity can be produced experimentally under appro
priate conditions.

them. 92.Higher concentrations of the vapour produce rapid distortion of the cells, which disintegrate without coagulation ; low concentrations, like those of mustard gas vapour, slowly produce even more fantastic distortions of the fibroblasts, lead ing to their complete breakdown. 3. When chlorethylamine is added to the culture medium, its lethal concentration for cells in vitro
is rather less than 92007 per cc., but if the agent is

incubated in plasma at body-temperature for 924 hours before being added to the cultures, it loses much of its toxicity at this concentration. At higher concentrations the detoxication by incuba tion in plasma is less complete. 4. Incubation of the chlorethylamine in saline increases its toxicity; this toxicity is partially de stroyed by further incubation in serum.
5. 924 hours' incubation in plasma is necessary

to complete the detoxication of low concentrations of the chiorethylamine; the detoxication may therefore proceed through the hydrolysis products.
6. The second hydrolysis product of methyl bis(fl-chlorethyl)amine, methyl diethanolamine, is

The action of the liquid agent and its saturated vapour is less drastic than that of mustard gas in the same forms, but more obvious cell damage is produced since the cells are not immediately fixed the nitrogen mustard. With low concen by
trations of the vapour, the effects of the two agents

much less toxic to cells in vitro than is the parent compound ; the half-hydrolysisproduct is at least as toxic and perhaps more so ; the piperazin ium dimer is non-toxic. Since all these substances are present in aqueous solutions of the free chlor
ethylamine base, the toxicity of the compound

are very similar. The lethal concentration of methyl bis($-chlorethyl)amine for cells in viiro is rather less than that of @$-dichlordiethylsul phide, since cultures show some outgrowth in a concentration of 92007 per cc. of the former sub stance while growth is completely inhibited in the
same concentration of the latter.

may be due to hydrolysis of the mono-hydroxy derivative. This substance also appears to be par tially detoxicated by contact with plasma.
7. Sublethal concentrations of methyl bis(fi

chlorethyl)amine
similar to those

cause

mitotic
by

abnormalities
mustard gas,

produced

carcinogenic

substances,

and x-rays.

In sublethal concentrations the chlorethylamine, like mustard gas, causes severe mitotic disturb ances including clumping and fragmentation of the chromosomes, multipolar figures and multinucle

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Embryonic

Cancer Research
The Effects of Nitrogen
Part

Mustard
I. Ectodermal

on

6. HADDOW, . The Chemotherapy of Cancer. Brit. Med. A

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Amphibian

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8. HUGHES, A. F. W. and FELL, H. B. Studies Mitosis Induced in Chick Tissue Cultures on Abnormal by Mustard

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Sci., In press. auf in vitro gezuchtete Zellen.

Quart. J. Microscop.
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Hoppe-Seyler's

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physiol. Chem., 279:206, 1943. 10. POMERAT, M. and EMERSON, A. Induction of aque C. G. ous Vacuolization in Cells Grown in Tissue Culture by Various Agents. Proc. and Trans. Texas Acad. Sci., 28:91,
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